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Developmental causes of the most common arrhythmia, atrial fibrillation (AF), are poorly defined, with compensation potentially masking arrhythmic risk. Here, we delete 9 amino acids (Δ9) within a conserved domain of the giant protein titin's A-band in zebrafish and human-induced pluripotent stem cell-derived atrial cardiomyocytes (hiPSC-aCMs). We find that ttna Δ9/Δ9 zebrafish embryos' cardiac morphology is perturbed and accompanied by reduced functional output, but ventricular function recovers within days. Despite normal ventricular function, ttna Δ9/Δ9 adults exhibit AF and atrial myopathy, which are recapitulated in TTN Δ9/Δ9-hiPSC-aCMs. Additionally, action potential is shortened and slow delayed rectifier potassium current (I Ks) is increased due to aberrant atrial natriuretic peptide (ANP) levels. Strikingly, suppression of I Ks in both models prevents AF and improves atrial contractility. Thus, a small internal deletion in titin causes developmental abnormalities that increase the risk of AF via ion channel remodeling, with implications for patients who harbor disease-causing variants in sarcomeric proteins.

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Trogocytosis is an underappreciated phenomenon that shapes the immune microenvironment surrounding many types of solid tumors. The consequences of membrane-bound proteins being deposited from a donor immune cell to a recipient cancer cell via trogocytosis are still unclear. Here, we report that human clear cell renal carcinoma tumors stably express the lymphoid markers CD45, CD56, CD14, and CD16. Flow cytometry performed on fresh kidney tumors revealed consistent CD45 expression on tumor cells, as well as varying levels of the other markers mentioned previously. These results were consistent with our immunofluorescent analysis, which also revealed colocalization of lymphoid markers with carbonic anhydrase 9 (CAIX), a standard kidney tumor marker. RNA analysis showed a significant upregulation of genes typically associated with immune cells in tumor cells following trogocytosis. Finally, we show evidence of chromosomal DNA being transferred from immune cells to tumor cells during trogocytosis. This horizontal gene transfer has transcriptional consequences in the recipient tumor cell, resulting in a fusion phenotype that expressed both immune and cancer specific proteins. This work demonstrates a novel mechanism by which tumor cell protein expression is altered through the acquisition of surface membrane fragments and genomic DNA from infiltrating lymphocytes. These results alter the way in which we understand tumor-immune cell interactions and may reveal new insights into the mechanisms by which tumors develop. Additionally, further studies into trogocytosis will help push the field towards the next generation of immunotherapies and biomarkers for treating renal cell carcinoma and other types of cancers.

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Trogocytosis is the process by which a recipient cell siphons small membrane fragments and proteins from a donor cell and may be utilized by cancer cells to avoid immune detection. We observed lymphocyte specific protein expressed by TNBC cells via immunofluorescence imaging of patient samples. Image analysis of CD45RA expression, a T cell specific protein, revealed that all stages of TNBCs express CD45RA. Flow cytometry revealed TNBC cells trogocytose CD45 protein from T cells. We also showed that the acquisition of these lymphoid markers is contact dependent. Confocal and super-resolution imaging further revealed CD45 + spherical structures containing T cell genomic DNA inside TNBC cells after co-culture. Trogocytosis between T cells and TNBC cells altered cancer cell gene expression. Our results revealed that CD45 is obtained by TNBC cells from T cells via trogocytosis and that TNBC cells express CD45 intracellularly and on the membrane. Teaser: TNBC cells acquire small spherical structures from T cells containing lymphocyte-specific membrane proteins and genomic DNA.

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Analyses of ancient DNA typically involve sequencing the surviving short oligonucleotides and aligning to genome assemblies from related, modern species. Here, we report that skin from a female woolly mammoth (†Mammuthus primigenius) that died 52,000 years ago retained its ancient genome architecture. We use PaleoHi-C to map chromatin contacts and assemble its genome, yielding 28 chromosome-length scaffolds. Chromosome territories, compartments, loops, Barr bodies, and inactive X chromosome (Xi) superdomains persist. The active and inactive genome compartments in mammoth skin more closely resemble Asian elephant skin than other elephant tissues. Our analyses uncover new biology. Differences in compartmentalization reveal genes whose transcription was potentially altered in mammoths vs. elephants. Mammoth Xi has a tetradic architecture, not bipartite like human and mouse. We hypothesize that, shortly after this mammoth's death, the sample spontaneously freeze-dried in the Siberian cold, leading to a glass transition that preserved subfossils of ancient chromosomes at nanometer scale.

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Objective: The effectiveness of antidepressant treatment for mood disorders is often limited by either a poor response or the emergence of adverse effects. These complications often necessitate multiple drug trials. This clinical challenge intensifies during pregnancy, when medications must be selected to improve the likelihood of response and optimize reproductive outcomes. We determined the distribution of common pharmacogenetic variants, metabolizer phenotypes, past medication responses, and side effects in childbearing-aged individuals seeking treatment in a tertiary care perinatal mental health clinic.Methods: Sixty treatment-seeking women (based on sex at birth) with DSM-5- defined bipolar disorder (n = 28) or major depressive disorder (n = 32) provided DNA samples and completed psychiatric diagnostic and severity assessments between April 2014 and December 2017. Samples were genotyped for single-nucleotide variants in drug metabolizing enzyme genes of commonly prescribed antidepressants (cytochrome P450 [CYP] 1A2, 2B6, 2C9, 2C19, 2D6, 3A4, and 3A5), and the frequency of normative metabolizer status was compared to reference populations data from Clinical Pharmacogenetics Implementation Consortium (CPIC) guidelines. The Antidepressant Treatment History Form was used to record historic medication trials and side effects.Results: A significantly greater proportion of extensive metabolizers for CYP2B6 was observed in the study population when compared to CPIC population frequency databases in Caucasians (0.64 vs 0.43 [95% CI: 0.49-0.76]; P value = .006) and African Americans (0.71 vs 0.33 [95% CI: 0.29-0.96]; P value = .045). No significant association was found between metabolizer phenotype and the likelihood of a medication side effect.Conclusion: Pharmacogenomic testing may have value for personalized prescribing in individuals capable of or considering pregnancy.

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Background: Prior reports have suggested a possible increase in the frequency of invasive fungal infections (IFIs) with use of a Bruton tyrosine kinase inhibitor (BTKi) for treatment of chronic lymphoid malignancies such as chronic lymphocytic leukemia (CLL), but precise estimates are lacking. We aim to characterize the prevalence of IFIs among patients with CLL, for whom a BTKi is now the first-line recommended therapy. Methods: We queried TriNetX, a global research network database, to identify adult patients with CLL using the International Classification of Diseases, Tenth Revision code (C91.1) and laboratory results. We performed a case-control propensity score-matched analysis to determine IFIs events by BTKi use. We adjusted for age, sex, ethnicity, and clinical risk factors associated with an increased risk of IFIs. Results: Among 5358 matched patients with CLL, we found an incidence of 4.6% of IFIs in patients on a BTKi versus 3.5% among patients not on a BTKi at 5 years. Approximately 1% of patients with CLL developed an IFI while on a BTKi within this period. Our adjusted IFI event analysis found an elevated rate of Pneumocystis jirovecii pneumonia (PJP) (0.5% vs 0.3%, P = .02) and invasive candidiasis (3.5% vs 2.7%, P = .012) with the use of a BTKi. The number needed to harm for patients taking a BTKi was 120 and 358 for invasive candidiasis and PJP, respectively. Conclusions: We found an adjusted elevated rate of PJP and invasive candidiasis with BTKi use. The rates are, however, low with a high number needed to harm. Additional studies stratifying other IFIs with specific BTKis are required to identify at-risk patients and preventive, cost-effective interventions.

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The ubiquitin-proteasome system mediates the degradation of a wide variety of proteins. Proteasome dysfunction is associated with neurodegenerative diseases and neurodevelopmental disorders in humans. Here we identified mutations in PSMC5, an AAA ATPase subunit of the proteasome 19S regulatory particle, in individuals with neurodevelopmental disorders, which were initially considered as variants of unknown significance. We have now found heterozygotes with the following mutations: P320R (6 individuals), R325W, Q160A, and one nonsense mutation at Q69. We focused on understanding the functional consequence of PSMC5 insufficiency and the P320R mutation in cells and found that both impair proteasome function and activate apoptosis. Interestingly, the P320R mutation impairs proteasome function by weakening the association between the 19S regulatory particle and the 20S core particle. Our study supports that proteasome dysfunction is the pathogenic cause of neurodevelopmental disorders in individuals carrying PSMC5 variants.

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Pathogenic variants in SCN8A, which encodes the voltage-gated sodium (NaV) channel NaV1.6, associate with neurodevelopmental disorders, including developmental and epileptic encephalopathy. Previous approaches to determine SCN8A variant function may be confounded by use of a neonatally expressed, alternatively spliced isoform of NaV1.6 (NaV1.6N) and engineered mutations rendering the channel tetrodotoxin (TTX) resistant. We investigated the impact of SCN8A alternative splicing on variant function by comparing the functional attributes of 15 variants expressed in 2 developmentally regulated splice isoforms (NaV1.6N, NaV1.6A). We employed automated patch clamp recording to enhance throughput, and developed a neuronal cell line (ND7/LoNav) with low levels of endogenous NaV current to obviate the need for TTX-resistance mutations. Expression of NaV1.6N or NaV1.6A in ND7/LoNav cells generated NaV currents with small, but significant, differences in voltage dependence of activation and inactivation. TTX-resistant versions of both isoforms exhibited significant functional differences compared with the corresponding WT channels. We demonstrated that many of the 15 disease-associated variants studied exhibited isoform-dependent functional effects, and that many of the studied SCN8A variants exhibited functional properties that were not easily classified as either gain- or loss-of-function. Our work illustrates the value of considering molecular and cellular context when investigating SCN8A variants.

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We present an annotated checklist of fleas (Siphonaptera) known to occur in the state of Delaware based on an examination of Siphonaptera collections at the University of Delaware and the Carnegie Museum of Natural History, as well as new specimens of fleas we collected from wildlife, other hosts, and tick flags. We review published records and compile them herein with our new records, which include 3 species previously unreported from Delaware. With these additions, there are now 18 flea species from 19 avian and mammalian hosts documented from Delaware.

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BACKGROUND: Administration of anti-CD19 chimeric antigen receptor T-cell (CART19) immunotherapy for large B-cell lymphomas (LBCLs), a subset of non-Hodgkin lymphoma (NHL), involves high costs and access to specialized tertiary care centers. We investigated whether minority health populations (MHPs) have equal access to CART19 and whether their outcomes are similar to those of non-MHPs. METHODS: We analyzed the prevalence and clinical outcomes of patients treated with commercial CART19 at two geographically and socioeconomically different institutions: the Abramson Cancer Center (ACC, Philadelphia, Pennsylvania) and the Knight Cancer Institute (KCI, Portland, Oregon). RESULTS: In the ACC catchment area, 8956 patients were diagnosed with NHL between 2015 and 2019 (latest available data from the state registry), including 17.9% MHPs. In the ACC, between 2018 and 2022 (CART became available in 2018), 1492 patients with LBCL were treated, and 194 received CART19. The proportion of MHPs was 15.7% for the entire LBCL cohort but only 6.7% for the CART19 cohort. During the same time, in the KCI catchment area, 4568 patients were diagnosed with NHL, including 4.2% MHPs. In the KCI, 396 patients with LBCL were treated, and 47 received CART19. The proportion of MHPs was 6.6% for the entire LBCL cohort and 4.2% for the CART19 cohort. The 3-month response, survival, and toxicities after CART19 infusion showed similar results, although the number of patients who were treated was limited. CONCLUSIONS: This study shows that the access of MHPs to tertiary centers for LBCL care was preserved but appeared reduced for commercial CART19 immunotherapy. Although clinical outcomes of MHPs seemed similar to those of non-MHPs, the small sample size precludes drawing firm conclusions. Further studies are needed. (Funded by the Laffey McHugh Foundation and others.).

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BACKGROUND: Many cardiomyopathy-associated FLNC pathogenic variants are heterozygous truncations, and FLNC pathogenic variants are associated with arrhythmias. Arrhythmia triggers in filaminopathy are incompletely understood. METHODS AND RESULTS: We describe an individual with biallelic FLNC pathogenic variants, p.Arg650X and c.970-4A>G, with peripartum cardiomyopathy and ventricular arrhythmias. We also describe clinical findings in probands with FLNC variants including Val2715fs87X, Glu2458Serfs71X, Phe106Leu, and c.970-4A>G with hypertrophic and dilated cardiomyopathy, atrial fibrillation, and ventricular tachycardia. Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were generated. The FLNC truncation, Arg650X/c.970-4A>G, showed a marked reduction in filamin C protein consistent with biallelic loss of function mutations. To assess loss of filamin C, gene editing of a healthy control iPSC line was used to generate a homozygous FLNC disruption in the actin binding domain. Because filamin C has been linked to protein quality control, we assessed the necessity of filamin C in iPSC-CMs for response to the proteasome inhibitor bortezomib. After exposure to low-dose bortezomib, FLNC-null iPSC-CMs showed an increase in the chaperone proteins BAG3, HSP70 (heat shock protein 70), and HSPB8 (small heat shock protein B8) and in the autophagy marker LC3I/II. FLNC null iPSC-CMs had prolonged electric field potential, which was further prolonged in the presence of low-dose bortezomib. FLNC null engineered heart tissues had impaired function after low-dose bortezomib. CONCLUSIONS: FLNC pathogenic variants associate with a predisposition to arrhythmias, which can be modeled in iPSC-CMs. Reduction of filamin C prolonged field potential, a surrogate for action potential, and with bortezomib-induced proteasome inhibition, reduced filamin C led to greater arrhythmia potential and impaired function.

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Multiple myeloma is a largely incurable and life-threatening malignancy of antibody-secreting plasma cells. An effective and widely available animal model that recapitulates human myeloma and related plasma cell disorders is lacking. We show that busulfan-conditioned human IL-6-transgenic (hIL-6-transgenic) NSG (NSG+hIL6) mice reliably support the engraftment of malignant and premalignant human plasma cells, including from patients diagnosed with monoclonal gammopathy of undetermined significance, pre- and postrelapse myeloma, plasma cell leukemia, and amyloid light chain amyloidosis. Consistent with human disease, NSG+hIL6 mice engrafted with patient-derived myeloma cells developed serum M spikes, and a majority developed anemia, hypercalcemia, and/or bone lesions. Single-cell RNA sequencing showed nonmalignant and malignant cell engraftment, the latter expressing a wide array of mRNAs associated with myeloma cell survival and proliferation. Myeloma-engrafted mice given CAR T cells targeting plasma cells or bortezomib experienced reduced tumor burden. Our results establish NSG+hIL6 mice as an effective patient-derived xenograft model for study and preclinical drug development of multiple myeloma and related plasma cell disorders.

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Myocarditis is clinically characterized by chest pain, arrhythmias, and heart failure, and treatment is often supportive. Mutations in DSP, a gene encoding the desmosomal protein desmoplakin, have been increasingly implicated in myocarditis. To model DSP-associated myocarditis and assess the role of innate immunity, we generated engineered heart tissues (EHTs) using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from patients with heterozygous DSP truncating variants (DSPtvs) and a gene-edited homozygous deletion cell line (DSP-/-). At baseline, DSP-/- EHTs displayed a transcriptomic signature of innate immune activation, which was mirrored by cytokine release. Importantly, DSP-/- EHTs were hypersensitive to Toll-like receptor (TLR) stimulation, demonstrating more contractile dysfunction compared with isogenic controls. Relative to DSP-/- EHTs, heterozygous DSPtv EHTs had less functional impairment. DSPtv EHTs displayed heightened sensitivity to TLR stimulation, and when subjected to strain, DSPtv EHTs developed functional deficits, indicating reduced contractile reserve compared with healthy controls. Colchicine or NF-κB inhibitors improved strain-induced force deficits in DSPtv EHTs. Genomic correction of DSP p.R1951X using adenine base editing reduced inflammatory biomarker release from EHTs. Thus, EHTs replicate electrical and contractile phenotypes seen in human myocarditis, implicating cytokine release as a key part of the myogenic susceptibility to inflammation. The heightened innate immune activation and sensitivity are targets for clinical intervention.

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Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) recapitulate numerous disease and drug response phenotypes, but cell immaturity may limit their accuracy and fidelity as a model system. Cell culture medium modification is a common method for enhancing maturation, yet prior studies have used complex media with little understanding of individual component contribution, which may compromise long-term hiPSC-CM viability. Here, we developed high-throughput methods to measure hiPSC-CM maturation, determined factors that enhanced viability, and then systematically assessed the contribution of individual maturation medium components. We developed a medium that is compatible with extended culture. We discovered that hiPSC-CM maturation can be sub-specified into electrophysiological/EC coupling, metabolism, and gene expression and that induction of these attributes is largely independent. In this work, we establish a defined baseline for future studies of cardiomyocyte maturation. Furthermore, we provide a selection of medium formulae, optimized for distinct applications and priorities, that promote measurable attributes of maturation.

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Bendamustine has been retrospectively shown to be an effective and safe lymphodepletion regimen prior to the anti-CD19 chimeric antigen receptor T cell (CART) products tisagenlecleucel and axicabtagene ciloleucel, as well as the anti-BCMA CART products idecabtagene vicleucel and ciltacabtagene autoleucel. However, bendamustine as lymphodepletion prior to lisocabtagene maraleucel (liso-cel), a 4-1BB co-stimulated, fixed CD4:CD8 ratio anti-CD19 CART product, has not been described yet. Thus, we studied a cohort of sequentially-treated patients with large B-cell lymphomas who received bendamustine lymphodepletion before liso-cel at the University of Pennsylvania between 5/2021 and 12/2023 (n = 31). Patients were evaluated for toxicities and responses. Of note, 7 patients (22.6%) would have dnot met the inclusion criteria for the registrational liso-cel clinical trials, mostly due to older age. Overall and complete response rates were 76.9% and 73.1%, respectively. At a median follow-up of 6.3 months, the 6-month progression-free and overall survival were 59.9% and 91.1%, respectively. Rates of cytokine-release syndrome (CRS) and neurotoxicity (ICANS) of any grade were 9.7% and 9.7%, respectively, with no grade ≥ 3 events. No infections were reported during the first 30 days following liso-cel infusion. Neutropenia ≥ grade 3 was observed in 29.0% of patients; thrombocytopenia ≥ grade 3 occurred in 9.7%. In conclusion, bendamustine lymphodepletion before liso-cel appears to be a strategy that can drive tumor responses while ensuring a mild toxicity profile.

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SCN2A-related disorders secondary to altered function in the voltage-gated sodium channel Nav1.2 are rare, with clinically heterogeneous expressions that include epilepsy, autism and multiple severe to profound impairments and other conditions. To advance understanding of the clinical phenotypes and their relationship to channel function, 81 patients (36 female, 44%, median age 5.4 years) with 69 unique SCN2A variants were systematically phenotyped and their Nav1.2 channel function systematically assessed. Participants were recruited through the FamileSCN2A Foundation. Primary phenotype (epilepsy of neonatal onset, n = 27; infant onset, n = 18; and later onset n = 24; and autism without seizures, n = 12) was strongly correlated with a non-seizure severity index (P = 0.002), which was based on presence of severe impairments in gross motor, fine motor, communication abilities, gastrostomy tube dependence and diagnosis of cortical visual impairment and scoliosis. Non-seizure severity was greatest in the neonatal-onset group and least in the autism group (P = 0.002). Children with the lowest severity indices were still severely impaired, as reflected by an average Vineland Adaptive Behavior composite score of 49.5 (>3 standard deviations below the norm-referenced mean of the test). Epileptic spasms were significantly more common in infant-onset (67%) than in neonatal (22%) or later-onset (29%) epilepsy (P = 0.007). Primary phenotype was also strongly correlated with variant function (P < 0.0001); gain-of-function and mixed function variants predominated in neonatal-onset epilepsy, shifting to moderate loss of function in infant-onset epilepsy and to severe and complete loss of function in later-onset epilepsy and autism groups. Exploratory cluster analysis identified five groups, representing: (i) primarily later-onset epilepsy with moderate loss-of-function variants and low severity indices; (ii) mostly infant-onset epilepsy with moderate loss-of-function variants but higher severity indices; and (iii) late-onset and autism only, with the lowest severity indices (mostly zero) and severe/complete loss-of-function variants. Two exclusively neonatal clusters were distinguished from each other largely on non-seizure severity scores and secondarily on variant function. The relationship between primary phenotype and variant function emphasizes the role of developmental factors in the differential clinical expression of SCN2A variants based on their effects on Nav1.2 channel function. The non-seizure severity of SCN2A disorders depends on a combination of the age at seizure onset (primary phenotype) and variant function. As precision therapies for SCN2A-related disorders advance towards clinical trials, knowledge of the relationship between variant function and clinical disease expression will be valuable for identifying appropriate patients for these trials and in selecting efficient clinical outcomes.

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Metastasis is the leading cause of cancer-related mortality. Paneth cells provide stem cell niche factors in homeostatic conditions, but the underlying mechanisms of cancer stem cell niche development are unclear. Here we report that Dickkopf-2 (DKK2) is essential for the generation of cancer cells with Paneth cell properties during colon cancer metastasis. Splenic injection of Dkk2-knockout (KO) cancer organoids into C57BL/6 mice resulted in a significant reduction of liver metastases. Transcriptome analysis showed reduction of Paneth cell markers such as lysozymes in KO organoids. Single cell RNA sequencing analyses of murine metastasized colon cancer cells and patient samples identified the presence of lysozyme positive cells with Paneth cell properties including enhanced glycolysis. Further analyses of transcriptome and chromatin accessibility suggested Hepatocyte nuclear factor 4-alpha (HNF4A) as a downstream target of DKK2. Chromatin immunoprecipitation followed by sequencing analysis revealed that HNF4A binds to the promoter region of Sox9, a well-known transcription factor for Paneth cell differentiation. In the liver metastatic foci, DKK2 knockout rescued HNF4A protein levels followed by reduction of lysozyme positive cancer cells. Taken together, DKK2-mediated reduction of HNF4A protein promotes the generation of lysozyme positive cancer cells with Paneth cell properties in the metastasized colon cancers.

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Full understanding of proteostasis and energy utilization in cells will require knowledge of the fraction of cell proteins being degraded with different half-lives and their rates of synthesis. We therefore developed a method to determine such information that combines mathematical analysis of protein degradation kinetics obtained in pulse-chase experiments with Bayesian data fitting using the maximum entropy principle. This approach will enable rapid analyses of whole-cell protein dynamics in different cell types, physiological states, and neurodegenerative disease. Using it, we obtained surprising insights about protein stabilities in cultured cells normally and upon activation of proteolysis by mTOR inhibition and increasing cAMP or cGMP. It revealed that >90% of protein content in dividing mammalian cell lines is long-lived, with half-lives of 24 to 200 h, and therefore comprises much of the proteins in daughter cells. The well-studied short-lived proteins (half-lives < 10 h) together comprise <2% of cell protein mass, but surprisingly account for 10 to 20% of measurable newly synthesized protein mass. Evolution thus appears to have minimized intracellular proteolysis except to rapidly eliminate misfolded and regulatory proteins.

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