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Cryptococcus neoformans is the leading cause of fungal meningitis and is characterized by pathogenic eosinophil accumulation in the context of type-2 inflammation. The chemoattractant receptor GPR35 is expressed by granulocytes and promotes their migration to the inflammatory mediator 5-hydroxyindoleacetic acid (5-HIAA), a serotonin metabolite. Given the inflammatory nature of cryptococcal infection, we examined the role of GPR35 in the circuitry underlying cell recruitment to the lung. GPR35 deficiency dampened eosinophil recruitment and fungal growth, whereas overexpression promoted eosinophil homing to airways and fungal replication. Activated platelets and mast cells were the sources of GPR35 ligand activity and pharmacological inhibition of serotonin conversion to 5-HIAA, or genetic deficiency in 5-HIAA production by platelets and mast cells resulted in more efficient clearance of Cryptococcus. Thus, the 5-HIAA-GPR35 axis is an eosinophil chemoattractant receptor system that modulates the clearance of a lethal fungal pathogen, with implications for the use of serotonin metabolism inhibitors in the treatment of fungal infections.
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Criptococose , Infecções Fúngicas Invasivas , Humanos , Eosinófilos , Ácido Hidroxi-Indolacético , Mastócitos , Plaquetas , Ligantes , Receptores de Formil Peptídeo , Serotonina , Criptococose/microbiologia , Criptococose/patologia , Receptores Acoplados a Proteínas G/genéticaRESUMO
PURPOSE: DOTATATE PET/CT (DOTATATE) is superior to conventional imaging in detecting metastasis for gastroenteropancreatic neuroendocrine tumors (GEP-NETs). However, limited availability, high-cost, and additive radiation exposure necessitate guidelines for its use. This study seeks to investigate the relationship between clinical characteristics and metastasis on DOTATATE. METHODS: This was a retrospective analysis of 815 patients who underwent DOTATATE at UCLA from 2014 to 2022. After applying inclusion and exclusion criteria, the study cohort consisted of 163 patients with pathologically diagnosed GEP-NETs, who either underwent primary tumor resection within 1-year prior, or had not undergone resection at the time of DOTATATE imaging. The presence of metastasis was determined using DOTATATE. Fisher's exact test, chi-squared test, and Mann-Whitney test were conducted to compare intergroup difference. Multivariate analysis was performed to identify clinical characteristics associated with metastasis on DOTATATE. RESULTS: Of patients with GEP-NETs, 40.5% (n = 66) were diagnosed with metastases by using DOTATATE. Those with metastatic disease were more likely to exhibit a larger primary tumor size (median 3.4 vs. 1.2, cm, P < 0.001), elevated serum chromogranin A level (CgA, median 208 vs. 97, mg/ml, P = 0.005), and higher tumor grade (P < 0.001). Primary tumor size ≥2 cm and serum CgA level ≥150 ng/mL for metastatic disease had a sensitivity and specificity of 64% and 89%, and 72% and 59%, respectively. Multivariate analysis demonstrated that primary tumor size (≥2/<2, cm, odds ratio [OR] 47.90, P < 0.001), tumor functionality (functional/nonfunctional, adjusted OR 10.17 P = 0.008), serum CgA level (≥150/<150, ng/ml, OR 6.25, P = 0.005), and tumor grade G2 (G2/G1, OR 9.6, P < 0.001) were independently associated with metastases on DOTATATE. CONCLUSIONS: Among patients with GEP-NETs, primary tumor size ≥2 cm, serum CgA level ≥150 ng/mL, and tumor grade G2 are associated with an increased risk of metastases on DOTATATE, and these predictors may be helpful to identify patients where DOTATATE is indicated for complete staging.
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T cell activation is initiated by T cell receptor (TCR) phosphorylation. This requires the local depletion of large receptor-type phosphatases from "close contacts" formed when T cells interact with surfaces presenting agonistic TCR ligands, but exactly how the ligands potentiate signaling is unclear. It has been proposed that TCR ligands could enhance receptor phosphorylation and signaling just by holding TCRs in phosphatase-depleted close contacts, but this has not been directly tested. We devised simple methods to move the TCR in and out of close contacts formed by T cells interacting with supported lipid bilayers (SLBs) and to slow the receptor's diffusion in the contacts, using a series of anti-CD3ε Fab- and ligand-based adducts of the receptor. TCRs engaging a Fab extended with the large extracellular region of CD45 were excluded from contacts and produced no signaling. Conversely, allowing the extended Fab to become tethered to the SLB trapped the TCR in the close contacts, leading to very strong signaling. Importantly, attaching untethered anti-CD3ε Fab or peptide/MHC ligands, each of which were largely inactive in solution but both of which reduced TCR diffusion in close contacts approximately fivefold, also initiated signaling during cell/SLB contact. Our findings indicate that holding TCRs in close contacts or simply slowing their diffusion in phosphatase-depleted regions of the cell surface suffices to initiate signaling, effects we could reproduce in single-particle stochastic simulations. Our study shows that the TCR is preconfigured for signaling in a way that allows it to be triggered by ligands acting simply as receptor "traps."
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Comunicação Celular , Membrana Celular/metabolismo , Bicamadas Lipídicas/metabolismo , Ativação Linfocitária , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismo , Humanos , Ligantes , Fosforilação , Linfócitos T/citologiaRESUMO
PURPOSE: Distal radius fractures (DRFs) are one of the most common conditions that musculoskeletal providers treat. As the frequency of solid organ transplants (SOT) increases, these providers are often called upon to manage DRFs in these patients. These patients are at increased risk for osteopenia and osteoporosis, given the altered bone metabolism after SOT and frequent use of glucocorticoid and immunosuppressive medications. This study aimed to examine both surgical and nonsurgical treatment outcomes of DRFs in the SOT population and the prevalence of decreased bone mineral density. METHODS: A retrospective review of patients treated at a single institution who had previously undergone SOT and subsequently sustained DRF between 2013 and 2022 was completed. Patients were excluded for incomplete documentation and treatment initiation at an outside institution. Demographic variables, clinical outcomes, organ transplant, steroid use, and second metacarpal cortical percentage were collected for both groups. A telephone survey with the QuickDASH questionnaire was conducted for all available patients. RESULTS: A total of 34 DRFs in 33 patients were included in the analysis. Of these, 15 fractures in 14 patients underwent surgical intervention, and 19 fractures in 19 patients were managed nonsurgically. The following three adverse events were observed in the patients managed operatively: hardware failure, postoperative carpal tunnel syndrome, and tendon irritation. No reported treatment complications were recorded in the patients managed nonsurgically. Of the 33 included patients, 32 had radiographic evidence of decreased bone mineral density and five were receiving treatment for osteoporosis. CONCLUSIONS: Management of DRFs in SOT patients is challenging, given their increased medical complexity. Decreased bone mineral density was nearly universal and undertreated in this patient population. Most of these patients in both groups had good or excellent functional outcomes with both surgical and nonsurgical management. Additionally, surgery was well tolerated with no reported anesthesia complications, wound-healing issues, or infections. TYPE OF STUDY/LEVEL OF EVIDENCE: Therapeutic IV.
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Addition of plerixafor (P) to granulocyte colony stimulating factor (G-CSF) during peripheral blood mobilization of hematopoietic stem cells (HSC) increases the number of patients meeting collection goals prior to autologous stem cell transplant (aSCT). However, use of P is not universal among transplant centers due to cost. This study aims to compare clinical and financial impacts of using an algorithm-based P mobilization strategy versus use in all patients. This was a single center, retrospective analysis of adult patients with myeloma or amyloidosis receiving aSCT who received apheresis of their HSC between 3/1/2017 and 3/1/2019. Patients prior to 3/1/2018 were classified as receiving P "per algorithm" and those after this date were classified as "up-front" P. For the per-algorithm group, P was given for a pre-apheresis CD34+ cell count of <20 cells/µL on mobilization day 5 and patients returned on day 6 for apheresis. Of the 129 patients included, 55 received P per-algorithm and 74 received up-front P. There was a reduction in median number of apheresis days (1.5 vs 1 day, p < 0.001) and an increase in median number of CD34+ cells collected (6.6 vs 8.5 × 106 cells/kg, p < 0.001) with up-front P. Up-front P increased drug cost but reduced apheresis costs, which resulted in a net savings of $121 per patient in total mobilization costs. These findings suggest that use of up-front P for mobilization significantly reduces apheresis days and increases HSC collection yield without increasing overall cost per patient.
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Ciclamos , Transplante de Células-Tronco Hematopoéticas , Compostos Heterocíclicos , Mieloma Múltiplo , Adulto , Antígenos CD34 , Benzilaminas , Fator Estimulador de Colônias de Granulócitos , Mobilização de Células-Tronco Hematopoéticas/métodos , Compostos Heterocíclicos/efeitos adversos , Humanos , Mieloma Múltiplo/terapia , Estudos Retrospectivos , Transplante AutólogoRESUMO
Much of what we know about the early stages of T cell activation has been obtained from studies of T cells interacting with glass-supported lipid bilayers that favor imaging but are orders of magnitude stiffer than typical cells. We developed a method for attaching lipid bilayers to polydimethylsiloxane polymer supports, producing "soft bilayers" with physiological levels of mechanical resistance (Young's modulus of 4 kPa). Comparisons of T cell behavior on soft and glass-supported bilayers revealed that whereas late stages of T cell activation are thought to be substrate-stiffness dependent, early calcium signaling was unaffected by substrate rigidity, implying that early steps in T cell receptor triggering are not mechanosensitive. The exclusion of large receptor-type phosphatases was observed on the soft bilayers, however, even though it is yet to be demonstrated at authentic cell-cell contacts. This work sets the stage for an imaging-based exploration of receptor signaling under conditions closely mimicking physiological cell-cell contact.
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Bicamadas Lipídicas , Linfócitos T , Comunicação Celular , Dimetilpolisiloxanos , Módulo de ElasticidadeRESUMO
The transferrin (Tf) trafficking pathway is a promising mechanism for use in targeted cancer therapy due to the overexpression of transferrin receptors (TfRs) on cancerous cells. We have previously developed a mathematical model of the Tf/TfR trafficking pathway to improve the efficiency of Tf as a drug carrier. By using diphtheria toxin (DT) as a model toxin, we found that mutating the Tf protein to change its iron release rate improves cellular association and efficacy of the drug. Though this is an improvement upon using wild-type Tf as the targeting ligand, conjugated toxins like DT are unfortunately still highly cytotoxic at off-target sites. In this work, we address this hurdle in cancer research by developing a mathematical model to predict the efficacy and selectivity of Tf conjugates that use an alternative toxin. For this purpose, we have chosen to study a mutant of DT, cross-reacting material 107 (CRM107). First, we developed a mathematical model of the Tf-DT trafficking pathway by extending our Tf/TfR model to include intracellular trafficking via DT and DT receptors. Using this mathematical model, we subsequently investigated the efficacy of several conjugates in cancer cells: DT and CRM107 conjugated to wild-type Tf, as well as to our engineered mutant Tf proteins (K206E/R632A Tf and K206E/R534A Tf). We also investigated the selectivity of mutant Tf-CRM107 against non-neoplastic cells. Through the use of our mathematical model, we predicted that (i) mutant Tf-CRM107 exhibits a greater cytotoxicity than wild-type Tf-CRM107 against cancerous cells, (ii) this improvement was more drastic with CRM107 conjugates than with DT conjugates, and (iii) mutant Tf-CRM107 conjugates were selective against non-neoplastic cells. These predictions were validated with in vitro cytotoxicity experiments, demonstrating that mutant Tf-CRM107 conjugates is indeed a more suitable therapeutic agent. Validation from in vitro experiments also confirmed that such whole-cell kinetic models can be useful in cancer therapeutic design.
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Toxinas Bacterianas/genética , Modelos Teóricos , Neoplasias/tratamento farmacológico , Transferrina/genética , Antineoplásicos/uso terapêutico , Toxinas Bacterianas/uso terapêutico , Toxina Diftérica , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Mutagênese Sítio-Dirigida , Mutação , Transferrina/análogos & derivados , Transferrina/uso terapêuticoRESUMO
Purpose: Unplanned excisions are defined as excisions of malignant tumors performed without preoperative cross-sectional imaging or diagnostic biopsy, frequently resulting in residual disease and re-excision secondary to positive surgical margins. The purpose of this study was to compare the relative morbidity of planned versus unplanned upper-extremity sarcoma excisions. Methods: A single tertiary referral hospital pathology database was queried from January 2015 through 2022 for primary upper-extremity sarcomas (forearm, wrist, hand, and finger). Demographics, tumor features, survival characteristics, and outcomes were retrospectively reviewed. Results: Forty-two upper-extremity sarcoma patients were identified, two-thirds of whom had unplanned excisions. Those with unplanned excisions were more likely to be female (relative risk [RR]: 1.9; P = .002), undergo initial excision at a nonsarcoma center (RR: 14.0; P < .001), have masses distal to the forearm (RR: 1.6; P = .02), and have smaller masses (4.8 vs 7.4 cm, P = .03). 71.4% of tumors were high grade, and 60.7% less than 5 cm in size.Unplanned excisions had positive margins in 96.4% of cases and were more likely to undergo re-excision (odds ratio [OR]: 20.0; P = .001), more total resections (2.7 vs 1.4, P = .009), sacrifice of neurovascular structures (OR: 6.1; P = .04), adjuvant radiation therapy (OR: 4.5; P = .05), adjuvant systemic therapy (OR: 10.9; P = .03), or experience a complication (OR: 17.6; P = .002) at an average of 38.0 months of follow-up.Nearly half of all unplanned excision patients developed a local recurrence or metastatic disease. Six patients required an amputation versus one in the planned cohort (P = .17), and 26.5% of patients died at an average of 32.5 months from presentation. Conclusions: Distal upper-extremity sarcoma excisions are frequently unplanned, with high rates of morbidity compared with planned excisions. Surgeons should have a low threshold for cross-sectional imaging and core needle biopsy of atypical lesions, irrespective of size, with referral to a sarcoma center. Type of study/level of evidence: Prognostic IV.
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Peyer's patches (PPs) are lymphoid structures situated adjacent to the intestinal epithelium that support B cell responses that give rise to many intestinal IgA-secreting cells. Induction of isotype switching to IgA in PPs requires interactions between B cells and TGFß-activating conventional dendritic cells type 2 (cDC2s) in the subepithelial dome (SED). However, the mechanisms promoting cDC2 positioning in the SED are unclear. Here, we found that PP cDC2s express GPR35, a receptor that promotes cell migration in response to various metabolites, including 5-hydroxyindoleacetic acid (5-HIAA). In mice lacking GPR35, fewer cDC2s were found in the SED, and frequencies of IgA+ germinal center (GC) B cells were reduced. IgA plasma cells were reduced in both the PPs and lamina propria. These phenotypes were also observed in chimeric mice that lacked GPR35 selectively in cDCs. GPR35 deficiency led to reduced coating of commensal bacteria with IgA and reduced IgA responses to cholera toxin. Mast cells were present in the SED, and mast cell-deficient mice had reduced PP cDC2s and IgA+ cells. Ablation of tryptophan hydroxylase 1 (Tph1) in mast cells to prevent their production of 5-HIAA similarly led to reduced PP cDC2s and IgA responses. Thus, mast cell-guided positioning of GPR35+ cDC2s in the PP SED supports induction of intestinal IgA responses.
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Linfócitos B , Mastócitos , Animais , Camundongos , Ácido Hidroxi-Indolacético , Movimento Celular , Imunoglobulina A Secretora , Nódulos Linfáticos Agregados , Receptores Acoplados a Proteínas G/genéticaRESUMO
Mucosal melanoma (MM) is a deadly cancer derived from mucosal melanocytes. To test the consequences of MM genetics, we developed a zebrafish model in which all melanocytes experienced CCND1 expression and loss of PTEN and TP53. Surprisingly, melanoma only developed from melanocytes lining internal organs, analogous to the location of patient MM. We found that zebrafish MMs had a unique chromatin landscape from cutaneous melanoma. Internal melanocytes could be labeled using a MM-specific transcriptional enhancer. Normal zebrafish internal melanocytes shared a gene expression signature with MMs. Patient and zebrafish MMs have increased migratory neural crest gene and decreased antigen presentation gene expression, consistent with the increased metastatic behavior and decreased immunotherapy sensitivity of MM. Our work suggests the cell state of the originating melanocyte influences the behavior of derived melanomas. Our animal model phenotypically and transcriptionally mimics patient tumors, allowing this model to be used for MM therapeutic discovery.
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Introduction: Distal third forearm fractures are common fractures in children. While outcomes are generally excellent, some patients fail initial non-operative management and require intervention. The purpose of this study is to identify independent risk factors associated with failure of closed reduction. Methods: We conducted a retrospective review of distal third forearm fractures in children treated with closed reduction and casting. Patients were divided into two cohorts-those who were successfully closed reduced and those who failed initial non-operative management. Demographic characteristics, cast type, cast index, radiographic fracture, soft tissue characteristics, and quality of reduction were analyzed between groups. Results: A total of 207 children treated for distal third forearm fractures were included for analysis. A total of 190 (91.8%) children maintained their reduction while 17 (8.2%) failed initial non-operative management. Modifiable risk factors associated with loss of reduction on univariate analysis included the use of a long arm cast (p = 0.003), increased post-reduction displacement (p = 0.02), and increased post-reduction angular deformity (p = 0.01). Non-modifiable risk factors included increased body mass index (p = 0.02), increased presenting fracture displacement (p = 0.002), and increased width of the soft tissue envelope at the fracture site (p = 0.0001). The use of long arm casts (13% vs 2%, odds ratio = 6.44) and soft tissue width (60.6 vs 50.4 mm, odds ratio = 1.1) remained significant risk factors for loss of reduction after multivariate analysis. Conclusion: Both larger soft tissue envelope at the site of the fracture and long arm cast immobilization are independently associated with an increased risk of failing initial closed reduction in distal third forearm fractures in the pediatric population. Level of evidence: level III Case Control Study.
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Nil.
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Colite Ulcerativa , Colite , Humanos , Estudos Retrospectivos , Nova Zelândia , Colite/diagnóstico , Biomarcadores , Colectomia , Infliximab , Resultado do Tratamento , Índice de Gravidade de DoençaRESUMO
T cells use finger-like protrusions called 'microvilli' to interrogate their targets, but why they do so is unknown. To form contacts, T cells must overcome the highly charged, barrier-like layer of large molecules forming a target cell's glycocalyx. Here, T cells are observed to use microvilli to breach a model glycocalyx barrier, forming numerous small (<0.5 µm diameter) contacts each of which is stabilized by the small adhesive protein CD2 expressed by the T cell, and excludes large proteins including CD45, allowing sensitive, antigen dependent TCR signaling. In the absence of the glycocalyx or when microvillar contact-size is increased by enhancing CD2 expression, strong signaling occurs that is no longer antigen dependent. Our observations suggest that, modulated by the opposing effects of the target cell glycocalyx and small adhesive proteins, the use of microvilli equips T cells with the ability to effect discriminatory receptor signaling.
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Antígenos , Linfócitos T , Antígenos/metabolismo , Transdução de Sinais , Microvilosidades/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Ativação LinfocitáriaRESUMO
RNA surveillance pathways detect and degrade defective transcripts to ensure RNA fidelity. We found that disrupted nuclear RNA surveillance is oncogenic. Cyclin-dependent kinase 13 (CDK13) is mutated in melanoma, and patient-mutated CDK13 accelerates zebrafish melanoma. CDK13 mutation causes aberrant RNA stabilization. CDK13 is required for ZC3H14 phosphorylation, which is necessary and sufficient to promote nuclear RNA degradation. Mutant CDK13 fails to activate nuclear RNA surveillance, causing aberrant protein-coding transcripts to be stabilized and translated. Forced aberrant RNA expression accelerates melanoma in zebrafish. We found recurrent mutations in genes encoding nuclear RNA surveillance components in many malignancies, establishing nuclear RNA surveillance as a tumor-suppressive pathway. Activating nuclear RNA surveillance is crucial to avoid accumulation of aberrant RNAs and their ensuing consequences in development and disease.
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Proteína Quinase CDC2 , Carcinógenos , Melanoma , Estabilidade de RNA , RNA Nuclear , Neoplasias Cutâneas , Animais , Proteína Quinase CDC2/genética , Melanoma/genética , Mutação , RNA Nuclear/genética , Neoplasias Cutâneas/genética , Peixe-Zebra , HumanosRESUMO
BACKGROUND: While the introduction of immune checkpoint inhibitors (ICI) such as pembrolizumab has significantly improved survival for patients with metastatic non-small cell lung cancer (NSCLC), there remains a need for improved predictive and prognostic biomarkers. PATIENTS AND METHODS: We conducted a retrospective, 3-center study using electronic medical record data for patients with stage IV NSCLC treated with first-line pembrolizumab, either as monotherapy or in combination with chemotherapy, between 2014 and 2019. We categorized variables as covariates or confounders. Covariates, which were the focus of analysis due to their emerging prognostic value, included pretreatment body mass index (BMI), neutrophil-to-lymphocyte ratio (NLR), albumin, and antibiotic exposure. Confounders, which highlighted characteristics for each patient and their cancer, included sex, age at start of immunotherapy, Programmed death-ligand 1 (PD-L1) expression, performance status (PS), tumor mutational burden and whether pembrolizumab was given as monotherapy or in combination with chemotherapy. The association between these variables with time to treatment failure (TTF) and overall survival (OS) was assessed using Kaplan-Meier method and Cox proportional hazards models. RESULTS: One hundred thirty-six patients were included in our study. Antibiotics usage, serum albumin, and NLR have univariate relationships with TTF. Serum albumin, NLR, and BMI were associated with OS in univariate analyses. In our multivariate analysis, antibiotic usage had a strong negative association with TTF when adjusting for all 6 confounders. CONCLUSION: Pretreatment usage of antibiotics, as well as albumin, NLR, and BMI have potential to predict treatment outcomes in patients with advanced NSCLC receiving first-line immunotherapy.
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Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Antibacterianos/uso terapêutico , Anticorpos Monoclonais Humanizados , Antígeno B7-H1 , Biomarcadores Tumorais/análise , Carcinoma Pulmonar de Células não Pequenas/patologia , Humanos , Neoplasias Pulmonares/patologia , Estudos Retrospectivos , Albumina Sérica/uso terapêutico , Resultado do TratamentoRESUMO
Elevated uptake of saturated fatty acid palmitate is associated with metastatic progression of cancer cells; however, the precise signaling mechanism behind the phenomenon is unclear. The loss of cell adhesion proteins, such as desmoplakin (DSP), is a key driving event in the transformation of cancer cells to more aggressive phenotypes. Here, we investigated the mechanism by which palmitate induces the loss of DSP in liver and breast cancer cells. We propose that palmitate activates the IRE1-XBP1 branch of the endoplasmic reticulum (ER) stress pathway to upregulate the ZEB transcription factor, leading to transcriptional repression of DSP. Using liver and breast cancer cells treated with palmitate, we found loss of DSP leads to increased cell migration independent of E-cadherin. We report that the ZEB family of transcription factors function as direct transcriptional repressors of DSP. CRISPR-mediated knockdown of IRE1 confirmed that the transcription of ZEB, loss of DSP, and enhanced migration in the presence of palmitate is dependent on the IRE1-XBP1 pathway. In addition, by analyzing the somatic expression and copy number variation profiles of over 11,000 tumor samples, we corroborate our hypothesis and establish the clinical relevance of DSP loss via ZEB in human cancers. IMPLICATIONS: Provides mechanistic link on palmitate-induced activation of IRE1α to cancer cell migration.
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Desmoplaquinas/metabolismo , Transição Epitelial-Mesenquimal/genética , Palmitatos/metabolismo , Resposta a Proteínas não Dobradas/genética , Proteína 1 de Ligação a X-Box/genética , Movimento Celular , Humanos , Transdução de SinaisRESUMO
Concept maps are graphical representations of how various concepts relate to one another. Assessment of concept maps developed by students in the pharmacy curriculum helps to evaluate student understanding of course material. However, providing feedback on concept maps can be time-consuming and often requires the grader to be a content expert. The purpose of this study was to develop and validate a software program to provide students with feedback on their concept map performance. Student maps for four different disease states were compared against expert concept maps. The analysis of the program compared favorably to a manual assessment of student maps for the maps' complexity and content but did not correlate for their organization. The value of using a software program to quickly and efficiently analyze concept maps is discussed.
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Chronic lymphocytic leukemia (CLL) is a hematologic malignancy that has seen significant advances in care over the last 5 years with the approval of oral agents such as ibrutinib and venetoclax for the treatment of this disease. As such, there has been a substantial shift away from the traditional chemotherapy infusions which have allowed patients greater autonomy with oral cancer therapies. This paradigm shift poses new challenges for the medical team, including drug-drug interactions, adherence counseling, and financial toxicity. Pharmacists are uniquely trained and equipped to help to manage the changing landscape of CLL care. From identifying common medications which may impair ibrutinib clearance to ensuring patients are on the appropriate anti-infective prophylaxis while receiving obinutuzumab, pharmacists can play a vital role in ensuring the highest quality of patient care. Furthermore, additional credentialing of clinical pharmacists in select states allows for independent visits with the pharmacists, allowing for greater involvement, particularly for initiation of venetoclax and management of ibrutinib-induced toxicities. Pharmacists are essential to both expanding and enhancing the care of patients with CLL and should be leveraged to improve patient outcomes whenever possible.
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In single molecule fluorescence enzymology, background fluorescence from labeled substrates in solution often limits fluorophore concentration to pico- to nanomolar ranges, several orders of magnitude less than many physiological ligand concentrations. Optical nanostructures called zero mode waveguides (ZMWs), which are 100-200 nm in diameter apertures fabricated in a thin conducting metal such as aluminum or gold, allow imaging of individual molecules at micromolar concentrations of fluorophores by confining visible light excitation to zeptoliter effective volumes. However, the need for expensive and specialized nanofabrication equipment has precluded the widespread use of ZMWs. Typically, nanostructures such as ZMWs are obtained by direct writing using electron beam lithography, which is sequential and slow. Here, colloidal, or nanosphere, lithography is used as an alternative strategy to create nanometer-scale masks for waveguide fabrication. This report describes the approach in detail, with practical considerations for each phase. The method allows thousands of aluminum or gold ZMWs to be made in parallel, with final waveguide diameters and depths of 100-200 nm. Only common lab equipment and a thermal evaporator for metal deposition are required. By making ZMWs more accessible to the biochemical community, this method can facilitate the study of molecular processes at cellular concentrations and rates.
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Microscopia de Fluorescência , Microtecnologia/métodos , Nanoestruturas/química , Imagem Individual de Molécula , Alumínio/química , Coloides/química , Cobre/química , Cristalização , Análise de Elementos Finitos , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/química , Ouro/química , Microesferas , Poliestirenos/química , PorosidadeRESUMO
Epigenetic regulators, when genomically altered, may become driver oncogenes that mediate otherwise unexplained pro-oncogenic changes lacking a clear genetic stimulus, such as activation of the WNT/ß-catenin pathway in melanoma. This study identifies previously unrecognized recurrent activating mutations in the G9a histone methyltransferase gene, as well as G9a genomic copy gains in approximately 26% of human melanomas, which collectively drive tumor growth and an immunologically sterile microenvironment beyond melanoma. Furthermore, the WNT pathway is identified as a key tumorigenic target of G9a gain-of-function, via suppression of the WNT antagonist DKK1. Importantly, genetic or pharmacologic suppression of mutated or amplified G9a using multiple in vitro and in vivo models demonstrates that G9a is a druggable target for therapeutic intervention in melanoma and other cancers harboring G9a genomic aberrations. SIGNIFICANCE: Oncogenic G9a abnormalities drive tumorigenesis and the "cold" immune microenvironment by activating WNT signaling through DKK1 repression. These results reveal a key druggable mechanism for tumor development and identify strategies to restore "hot" tumor immune microenvironments.This article is highlighted in the In This Issue feature, p. 890.