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Colorectal cancer (CRC) is the third most common cancer worldwide. In the United States alone, CRC was responsible for approximately 52,550 deaths in 2023, with an estimated 153,020 new cases. CRC presents with synchronous peritoneal spread in 5-10% of patients, and up to 20-50% of patients with recurrent disease will develop metachronous colorectal cancer peritoneal metastatic (CRC-PM) disease. Eradication of the tumor, tumor margins and microscopic residual disease is paramount, as microscopic residual disease is associated with local recurrences, with 5-year survival rates of less than 35%. The success of resection and reduction of residual disease depends on the accuracy with which cancer cells and normal tissue can be intra-operatively distinguished. Fluorescence Molecular Imaging (IFMI) and tumor-targeted contrast agents represent a promising approach for intraoperative detection and surgical intervention. Proper target selection, the development of scalable imaging agents and enhanced real-time tumor and tumor microenvironment imaging are critical to enabling enhanced surgical resection. LGR5 (leucine-rich repeat-containing G-protein-coupled receptor 5), a colonic crypt stem cell marker and the receptor for the R-spondins (RSPO) in the Wnt signaling pathway, is also expressed on colorectal cancer stem cells (CSC) and on CRC tumors and metastases, suggesting it could be a useful target for imaging of CRC. However, there are numerous diverging reports on the role of LGR5 in CRC therapy and outcomes. Herein, we report on the synthesis and validation of a 37 amino acid RSPO1-mimetic peptide, termed RC18, that was specifically designed to access the R-spondin binding site of LGR5 to potentially be used for interoperative imaging of CRC-PM. The receptor-binding capabilities of the RC18 indicate that direct interactions with LGR5 neither significantly increased LGR5 signaling nor blocked RSPO1 binding and signal transduction, suggesting that the RSPO1-mimetic is functionally inert, making it an attractive contrast agent for intraoperative CRC-PM imaging.
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Casein kinase 1α (CK1α) is a serine/threonine protein kinase that acts in various cellular processes affecting cell division and signal transduction. CK1α is present as multiple splice variants that are distinguished by the presence or absence of a long insert (L-insert) and a short carboxyl-terminal insert (S-insert). When overexpressed, zebrafish CK1α splice variants exhibit different biological properties, such as subcellular localization and catalytic activity. However, whether endogenous, alternatively spliced CK1α gene products also differ in their biological functions has yet to be elucidated. Here, we identify a panel of splice variant specific CK1α antibodies and use them to show that four CK1α splice variants are expressed in mammals. We subsequently show that the relative abundance of CK1α splice variants varies across distinct mouse tissues and between various cancer cell lines. Furthermore, we identify pathways whose expression is noticeably altered in cell lines enriched with select splice variants of CK1α. Finally, we show that the S-insert of CK1α promotes the growth of HCT 116 cells as cells engineered to lack the S-insert display decreased cell growth. Together, we provide tools and methods to identify individual CK1α splice variants, which we use to begin to uncover the differential biological properties driven by specific splice variants of mammalian CK1α.
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Empalme Alternativo , Caseína Quinasa Ialfa , Animales , Humanos , Ratones , Caseína Quinasa Ialfa/metabolismo , Caseína Quinasa Ialfa/genética , Línea Celular Tumoral , Proliferación Celular , Células HCT116 , Isoenzimas/genética , Isoenzimas/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologíaRESUMEN
Most children with medulloblastoma (MB) achieve remission, but some face very aggressive metastatic tumors. Their dismal outcome highlights the critical need to advance therapeutic approaches that benefit such high-risk patients. Minnelide, a clinically relevant analog of the natural product triptolide, has oncostatic activity in both preclinical and early clinical settings. Despite its efficacy and tolerable toxicity, this compound has not been evaluated in MB. Utilizing a bioinformatic data set that integrates cellular drug response data with gene expression, we predicted that Group 3 (G3) MB, which has a poor 5-year survival, would be sensitive to triptolide/Minnelide. We subsequently showed that both triptolide and Minnelide attenuate the viability of G3 MB cells ex vivo. Transcriptomic analyses identified MYC signaling, a pathologically relevant driver of G3 MB, as a downstream target of this class of drugs. We validated this MYC dependency in G3 MB cells and showed that triptolide exerts its efficacy by reducing both MYC transcription and MYC protein stability. Importantly, Minnelide acted on MYC to reduce tumor growth and leptomeningeal spread, which resulted in improved survival of G3 MB animal models. Moreover, Minnelide improved the efficacy of adjuvant chemotherapy, further highlighting its potential for the treatment of MYC-driven G3 MB.
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Diterpenos , Compuestos Epoxi , Meduloblastoma , Fenantrenos , Proteínas Proto-Oncogénicas c-myc , Ensayos Antitumor por Modelo de Xenoinjerto , Fenantrenos/farmacología , Diterpenos/farmacología , Compuestos Epoxi/farmacología , Meduloblastoma/tratamiento farmacológico , Meduloblastoma/genética , Meduloblastoma/patología , Meduloblastoma/metabolismo , Animales , Humanos , Ratones , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Línea Celular Tumoral , Profármacos/farmacología , Neoplasias Cerebelosas/tratamiento farmacológico , Neoplasias Cerebelosas/genética , Neoplasias Cerebelosas/patología , Neoplasias Cerebelosas/metabolismo , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , OrganofosfatosRESUMEN
Type 2 diabetes (T2D) is a complex chronic metabolic disorder characterized by hyperglycemia because of insulin resistance. Diabetes with chronic hyperglycemia may alter brain metabolism, including brain glucose and neurotransmitter levels; however, detailed, longitudinal studies of metabolic alterations in T2D are lacking. To shed insight, here, we characterized the consequences of poorly controlled hyperglycemia on neurochemical profiles that reflect metabolic alterations of the brain in both humans and animal models of T2D. Using in vivo 1 H magnetic resonance spectroscopy, we quantified 12 metabolites cross-sectionally in T2D patients and 20 metabolites longitudinally in T2D db/db mice versus db+ controls. We found significantly elevated brain glucose (91%, p < 0.001), taurine (22%, p = 0.02), glucose+taurine (56%, p < 0.001), myo-inositol (12%, p = 0.02), and choline-containing compounds (10%, p = 0.01) in T2D patients versus age- and sex-matched controls, findings consistent with measures in T2D db/db versus control db+ littermates. In mice, hippocampal and striatal neurochemical alterations in brain glucose, ascorbate, creatine, phosphocreatine, γ-aminobutyric acid, glutamate, glutamine, glutathione, glycerophosphoryl-choline, lactate, myo-inositol, and taurine persisted in db/db mice with chronic disease progression from 16 to 48 weeks of age, which were distinct from control db+ mice. Overall, our study demonstrates the utility of 1 H magnetic resonance spectroscopy as a non-invasive tool for characterizing and monitoring brain metabolic changes with T2D progression.
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The control of Wnt receptor abundance is critical for animal development and to prevent tumorigenesis, but the mechanisms that mediate receptor stabilization remain uncertain. We demonstrate that stabilization of the essential Wingless/Wnt receptor Arrow/LRP6 by the evolutionarily conserved Usp46-Uaf1-Wdr20 deubiquitylase complex controls signaling strength in Drosophila. By reducing Arrow ubiquitylation and turnover, the Usp46 complex increases cell surface levels of Arrow and enhances the sensitivity of target cells to stimulation by the Wingless morphogen, thereby increasing the amplitude and spatial range of signaling responses. Usp46 inactivation in Wingless-responding cells destabilizes Arrow, reduces cytoplasmic accumulation of the transcriptional coactivator Armadillo/ß-catenin, and attenuates or abolishes Wingless target gene activation, which prevents the concentration-dependent regulation of signaling strength. Consequently, Wingless-dependent developmental patterning and tissue homeostasis are disrupted. These results reveal an evolutionarily conserved mechanism that mediates Wnt/Wingless receptor stabilization and underlies the precise activation of signaling throughout the spatial range of the morphogen gradient.
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Proteínas de Drosophila , Vía de Señalización Wnt , Animales , Proteínas de Drosophila/metabolismo , Proteína Wnt1/genética , Proteína Wnt1/metabolismo , Drosophila/genética , Factores de Transcripción/metabolismoRESUMEN
The relative abundance of Wnt receptors plays a crucial role in controlling Wnt signaling in tissue homeostasis and human disease. While the ubiquitin ligases that ubiquitylate Wnt receptors are well-characterized, the deubiquitylase that reverses these reactions remains unclear. Herein, we identify USP46, UAF1, and WDR20 (USP46 complex) as positive regulators of Wnt signaling in cultured human cells. We find that the USP46 complex is similarly required for Wnt signaling in Xenopus and zebrafish embryos. We demonstrate that Wnt signaling promotes the association between the USP46 complex and cell surface Wnt coreceptor, LRP6. Knockdown of USP46 decreases steady-state levels of LRP6 and increases the level of ubiquitylated LRP6. In contrast, overexpression of the USP46 complex blocks ubiquitylation of LRP6 by the ubiquitin ligases RNF43 and ZNFR3. Size exclusion chromatography studies suggest that the size of the USP46 cytoplasmic complex increases upon Wnt stimulation. Finally, we show that USP46 is essential for Wnt-dependent intestinal organoid viability, likely via its role in LRP6 receptor homeostasis. We propose a model in which the USP46 complex increases the steady-state level of cell surface LRP6 and facilitates the assembly of LRP6 into signalosomes via a pruning mechanism that removes sterically hindering ubiquitin chains.
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Endopeptidasas , Vía de Señalización Wnt , beta Catenina , Animales , Humanos , beta Catenina/genética , beta Catenina/metabolismo , Ligasas/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismo , Receptores Wnt , Ubiquitina , Pez Cebra/metabolismo , Endopeptidasas/metabolismoRESUMEN
PURPOSE: To elicit feedback from participants who completed the eMOMSTM study, a feasibility randomized controlled trial (NCT04021602), on their perceptions of program strengths and weaknesses. STUDY DESIGN: Qualitative - Semi-structured, telephone interview guide using open-ended questions. SETTING: Rural Great Plains state, United States. PARTICIPANTS: Of 26 individuals who completed the eMOMSTM study, 24 consented to an interview. METHOD: Interviews were completed between October 2020 and May 2021. Audio-recordings were transcribed verbatim and organized in Microsoft 365. Data were analyzed using an exploratory, inductive thematic analysis. RESULTS: Participants' mean age was 27.5 (± 5.4) years and mean pre-pregnancy BMI was 29.5 kg/m2 (± 2.7). The majority (71%) were non-Hispanic White and 54% had a high school education/some college. Based on specific areas of inquiry, the following themes emerged: convenience of online program access using Facebook, importance of health coach's support and online interaction, positivity toward improving one's health, increased consciousness of health behaviors, diverse lactation educational needs, importance of educational materials on depression, and grief over the loss of birth expectations during COVID-19. CONCLUSION: Findings suggest participants' perceived value of a lifestyle change program coupled with lactation education and support delivered using social media. Findings inform future studies to further adapt lifestyle change programs.
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COVID-19 , Femenino , Embarazo , Humanos , Adulto , COVID-19/prevención & control , Conductas Relacionadas con la Salud , Estilo de Vida , Electrónica , LactanciaRESUMEN
The Wnt-ß-catenin signal transduction pathway is essential for embryonic development and adult tissue homeostasis. Wnt signaling converts TCF from a transcriptional repressor to an activator in a process facilitated by the E3 ligase XIAP. XIAP-mediated monoubiquitylation of the transcriptional corepressor Groucho (also known as TLE) decreases its affinity for TCF, thereby allowing the transcriptional coactivator ß-catenin to displace it on TCF. Through a genome-scale screen in cultured Drosophila melanogaster cells, we identified the deubiquitylase USP47 as a positive regulator of Wnt signaling. We found that USP47 was required for Wnt signaling during Drosophila and Xenopus laevis development, as well as in human cells, indicating evolutionary conservation. In human cells, knockdown of USP47 inhibited Wnt reporter activity, and USP47 acted downstream of the ß-catenin destruction complex. USP47 interacted with TLE3 and XIAP but did not alter their amounts; however, knockdown of USP47 enhanced XIAP-mediated ubiquitylation of TLE3. USP47 inhibited ubiquitylation of TLE3 by XIAP in vitro in a dose-dependent manner, suggesting that USP47 is the deubiquitylase that counteracts the E3 ligase activity of XIAP on TLE. Our data suggest a mechanism by which regulated ubiquitylation and deubiquitylation of TLE enhance the ability of ß-catenin to cycle on and off TCF, thereby helping to ensure that the expression of Wnt target genes continues only as long as the upstream signal is present.
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Vía de Señalización Wnt , beta Catenina , Animales , Humanos , beta Catenina/metabolismo , Drosophila , Drosophila melanogaster/metabolismo , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , XenopusRESUMEN
The Hedgehog signaling pathway functions in both embryonic development and adult tissue homeostasis. Importantly, its aberrant activation is also implicated in the progression of multiple types of cancer, including basal cell carcinoma and medulloblastoma. GLI transcription factors function as the ultimate effectors of the Hedgehog signaling pathway. Their activity is regulated by this signaling cascade via their mRNA expression, protein stability, subcellular localization, and ultimately their transcriptional activity. Further, GLI proteins are also regulated by a variety of non-canonical mechanisms in addition to the canonical Hedgehog pathway. Recently, with an increased understanding of epigenetic gene regulation, novel transcriptional regulators have been identified that interact with GLI proteins in multi-protein complexes to regulate GLI transcriptional activity. Such complexes have added another layer of complexity to the regulation of GLI proteins. Here, we summarize recent work on the regulation of GLI transcriptional activity by these novel protein complexes and describe their relevance to cancer, as such GLI regulators represent alternative and innovative druggable targets in GLI-dependent cancers.
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Background: Medulloblastoma (MB) is the most common pediatric brain tumor. Although standard-of-care treatment generally results in good prognosis, many patients exhibit treatment-associated lifelong disabilities. This outcome could be improved by employing therapies targeting the molecular drivers of this cancer. Attempts to do so in the SONIC HEDGEHOG MB subgroup (SHH-MB) have largely focused on the SHH pathway's principal activator, smoothened (SMO). While inhibitors targeting SMO have shown clinical efficacy, recurrence and resistance are frequently noted, likely resulting from mutations in or downstream of SMO. Therefore, identification of novel SHH regulators that act on the pathway's terminal effectors could be used to overcome or prevent such recurrence. We hypothesized that protein arginine methyltransferase 5 (PRMT5) is one such regulator and investigated its role and potential targeting in SHH-MB. Methods: PRMT5 expression in SHH-MB was first evaluated. Knockdown and pharmacological inhibitors of PRMT5 were used in SHH-MB sphere cultures to determine its effect on viability and SHH signaling. GLI1 arginine methylation was then characterized in primary SHH-MB tissue using LC-MS/MS. Finally, PRMT5 inhibitor efficacy was evaluated in vivo. Results: PRMT5 is overexpressed in SHH-MB tissue. Furthermore, SHH-MB viability and SHH activity is dependent on PRMT5. We found that GLI1 isolated from SHH-MB tissues is highly methylated, including three PRMT5 sites that affect SHH-MB cell viability. Importantly, tumor growth is decreased and survival increased in mice given PRMT5 inhibitor. Conclusions: PRMT5 is a requisite driver of SHH-MB that regulates tumor progression. A clinically relevant PRMT5 inhibitor represents a promising candidate drug for SHH-MB therapy.
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Dysregulation of Sonic hedgehog (SHH) signaling drives the growth of distinct cancer subtypes, including medulloblastoma (MB). Such cancers have been treated in the clinic with a number of clinically relevant SHH inhibitors, the majority of which target the upstream SHH regulator, Smoothened (SMO). Despite considerable efficacy, many of these patients develop resistance to these drugs, primarily due to mutations in SMO. Therefore, it is essential to identify druggable, signaling components downstream of SMO to target in SMO inhibitor resistant cancers. We utilized an integrated functional genomics approach to identify epigenetic regulators of SHH signaling and identified a novel complex of Ubiquitin-like with PHD and RING finger domains 1 (UHRF1), DNA methyltransferase 1 (DNMT1), and GLI proteins. We show that this complex is distinct from previously described UHRF1/DNMT1 complexes, suggesting that it works in concert to regulate GLI activity in SHH driven tumors. Importantly, we show that UHRF1/DNMT1/GLI complex stability is targeted by a repurposed FDA-approved therapy, with a subsequent reduction in the growth of SHH-dependent MB ex vivo and in vivo. IMPLICATIONS: This work describes a novel, druggable UHRF1/DNMT1/GLI complex that regulates SHH-dependent tumor growth, and highlights an FDA-approved drug capable of disrupting this complex to attenuate tumor growth.
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Neoplasias Cerebelosas , Meduloblastoma , Humanos , Proteínas Hedgehog/metabolismo , Receptor Smoothened/genética , Receptor Smoothened/metabolismo , Meduloblastoma/tratamiento farmacológico , Meduloblastoma/genética , Meduloblastoma/metabolismo , Transducción de Señal/genética , Neoplasias Cerebelosas/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
The Cullin-RING ligase 4 E3 ubiquitin ligase component Cereblon (CRBN) is a well-established target for a class of small molecules termed immunomodulatory drugs (IMiDs). These drugs drive CRBN to modulate the degradation of a number of neosubstrates required for the growth of multiple cancers. Whereas the mechanism underlying the activation of CRBN by IMiDs is well described, the normal physiological regulation of CRBN is poorly understood. We recently showed that CRBN is activated following exposure to Wnt ligands and subsequently mediates the degradation of a subset of physiological substrates. Among the Wnt-dependent substrates of CRBN is Casein kinase 1α (CK1α), a known negative regulator of Wnt signaling. Wnt-mediated degradation of CK1α occurs via its association with CRBN at a known IMiD binding pocket. Herein, we demonstrate that a small-molecule CK1α agonist, pyrvinium, directly prevents the Wnt-dependent interaction of CRBN with CK1α, attenuating the consequent CK1α degradation. We further show that pyrvinium disrupts the ability of CRBN to interact with CK1α at the IMiD binding pocket within the CRBN-CK1α complex. Of note, this function of pyrvinium is independent of its previously reported ability to enhance CK1α kinase activity. Furthermore, we also demonstrate that pyrvinium attenuates CRBN-induced Wnt pathway activation in vivo. Collectively, these results reveal a novel dual mechanism through which pyrvinium inhibits Wnt signaling by both attenuating the CRBN-mediated destabilization of CK1α and activating CK1α kinase activity.
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Caseína Quinasa Ialfa , Compuestos de Pirvinio , Caseína Quinasa Ialfa/metabolismo , Compuestos de Pirvinio/farmacología , Ubiquitina-Proteína Ligasas/metabolismo , Vía de Señalización WntRESUMEN
The manufacturing of therapeutic biologics can result in a heterogeneous population of charge variants, encompassing many quality attributes which could impact activity and pharmacokinetics. Monitoring the relative abundance of these charge variants to demonstrate process consistency is an expectation of regulatory agencies. Control of the relative abundance of charge variants is also necessary to ensure product comparability across the product lifecycle. We have observed a significant shift in the relative abundance of charged species, as measured by capillary isoelectric focusing, during clarified cell culture fluid holds for several monoclonal antibodies. This lack of stability requires that the hold time for this process intermediate be significantly curtailed, eliminating manufacturing flexibility. We have identified the cause of this shift in relative abundance of charged species as changes in glycation levels, focused predominantly on three conserved, solvent accessible, lysine residues. Mutants of a model protein were generated that show increased charge state stability can be gained by eliminating these reactive lysines. Further, characterization studies were conducted on these mutants to determine the impact to biological activity and stability of the molecule, with no detrimental effects observed. Incorporating this knowledge into the assessments of candidate drugs could allow for the selection of molecules less susceptible to this product degradation pathway, allowing for greater manufacturing flexibility. This process of identifying and removing reactive lysine residues could be useful in the design of drug candidates with improved charge state stability, across a range of modalities.
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Anticuerpos Monoclonales , Lisina , Anticuerpos Monoclonales/genética , Técnicas de Cultivo de Célula , GlicosilaciónRESUMEN
Background and study aims Surgical gastroenterostomy (SGE) has been the mainstay treatment for gastric outlet obstruction (GOO). The emergence of endoscopic ultrasound-guided gastroenterostomy (EUS-GE) presents a less invasive alternative for palliation of GOO. We conducted a comprehensive review and meta-analysis to compare the effectiveness and safety of EUS-GE compared to SGE. Methods Multiple electronic databases and conference proceedings up to April 2021 were searched to identify studies that reported on safety and effectiveness of EUS-GE in comparison to SGE. Pooled odds ratios (ORs) of technical success, clinical success, adverse events (AE) and recurrence, and pooled standardized mean difference (SMD) of procedure time and post-procedure length of stay (LOS) were calculated. Study heterogeneity was assessed using I 2 and Cochran Q statistics. Results Seven studies including 625 patients (372 EUS-GE and 253 SGE) were included. EUS-GE had lower pooled odds of technical success compared with SGE (OR 0.19, 95â% confidence interval [CI] 0.06-0.60, I 2 0â%). Among the technically successful cases, EUS-GE was superior in terms of clinical success (OR 4.73, 95â% CI 1.83-12.25, I 2 18â%), lower overall AE (OR 0.20, 95â% CI 0.10-0.37, I 2 39â%), and shorter procedure time (SMD -2.4, 95â% CI -4.1, -0.75, I 2 95â%) and post-procedure LOS (SMD -0.49, 95â% CI -0.94, -0.03, I 2 78%). Rates of severe AE (0.89, 95â% CI 0.11-7.36, I 2 67â%) and recurrence (OR 0.49, 95â% CI 0.18-1.38, I 2 49â%) were comparable. Conclusions Our results suggest EUS-GE is a promising alternative to SGE due to its superior clinical success, overall safety, and efficiency. With further evolution EUS-GE could become the intervention of choice in GOO.
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Type 1 diabetes (T1D) is characterized by the loss of immune self-tolerance, resulting in an aberrant immune responses against self-tissue. A few therapeutics have been partially successful in reverting or slowing down T1D progression in patients, and the infusion of autologous hematopoietic stem cells (HSCs) is emerging as an option to be explored. In this study, we proposed to pharmacologically enhance by ex vivo modulation with small molecules the immunoregulatory and trafficking properties of HSCs to provide a safer and more efficacious treatment option for patients with T1D and other autoimmune disorders. A high-throughput targeted RNA sequencing screening strategy was used to identify a combination of small molecules (16,16-dimethyl PGE2 and dexamethasone), which significantly upregulate key genes involved in trafficking (e.g., CXCR4) and immunoregulation (e.g., programmed death ligand 1). The pharmacologically enhanced, ex vivo-modulated HSCs (regulatory HSCs [HSC.Regs]) have strong trafficking properties to sites of inflammation in a mouse model of T1D, reverted autoimmune diabetes in NOD mice, and delayed experimental multiple sclerosis and rheumatoid arthritis in preclinical models. Mechanistically, HSC.Regs reduced lymphocytic infiltration of pancreatic ß cells and inhibited the activity of autoreactive T cells. Moreover, when tested in clinically relevant in vitro autoimmune assays, HSC.Regs abrogated the autoimmune response. Ex vivo pharmacological modulation enhances the immunoregulatory and trafficking properties of HSCs, thus generating HSC.Regs, which mitigated autoimmune diabetes and other autoimmune disorders.
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Enfermedades Autoinmunes , Diabetes Mellitus Tipo 1 , Trasplante de Células Madre Hematopoyéticas , Animales , Enfermedades Autoinmunes/terapia , Diabetes Mellitus Tipo 1/terapia , Trasplante de Células Madre Hematopoyéticas/métodos , Células Madre Hematopoyéticas , Humanos , Ratones , Ratones Endogámicos NODRESUMEN
PURPOSE: Proximal femoral bone stress injuries (BSI), especially those involving the femoral neck (FNBSI), pose a risk to military medical readiness. There is currently no optimal physical examination technique or test item cluster that substantially influences the clinical diagnosis of FNBSI. Consequently, a lower threshold to order diagnostic imaging is employed by clinicians who manage military populations at risk for FNBSI. A viable physical examination technique or cluster of techniques is needed to better inform this clinical decision process and reduce the associated diagnostic imaging burden. This project assessed the perceived clinical utility of several novel physical examination techniques intended to identify proximal femoral bone stress injuries. METHODS: Thirteen FNBSI-specific physical examination techniques were evaluated using standardized grading criteria, evaluating safety, reliability, and credibility. Based on group consensus, two weight-bearing techniques- forward lunge and tap (FLT), rear lunge reach and tap (RLRT)-and three non-weight-bearing techniques- proximal femoral shear test, 45-degree compress and percuss, and the side-lying scissor test-were each determined to possess a parsimonious cluster of desirable examination properties. A one-hour, multimedia presentation accompanied by live demonstrations was presented to 13 clinicians. Each clinician rated the physical examination techniques based on the following five criteria: patient safety, likely to identify only bone pathology, accuracy regardless of symptom duration or acuity, performed in the mid-range of available motion, and reliability. These criteria were individually weighted from 1 (strongly disagree) to 5 (strongly agree), yielding a possible maximum score of 25. Each physical examination technique was also given a yes or no rating for overall credibility. The minimum acceptable value was set a priori at 80% yes votes. RESULTS: All clinicians in attendance were physical therapists with an average of 5.9 (SD: 4.4) years of experience managing patients with FNBSI. All attendees either agreed or strongly agreed all techniques would be safe to use with patients suspected of having a FNBSI. The highest overall scoring test based on the five criteria was the FLT with a score of 21. The only two tests to exceed the 80% benchmark for overall credibility were the FLT (92.3%) and the RLRT (83.3%). There were no overall statistically significant differences within each individual criterion except for the safety criterion. However, post hoc pairwise comparisons revealed no statistically significant differences. CONCLUSIONS: A minimum of two of the novel physical examination techniques (FLT, RLRT) appear to have sufficient credibility to warrant further evaluation based on voting results from an experienced group of clinicians. A concurrent criterion validity study to assess the diagnostic accuracy properties associated with these techniques is now indicated. CLINICAL RELEVANCE: This line of research may assist future clinicians to determine the need for diagnostic imaging procedures in patients with a suspected FNBSI.
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Fémur , Examen Físico , Fémur/diagnóstico por imagen , Humanos , Reproducibilidad de los ResultadosRESUMEN
In this paper, we evaluate how employing fraction collection and multistep gradients with RoboColumns® (Repligen, formally Atoll) affects both comparison to benchtop experimental data and column simulation parameter estimation. These operational differences arise from the RoboColumn® system (operated on an automated liquid handling device) requiring offline analysis for determination of elution profiles rather than the continuous in-line UV curves obtained with larger scale systems. In addition, multistep gradients are used to model the smooth linear gradients of larger scale systems because sequential injections are used to provide liquid flow. Comparisons of two sets of column simulations was first carried out to demonstrate that fraction collection reduced the first moments of the elution peaks by 1/2 of the fraction volumes. Additional column simulations determined that the effect of a multistep gradient approximation on retention volume was dependent upon the gradient step length. An empirical transformation was then developed to correct the first moments obtained from gradient experimental data using the RoboColumn® system. These corrected values provided a more direct comparison of the experimental data at different scales and resulted in a significant improvement in agreement with results obtained using a 20 mL benchtop column. Linear steric mass-action (SMA) parameters were then estimated using the corrected values and employed to successfully predict the performance of the benchtop system data. Finally, these parameters were demonstrated to be well suited for modeling the RoboColumn® gradient data when properly accounting for multistep gradients and fraction collection. This work continues previous investigations into understanding system differences associated with robotic liquid handling devices and proposes a methodology for properly accounting for operational differences to predict operation at larger scales using conventional chromatography systems.
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Cromatografía , Simulación por ComputadorRESUMEN
BACKGROUND: Notch signaling drives many aspects of neoplastic phenotype. Here, we report that the Integrator complex (INT) is a new component of the Notch transcriptional supercomplex. Together with Notch Activation Complex Kinase (NACK), INT activates Notch1 target genes by driving RNA polymerase II (RNAPII)-dependent transcription, leading to tumorigenesis. METHODS: Size exclusion chromatography and CBF-1/RBPJ/Suppressor of Hairless/Lag-1 (CSL)-DNA affinity fast protein liquid chromatography (FPLC) was used to purify Notch/CSL-dependent complexes for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Chromatin immunoprecipitation (ChIP) and quantitative polymerase chain reaction (qPCR) were performed to investigate transcriptional regulation of Notch target genes. Transfection of Notch Ternary Complex components into HEK293T cells was used as a recapitulation assay to study Notch-mediated transcriptional mechanisms. Gene knockdown was achieved via RNA interference and the effects of protein depletion on esophageal adenocarcinoma (EAC) proliferation were determined via a colony formation assay and murine xenografts. Western blotting was used to examine expression of INT subunits in EAC cells and evaluate apoptotic proteins upon INT subunit 11 knockdown (INTS11 KD). Gene KD effects were further explored via flow cytometry. RESULTS: We identified the INT complex as part of the Notch transcriptional supercomplex. INT, together with NACK, activates Notch-mediated transcription. While NACK is required for the recruitment of RNAPII to a Notch-dependent promoter, the INT complex is essential for RNAPII phosphorylated at serine 5 (RNAPII-S5P), leading to transcriptional activation. Furthermore, INT subunits are overexpressed in EAC cells and INTS11 KD results in G2/M cell cycle arrest, apoptosis, and cell growth arrest in EAC. CONCLUSIONS: This study identifies the INT complex as a novel co-factor in Notch-mediated transcription that together with NACK activates Notch target genes and leads to cancer cell proliferation. Video abstract.
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Carcinogénesis/genética , Endorribonucleasas/genética , Neoplasias/genética , Receptor Notch1/genética , Apoptosis/genética , Puntos de Control del Ciclo Celular/genética , Proliferación Celular/genética , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/genética , Complejos Multiproteicos/genética , Neoplasias/patología , Interferencia de ARN , ARN Polimerasa II/genéticaRESUMEN
Immunomodulatory drugs (IMiDs) are important for the treatment of multiple myeloma and myelodysplastic syndrome. Binding of IMiDs to Cereblon (CRBN), the substrate receptor of the CRL4CRBN E3 ubiquitin ligase, induces cancer cell death by targeting key neo-substrates for degradation. Despite this clinical significance, the physiological regulation of CRBN remains largely unknown. Herein we demonstrate that Wnt, the extracellular ligand of an essential signal transduction pathway, promotes the CRBN-dependent degradation of a subset of proteins. These substrates include Casein kinase 1α (CK1α), a negative regulator of Wnt signaling that functions as a key component of the ß-Catenin destruction complex. Wnt stimulation induces the interaction of CRBN with CK1α and its resultant ubiquitination, and in contrast with previous reports does so in the absence of an IMiD. Mechanistically, the destruction complex is critical in maintaining CK1α stability in the absence of Wnt, and in recruiting CRBN to target CK1α for degradation in response to Wnt. CRBN is required for physiological Wnt signaling, as modulation of CRBN in zebrafish and Drosophila yields Wnt-driven phenotypes. These studies demonstrate an IMiD-independent, Wnt-driven mechanism of CRBN regulation and provide a means of controlling Wnt pathway activity by CRBN, with relevance for development and disease.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Péptido Hidrolasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Vía de Señalización Wnt/fisiología , Proteínas de Pez Cebra/genética , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Caseína Quinasa Ialfa/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Embrión no Mamífero , Evolución Molecular , Células HEK293 , Humanos , Factores Inmunológicos/química , Factores Inmunológicos/farmacología , Lenalidomida/química , Lenalidomida/farmacología , Ratones , Organoides , Péptido Hidrolasas/metabolismo , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación , Pez Cebra/embriología , Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
The immunotoxic impacts of mercury during early life is poorly understood. We investigated the associations between gestational mercury exposure and frequency of cord blood T cells as well as placental gene expression. Frequency of natural Treg cells was positively associated with prenatal and postpartum mercury toenail concentrations. Frequency of NKT and activated naïve Th cells was positively associated with prenatal toenail mercury concentrations and number of maternal silver-mercury dental amalgams, respectively. Placental gene expression analyses revealed distinct gene signatures associated with mercury exposure. Decreased placental expression of a histone demethylase, KDM4DL, was associated with both higher prenatal and postpartum maternal toenail mercury levels among male infants and remained statistically significant after adjustment for fish and seafood consumption. The results suggest that gestational exposure to mercury concentrations contribute to alterations in both T cells and gene expression in placenta at birth. These alterations may inform mechanisms of mercury immunotoxicity.