Early Steps Research Articles

Early steps of protein disaggregation by Hsp70 chaperone and class B J-domain proteins are shaped by Hsp110.

Hsp70 is a key cellular system counteracting protein misfolding and aggregation, associated with stress, ageing, and disease. Hsp70 solubilises aggregates and aids protein refolding through substrate binding and release cycles regulated by co-chaperones: J-domain proteins (JDPs) and nucleotide exchange factors (NEFs). Here, we elucidate the collaborative impact of Hsp110 NEFs and different JDP classes throughout Hsp70-dependent aggregate processing. We show that Hsp110 plays a major role at initial stages of disaggregation, determining its final efficacy. The NEF catalyses the recruitment of thick Hsp70 assemblies onto aggregate surface, which modifies aggregates into smaller species more readily processed by chaperones. Hsp70 stimulation by Hsp110 is much stronger with class B than class A JDPs and requires the auxiliary interaction between class B JDP and the Hsp70 EEVD motif. Furthermore, we demonstrate for the first time that Hsp110 disrupts the JDP-Hsp70 interaction. Such destabilisation of chaperone complexes at the aggregate surface might improve disaggregation, but also lead to the inhibition above the sub-stoichiometric Hsp110 optimum. Thus, balanced interplay between the co-chaperones and Hsp70 is critical to unlock its disaggregating potential.

Regulation of Cx36 trafficking through the early secretory pathway by COPII cargo receptors and Grasp55

Gap junctions formed by the major neuronal connexin Cx36 function as electrical synapses in the nervous system and provide unique functions such as synchronizing neuron activities or supporting network oscillations. Although the physiological significance of electrical synapses for neuronal networks is well established, little is known about the pathways that regulate the transport of its main component: Cx36. Here we have used HEK293T cells as an expression system in combination with siRNA and BioID screens to study the transition of Cx36 from the ER to the cis Golgi. Our data indicate that the C-terminal tip of Cx36 is a key factor in this process, mediating binding interactions with two distinct components in the early secretory pathway: the COPII complex and the Golgi stacking protein Grasp55. The C-terminal amino acid valine serves as an ER export signal to recruit COPII cargo receptors Sec24A/B/C at ER exit sites, whereas the PDZ binding motif “SAYV” mediates an interaction with Grasp55. These two interactions have opposing effects in their respective compartments. While Sec24 subunits carry Cx36 out of the ER, Grasp55 stabilizes Cx36 in the Golgi as shown in over expression experiments. These early regulatory steps of Cx36 are expected to be essential for the formation, function, regulation and plasticity of electrical synapses in the developing and mature nervous system.

Endosomal actin branching, fission, and receptor recycling require FCHSD2 recruitment by MICAL-L1.

Endosome fission is required for the release of carrier vesicles and the recycling of receptors to the plasma membrane. Early events in endosome budding and fission rely on actin branching to constrict the endosomal membrane, ultimately leading to nucleotide hydrolysis and enzymatic fission. However, our current understanding of this process is limited, particularly regarding the coordination between the early and late steps of endosomal fission. Here we have identified a novel interaction between the endosomal scaffolding protein, MICAL-L1, and the human homologue of the Drosophila Nervous Wreck (Nwk) protein, FCH and double SH3 domains protein 2 (FCHSD2). We demonstrate that MICAL-L1 recruits FCHSD2 to the endosomal membrane, where it is required for ARP2/3-mediated generation of branched actin, endosome fission and receptor recycling to the plasma membrane. Because MICAL-L1 first recruits FCHSD2 to the endosomal membrane, and is subsequently responsible for recruitment of the ATPase and fission protein EHD1 to endosomes, our findings support a model in which MICAL-L1 orchestrates endosomal fission by connecting between the early actin-driven and subsequent nucleotide hydrolysis steps of the process.

Activation and modulation of the host response to DNA damage by an integrative and conjugative element.

Mobile genetic elements help drive horizontal gene transfer and bacterial evolution. Conjugative elements and temperate bacteriophages can be stably maintained in host cells. They can alter host physiology and regulatory responses and typically carry genes that are beneficial to their hosts. We found that ICE Bs1 , an integrative and conjugative element of Bacillus subtilis , inhibits the host response to DNA damage (the SOS response). Activation of ICE Bs1 before DNA damage reduced host cell lysis that was caused by SOS-mediated activation of two resident prophages. Further, activation of ICE Bs1 itself activated the SOS response in a subpopulation of cells, and this activation was attenuated by the functions of the ICE Bs1 genes ydcT and yddA (now ramT and ramA , for RecA modulator). Double mutant analyses indicated that RamA functions to inhibit and RamT functions to both inhibit and activate the SOS response. Both RamT and RamA caused a reduction in RecA filaments, one of the early steps in activation of the SOS response. We suspect that there are several different mechanisms by which mobile genetic elements that generate ssDNA during their lifecycle inhibit the host SOS response and RecA function, as RamT and RamA differ from the known SOS inhibitors encoded by conjugative elements.

8469 Endocrinologists Underutilize Virtual Care Services Resulting in Suboptimal Clinical and Financial Outcomes

Abstract Disclosure: D. Terry: Employee; Self; Valendo Health. Less than 150,000 Medicare patients with diabetes (PWD) receive billable telehealth and remote monitoring services, even though over 12 million PWDs are eligible for these services. This is a missed quality opportunity, as studies show that PWDs who receive these services experience reductions in A1C levels and improvements in TIR. It is also a missed financial opportunity for Endocrinologists, who are among the lowest paid specialists in the US. The economics of Endocrinology are important, because inadequate compensation is leading to access constraints when the diabetes population is growing, and care complexity is increasing. We analyzed billed virtual health claims for every Medicare PWD in 2022. The types of services we examined included: Chronic Care Management (CCM & PCM), Continuous Glucose Monitoring (CGM & RPM), Medical Nutrition Therapy (MNT), and Diabetes Self-Management Training (DSMT). When we looked by Endocrinologist by virtual care service type, we found the following levels of service delivery for Endocrinologists at the median levels of utilization:4.8% of eligible PWDs received remote Continuous Glucose Monitoring support.4.2% received virtual Chronic Care Management services.6.2% received virtual Diabetes Self-Management Support.<1% received Medical Nutrition Therapy This is clearly a missed clinical quality opportunity as a study published in JAMA in October 2023, Comprehensive Telehealth Model to Support Diabetes Self-Management, reported the following improvements for PWDs who received virtual care for 6 months:Mean change in A1C levels: Type 1: -0.6%; Type 2: -1.0%Mean TIR Improvement: Type 1: 16%; Type 2: 18% It is also a missed financial opportunity for Endocrinology practices. Each patient with diabetes is potentially eligible for $100 or more of virtual care services per month. We’ve looked at every Endocrinology practice in the US and many are missing out on over a million dollars of annual revenue opportunity. This is important as Endocrinology organizations everywhere, independent and hospital based, are struggling to retain the doctors & staff necessary to provide adequate access to care - leading to months of wait time to see an Endocrinologist. The prevalence of diabetes is growing significantly, while Endocrinologist capacity is shrinking. Value added and billable virtual care services create new opportunities for Endocrinologist to generate the revenue necessary to increase clinical capacity and improve access by creating diverse clinical teams led by Endocrinologists and supported by Diabetes Educators, Dietitians, and others with expertise in diabetes who can care for many more PWDs. Creating virtual care capabilities is also an early step in moving Endocrinology into Value-Based Care arrangements that reward providers for improving care and reducing costs rather than for generating more visits. Presentation: 6/1/2024

A Minimal Hybrid Sterility Genome Assembled by Chromosome Swapping Between Mouse Subspecies (Mus musculus).

Hybrid sterility is a reproductive isolation barrier between diverging taxa securing the early steps of speciation. Hybrid sterility is ubiquitous in the animal and plant kingdoms, but its genetic control is poorly understood. In our previous studies, we have uncovered the sterility of hybrids between musculus and domesticus subspecies of the house mouse, which is controlled by the Prdm9 gene, the X-linked Hstx2 locus, and subspecific heterozygosity for genetic background. To further investigate this form of genic-driven chromosomal sterility, we constructed a simplified hybrid sterility model within the genome of the domesticus subspecies by swapping domesticus autosomes with their homologous partners from the musculus subspecies. We show that the "sterility" allelic combination of Prdm9 and Hstx2 can be activated by a musculus/domesticus heterozygosity of as few as two autosomes, Chromosome 17 (Chr 17) and Chr 18 and is further enhanced when another heterosubspecific autosomal pair is present, whereas it has no effect on meiotic progression in the pure domesticus genome. In addition, we identify a new X-linked hybrid sterility locus, Hstx3, at the centromeric end of Chr X, which modulates the incompatibility between Prdm9 and Hstx2. These results further support our concept of chromosomal hybrid sterility based on evolutionarily accumulated divergence between homologous sequences. Based on these and previous results, we believe that future studies should include more information on the mutual recognition of homologous chromosomes at or before the first meiotic prophase in interspecific hybrids, as this may serve as a general reproductive isolation checkpoint in mice and other species.

Aclarubicin Reduces the Nuclear Mobility of Human DNA Topoisomerase IIβ.

DNA topoisomerase II (TOP2) is an enzyme that resolves DNA topological problems arising in various nuclear processes, such as transcription. Aclarubicin, a member of the anthracyclines, is known to prevent the association of TOP2 with DNA, inhibiting the early step of TOP2 catalytic reactions. During our research on the subnuclear distribution of human TOP2B, we found that aclarubicin affects the mobility of TOP2B in the nucleus. FRAP analysis demonstrated that aclarubicin decreased the nuclear mobility of EGFP-tagged TOP2B in a concentration-dependent manner. Aclarubicin exerted its inhibitory effects independently of TOP2B enzymatic activities: TOP2B mutants defective for either ATPase or topoisomerase activity also exhibited reduced nuclear mobility in the presence of aclarubicin. Immunofluorescence analysis showed that aclarubicin antagonized the induction of DNA damage by etoposide. Although the prevention of the TOP2-DNA association is generally considered a primary action of aclarubicin in TOP2 inhibition, our findings highlight a previously unanticipated effect of aclarubicin on TOP2B in the cellular environment.

T-cell clones of uncertain significance. When is the rogue clone dangerous?

T cell large granular lymphocyte clones that persist over time and that exhibit molecular and immunophenotypic features closely resembling those of T-cell Large Granular Lymphocyte Leukemia (T-LGLL) may be detectable in individuals who lack any clinical or laboratory features supporting a diagnosis of T-cell malignancy. This condition represents a potential precursor state termed T cell clones of uncertain significance (T-CUS). T-CUS represents the even more benign extreme of the wide spectrum of clonal T- large granular lymphocyte proliferations, emphasizing the need for an appropriate multiparametric diagnostic assessment that avoids misdiagnosis of T-cell neoplasia. This approach should overcome the numerical cut-offs as the sole criteria to differentiate the benign condition from the related malignancies. In particular, genomic aberrancies might prospectively identify individuals who are at risk of progression to full blown T-cell malignancy. We herein discuss the significance of these T cell clones in both healthy and disease states suggesting molecular assays for tracking early steps of disease.

A step in the deep evolution of Alvinellidae (Annelida: Polychaeta): A phylogenomic comparative approach based on transcriptomes

The Alvinellidae are a family of worms that are endemic to deep-sea hydrothermal vents in the Pacific and Indian Oceans. These annelid worms, a sister group to the Ampharetidae, occupy a wide range of thermal habitats. The family includes the most thermotolerant marine animals described to date such as the Pompeii worm Alvinella pompejana, and other species living at much lower temperatures such as Paralvinella grasslei or Paralvinella pandorae. The phylogeny of this family has not been studied extensively. It is, however, a complex case where molecular phylogenies have given conflicting results, especially concerning the monophyletic or polyphyletic nature of the genus Paralvinella.We carried out a comprehensive study of the phylogeny of this family using the best molecular data currently available from RNAseq datasets, leading to several hundred orthologous transcripts for 11 of the 14 species currently described or in description. The results obtained by the two most popular phylogenetic inference models (using either gene concatenation with maximum likelihood, or a coalescent-based model from gene trees) were compared using a series of ampharetid and terebellid outgroups.Our study shows that the global phylogenetic signal favors the hypothesis of paraphyly for the Paralvinella genus, with P. pandorae being sister species of the other Alvinellidae.However, a high number of gene trees also supports the hypothesis of alternative trees in which the monophyly of the Paralvinella genus, as initially proposed by Desbruyères and Laubier, is valid with the species P. pandorae and Paralvinella unidentata being sister species. According to molecular dating, the radiation of the Alvinellidae was rapid and took place in a short period of time between 80 and 110 million years ago. This is reflected at the genomic scale by gene trees equally separated between different phylogenetic hypothesis, showing high rates of incomplete lineage sorting between the first lineages of the Alvinellidae and probable gene transfers. Although different genomic regions seem to have different phylogenetic stories in the early step of the alvinellid radiation, our study supports the view that the two P. pandorae species can be grouped into a separate genus (possibly Nautalvinella) and that the Miralvinella subgenus, defined by Desbruyères and Laubier, is not valid anymore.

Phosphorylation strongly affects the inhibition of human carbonic anhydrase I CO2 hydration activity

Human carbonic anhydrases (hCAs) have essential roles in respiration, acid-base balance, and fluid secretion, with implications in diseases such as glaucoma, epilepsy, obesity, and cancer. Of the fifteen known hCAs, human CA I (hCA I) is particularly abundant in erythrocytes, playing a critical role in CO2 transport. Despite extensive research on hCA I, the impact of post-translational modifications (PTMs), particularly phosphorylation, on its catalytic activity and inhibitor binding remains poorly understood. Although multiple phosphorylation sites have been identified in hCA I in vivo through high-throughput proteomics studies including at the highly conserved Ser51 residue, the functional consequences of these modifications are not well characterized. We investigated the effects of a phosphomimetic mutation at Ser51 on hCA I, examining its catalytic efficiency and susceptibility to inhibition by sulfonamides and anions. Using a recombinant expression system and a stopped-flow kinetic assay, we characterized the CO2 hydration activity and inhibition profiles of S51E hCA I compared to the wild type enzyme. Our results demonstrate that the S51E mutation increases the catalytic turnover rate (kcat) from 2.0 × 105 s−1 to 2.6 × 105 s−1 but significantly decreases substrate affinity, raising the Michaelis constant (KM) from 4.0 mM to 13.9 mM, reducing overall catalytic efficiency by over 50 %. Inhibition studies with a panel of 41 sulfonamides revealed that the S51E mutation dramatically alters inhibitor sensitivity, particularly for the most effective inhibitors. For example, 15 of the 16 most effective sulfonamide inhibitors for hCA I (with KIs <350 nM) were an average of over 35-fold less effective in inhibiting S51E hCA I than the wild type. The KI of the anticonvulsant zonisamide increased from 31 nM for the wild type hCA I to 4.0 μM. The inhibition profile with a panel of 37 small anions further indicated that the S51E mutant exhibited significantly reduced susceptibility to inhibition by 24 out of 37 tested anions, with some KI values increasing by up to 11,000-fold for inhibitors like hydrogen sulfide. This study underscores the significant impact that phosphorylation may have on hCA I function and inhibition. By characterizing the effects of phosphorylation on the CO2 hydration activity and inhibitor sensitivity of hCA I, these findings represent early steps in developing more selective proteoform-specific inhibitors, which could lead to more effective treatments for diseases involving carbonic anhydrases.

Racial discrepancies in initial presentation and diagnosis of patients with endometrial cancer.

89 Background: Endometrial cancer incidence and mortality are increasing, particularly in Black patients who have more aggressive non-endometrioid subtypes. Racial disparities in mortality may be partially contributable to early clinical factors including recognition of symptoms, type of evaluation, and referral timing. We evaluated differences in factors related to symptom appraisal and delays in diagnosis by race and histology. Methods: In this retrospective cohort study, we included patients diagnosed with endometrial cancer from 01/01/2019 to 09/01/2023 at a single institution who were enrolled in the Discovery and Evaluation of Testing for Endometrial Cancer in Tampons (DETECT) study. Demographic characteristics and factors regarding initial presentation and workup were obtained from the electronic medical record. We evaluated patient-reported length and type of symptoms at initial presentation, diagnostic imaging type, and time from initial presentation to time of imaging, tissue sampling, and referral to gynecologic oncology. We assessed differences by race and histology (endometrioid vs. non-endometroid) using chi-square and Kruskal-Wallis tests and analysis of variance. Results: Of the 335 patients included in our analysis, 237 (70.7%) had endometrioid and 98 (29.3%) had non-endometrioid histology. Patients with endometrioid histology were significantly more likely to be younger, White, non-smokers, and have higher obesity levels by body mass index than those with non-endometrioid histology (p=&lt;0.01). The length of symptoms at time of initial presentation was significantly longer in Black patients overall (p=0.01) and in Black patients with endometrioid histology compared to their White counterparts (p=0.04). A similar trend was seen in patients with non-endometrioid histology but was not statistically significant (p=0.15). There was no difference in type of symptoms reported. The type of imaging (p=0.005) differed significantly between endometrioid and non-endometrioid groups. White patients were more likely to have a transvaginal ultrasound performed at initial presentation (p=0.004). Black patients with endometrioid endometrial cancer were more likely to have a longer time period from initial presentation to referral to gynecologic oncology (p=0.04) despite having a shorter time between initial presentation and endometrial sampling (p=0.04). Conclusions: In our cohort, Black patients with endometrial cancer were more likely to have experienced symptoms for a longer time period prior to presentation, less likely to be evaluated with transvaginal ultrasound, and had a longer time period between initial presentation and referral to gynecologic oncology compared to White patients. Raising awareness and targeting these early steps in evaluation and detection of endometrial cancer may help decrease racial disparities in outcomes.

Hevea brasiliensis rubber particles' fluid interfaces reveal size impact on early coagulation steps

Natural rubber originates from the coagulation of rubber particles (RP) from Hevea brasiliensis latex. The size distribution of Hevea RP is bimodal with the presence of small rubber particles (SRP) and large rubber particles (LRP). This study aims at getting a better understanding of the early coagulation steps of Hevea RP taking into account the particle size. SRP and LRP were obtained by centrifugation of freshly tapped ammonia-free latex from RRIM600 clone. Size and zeta potential measurements showed that both RP fractions were efficiently separated and stable in basic buffer. SRP and LRP dispersions were placed in a Langmuir trough and RP were let to adsorb at the air-liquid interface to form interfacial films. Surface tension and ellipsometry indicate that the formation kinetics and the stabilization of the film at the air-liquid interface are faster for SRP than LRP. Moreover, the arrangement of RP at the interface differs between SRP and LRP, as shown by Brewster angle microscopy, atomic force microscopy and confocal laser scanning microscopy. First, the RP membrane and cis-1,4-polyisoprene core spread at the air-liquid interface before clustering. Then, while the SRP fuse, the LRP keep their structure in individual particles in floating aggregate. The role of the non-isoprene molecules on the different organization of SRP and LRP films is discussed, the one of the two major RP proteins, SRPP1 (Small Rubber Particle Protein) and Rubber Elongation Factor (REF1) in the early coagulation steps.

Accurate Visualization of C4d Complement Fragment in Immunohistochemistry by C-Terminal Linear Neoepitope-Specific Antibodies

C4d is the end degradation product of activated complement component C4b that appears during the early steps of the classical and lectin complement pathways. Within the primary sequence of C4d, there is a reactive thioester group that binds covalently to nearby surfaces, thus labeling the locations of complement activation. This feature makes C4d a target for immunohistochemical staining aimed to aid the diagnosis of, among others, the antibody-mediated rejection of transplanted organs, membranous glomerulonephritis, bullous pemphigoid, or inflammatory myopathies. However, the credibility of C4d immunostaining is debatable, as a high background in surrounding tissues and body fluids and diffused patterns of deposits in target structures are experienced with some of the available anti-C4d antibodies. Herein, we present an improved version of a rabbit anti-C4d antibody, originally raised against the C-terminal linear neoepitope of this complement fragment. Minor cross-reactivity with C4b and native C4 proteins, measured by ELISAs, as well as relatively low concentrations necessary for obtaining a specific signal in immunohistochemical analyses of formalin-fixed paraffin-embedded material, makes the improved antibody superior to commercially available rabbit monoclonal anti-C4d antibody SP91 dedicated to ex vivo diagnostics, as demonstrated by the staining of a panel of kidney transplant biopsies.

A candidate loss-of-function variant in SGIP1 causes synaptic dysfunction and recessive parkinsonism

Synaptic dysfunction is recognized as an early step in the pathophysiology of parkinsonism. Several genetic mutations affecting the integrity of synaptic proteins cause or increase the risk of developing disease. We have identified a candidate causative mutation in synaptic "SH3GL2 Interacting Protein 1" (SGIP1), linked to early-onset parkinsonism in a consanguineous Arab family. Additionally, affected siblings display intellectual, cognitive, and behavioral dysfunction. Metabolic network analysis of [18F]-fluorodeoxyglucose positron emission tomography scans shows patterns very similar to those of idiopathic Parkinson's disease. We show that the identified SGIP1 mutation causes a loss of protein function, and analyses in newly created Drosophila models reveal movement defects, synaptic transmission dysfunction, and neurodegeneration, including dopaminergic synapse loss. Histology and correlative light and electron microscopy reveal the absence of synaptic multivesicular bodies and the accumulation of degradative organelles. This research delineates a putative form of recessive parkinsonism, converging on defective synaptic proteostasis and opening avenues for diagnosis, genetic counseling, and treatment.

Stepping Back: How Should Pass/Fail Scoring Influence Step 1 Timing?

Although most students complete Step 1 before clerkships, some institutions delay the exam until after clerkships. The change to pass/fail grading adds additional complexity that should be considered when deciding about exam timing. Both early and late administration may affect learning outcomes, learner behavior, student well-being, and residency match success. Step 1 completion before clerkships promotes learning outcomes (e.g., integration and mastery of foundational material), may encourage students to focus on the curriculum, and may better prepare students for clinical science exams (CSEs). However, delaying the exam ensures that students maintain foundational knowledge and may encourage clinical educators to demonstrate basic science illustrations. An early Step 1 may affect learner behavior by allowing clerkship students to focus on clinical learning. The associated National Board of Medical Examiners performance report may also be used for Step 2 and CSE preparation. However, delaying Step 1 allows greater scheduling flexibility based on developmental milestones. Administration of Step 1 before clerkships removes a significant stressor from the clinical year and decompresses the residency application period. However, a delayed Step 1 reduces the pressure on students to engage in numerous extracurricular and research activities to distinguish themselves due to the pass/fail change. An early Step 1 exam may also lead to improved CSE performance, which is often linked to clerkship honors criteria, an increasingly valuable distinction for residency match success after the change to pass/fail. In contrast, delaying Step 1 is associated with higher first-time pass rates, which may be especially important for students at risk for failure. Medical educators and students should collaboratively approach the question of Step 1 timing, considering these factors within the context of the medical school program, curricular constraints and priorities, and students' individual needs and goals.

Limited population structure but signals of recent selection in introduced African Fig Fly (Zaprionus indianus) in North America.

Invasive species have devastating consequences for human health, food security, and the environment. Many invasive species adapt to new ecological niches following invasion, but little is known about the early steps of adaptation. Here we examine population genomics of a recently introduced drosophilid in North America, the African Fig Fly, Zaprionus indianus. This species is likely intolerant of subfreezing temperatures and recolonizes temperate environments yearly. We generated a new chromosome-level genome assembly for Z. indianus. Using resequencing of over 200 North American individuals collected over four years in temperate Virginia, plus a single collection from subtropical Florida, we tested for signatures of recolonization, population structure, and adaptation within invasive populations. We show founding populations are sometimes small and contain close genetic relatives, yet temporal population structure and differentiation of populations is mostly absent across recurrent recolonization events. Although we find limited signals of genome-wide spatial or temporal population structure, we identify haplotypes on the X chromosome that are repeatedly differentiated between Virginia and Florida populations. These haplotypes show signatures of natural selection and are not found in African populations. We also find evidence for several large structural polymorphisms segregating within North America populations and show X chromosome evolution in invasive populations is strikingly different from the autosomes. These results show that despite limited population structure, populations may rapidly evolve genetic differences early in an invasion. Further uncovering how these genomic regions influence invasive potential and success in new environments will advance our understanding of how organisms evolve in changing environments.

Visualizing nuclear pore complex plasticity with Pan-Expansion Microscopy.

Cell-type specific and environmentally-responsive plasticity in nuclear pore complex (NPC) composition and structure is an emerging area of investigation, but its molecular underpinnings remain ill defined. To understand the cause and consequence of NPC plasticity requires technologies to visualize differences within individual NPCs across the thousands in a given nucleus. We evaluate the utility of Pan Expansion Microscopy (Pan-ExM), which enables 16-20 fold isotropic cell enlargement while preserving the proteome, to reveal NPC plasticity. NPCs are robustly identified by deep learning-facilitated segmentation as tripartite structures corresponding to the nucleoplasmic ring, inner ring with central transport channel, and cytoplasmic ring, as confirmed by immunostaining. We demonstrate a range of NPC diameters with a bias for dilated NPCs at the basal nuclear surface, often in local clusters. These diameter biases are eliminated by disrupting linker of nucleoskeleton and cytoskeleton (LINC) complex-dependent connections between the nuclear envelope (NE) and the cytoskeleton, supporting that they reflect local variations in NE tension. Pan-ExM further reveals that the transmembrane nucleoporin/nup POM121 resides specifically at the nuclear ring in multiple model cell lines, surprising given the expectation that it would be a component of the inner ring like other transmembrane nups. Remarkably, however, POM121 shifts from the nuclear ring to the inner ring specifically in aged induced pluripotent stem cell derived neurons (iPSNs) from a patient with C9orf72 amyotrophic lateral sclerosis (ALS). Thus, Pan-ExM allows the visualization of changes in NPC architecture that may underlie early steps in an ALS pathomechanism. Taken together, Pan-ExM is a powerful and accessible tool to visualize NPC plasticity in physiological and pathological contexts at single NPC resolution.

Correlating NAD(P)H lifetime shifts to tamoxifen resistance in breast cancer cells: A metabolic screening study with time-resolved flow cytometry

Time-resolved flow cytometry (TRFC) was used to measure metabolic differences in estrogen receptor-positive breast cancer cells. This specialty cytometry technique measures fluorescence lifetimes as a single-cell parameter thereby providing a unique approach for high-throughput cell counting and screening. Differences in fluorescence lifetime were detected and this was associated with sensitivity to the commonly prescribed therapeutic tamoxifen. Differences in fluorescence lifetime are attributed to the binding states of the autofluorescent metabolite NAD(P)H. The function of NAD(P)H is well described and in general involves cycling from a reduced to oxidized state to facilitate electron transport for the conversion of pyruvate to lactate. NAD(P)H fluorescence lifetimes depend on the bound or unbound state of the metabolite, which also relates to metabolic transitions between oxidative phosphorylation and glycolysis. To determine if fundamental metabolic profiles differ for cells that are sensitive to tamoxifen compared to those that are resistant, large populations of MCF-7 breast cancer cells were screened and fluorescence lifetimes were quantified. Additionally, metabolic differences associated with tamoxifen sensitivity were measured with a Seahorse HS mini metabolic analyzer (Agilent Technologies Inc. Santa Clara, CA) and confocal imaging. Results show that tamoxifen-resistant breast cancer cells have increased utilization of glycolysis for energy production compared to tamoxifen-sensitive breast cancer cells. This work is impacting because it establishes an early step toward developing a reliable screening technology in which large cell censuses can be differentiated for drug sensitivity in a label-free fashion.

Correlation of spirometry findings and post-six-minute walk test oxygen desaturation in chronic simple silicosis patients with age, duration of silica exposure, smoking pack years, occupation and mean pulmonary artery pressure.

Silicosis is associated with preventable but irreversible lung damage. Early quantifiable assessment of silicosis workers would promote early interventional steps to reduce health deterioration. The objectives of this study were to correlate spirometry findings and post-six-minute walk test oxygen desaturation (post-6MWT OD) in chronic simple silicosis with age, duration of silica exposure (DSE), smoking pack years (SPY), occupation, and mean pulmonary artery pressure. Based on occupational exposure to silica and radiologic confirmation of chronic simple silicosis, 104 patients (all males) were enrolled and grouped based on SPY (nil, 1-10, 11-20, and >20) and occupation (drillers and dressers). They were further investigated with spirometry, post-6MWT OD, and transthoracic echocardiography (TTE), and findings were statistically analyzed. Abnormal spirometry findings were seen in 62.5% of total cases (65/104), with the highest percentage in the >20 SPY group (84%; 21/25) and drillers (68.3%; 28/41). The post-6MWT OD was seen in 50.96% of cases (53/104) with the highest percentage in the >20 SPY group (56%; 14/25) and drillers (63.4%; 26/41). Normal and restrictive patterns were predominant among <20 SPY groups, while obstructive and mixed patterns were prevalent in the >20 SPY group. Normal, obstructive, and restrictive patterns were predominant among dressers, while mixed patterns were in drillers. Mean age and mean DSE were higher for >20 SPY group and dressers; obstructive and mixed patterns; and patients with post-6MWT OD. Pulmonary hypertension was significantly associated with the presence of abnormal spirometry patterns (69.3%; 45/65) and post-6MWT OD (79.3%; 42/53). Mean forced expiratory volume in one second (FEV1)/forced vital capacity (FVC) was significantly lower for the >20 SPY group. Mean FEV1 had an inverse relation with SPY, and mean FVC was lower for drillers than dressers. Spirometry, post-6MWT OD, and TTE assessment give a complete overview of the cardiopulmonary exercise capacity of chronic silicosis patients and facilitate early interventions with special consideration for workers involved in machine-based mining activity.

Nuclear lamin A/C phosphorylation by loss of androgen receptor leads to cancer-associated fibroblast activation

Alterations in nuclear structure and function are hallmarks of cancer cells. Little is known about these changes in Cancer-Associated Fibroblasts (CAFs), crucial components of the tumor microenvironment. Loss of the androgen receptor (AR) in human dermal fibroblasts (HDFs), which triggers early steps of CAF activation, leads to nuclear membrane changes and micronuclei formation, independent of cellular senescence. Similar changes occur in established CAFs and are reversed by restoring AR activity. AR associates with nuclear lamin A/C, and its loss causes lamin A/C nucleoplasmic redistribution. AR serves as a bridge between lamin A/C and the protein phosphatase PPP1. Loss of AR decreases lamin-PPP1 association and increases lamin A/C phosphorylation at Ser 301, a characteristic of CAFs. Phosphorylated lamin A/C at Ser 301 binds to the regulatory region of CAF effector genes of the myofibroblast subtype. Expression of a lamin A/C Ser301 phosphomimetic mutant alone can transform normal fibroblasts into tumor-promoting CAFs.