Introduction: MDS are primarily diseases of the elderly, with median age at diagnosis of 72 years. Focused data on clinical and molecular characteristics, as well as outcomes, in younger pts under ...age 60 years are limited. We conducted this multicenter retrospective study to describe pt characteristics, cytogenetic and molecular profiles, treatment modalities, and outcomes in younger pts with MDS, including adolescent and young adults (AYA). Methods: The study included pts diagnosed with MDS between 2002-2023 at 7 academic centers. We compared baseline characteristics, treatment, and outcomes of AYA (18-39 years) and younger adult (YA) pts (40-59 years). Responses were assessed using the modified IWG 2006 response criteria which included complete remission (CR), marrow CR (mCR), and hematologic improvement (HI). Wilcoxon rank sum test and Fisher's exact (and Chi-square) test were used to compare pt and disease characteristics for continuous and categorical variables, respectively. Overall survival (OS) was estimated using the Kaplan-Meier method and compared using log-rank test. Results: A total of 173 pts were identified (AYA, N=50; YA, N=123). Median age at diagnosis was 50 years (range, 18-59), with 51% females. Baseline characteristics are shown in Table 1. Therapy-related MDS (t-MDS) was observed in 40 pts (23%) with the remaining classified as de novo MDS (N=133; 77%). Inherited bone marrow failure syndromes were documented in 15 pts (9%), with Shwachman-Diamond Syndrome (N=4) and Fanconi anemia (N=4) being the most frequent. Using the WHO 2016 classification, the most common subtypes of MDS in both the AYA and YA cohorts were MDS with multilineage dysplasia (38% vs 19%), MDS with excess blasts-2 (MDS-EB) (24% vs 29%), and MDS-EB-1 (16% vs 20%). Similarly, using the WHO 2022 classification, the most common subtypes were MDS with low blasts (50% vs 21%), MDS with increased blasts-2 (MDS-IB-2) (24% vs 24%), and MDS-IB-1 (16% vs 18%). Stratifying pts using the IPSS-R cytogenetic (CG) risk group, the most common CG risk category identified was good CG (34% vs 52%, p=0.012). Poor/very poor CG were present in 34% and 37% of the AYA and YA groups, respectively. STAG2 and NRAS mutations occurred more frequently in the AYA pts compared to the YA group, while the YA cohort was enriched for the following mutations: TP53, DNMT3A, SF3B1, SRSF2, and TET2 (Figure 1). In the AYA population, 18 pts (38%) had very low/low, 10 (21%) intermediate, and 20 pts (42%) had high/very high risk MDS using IPSS-R. In the YA population, 32 pts (26%) had very low/low, 32 (26%) intermediate, and 57 pts (47%) had high/very high risk MDS using IPSS-R. Twenty-seven percent of pts with very low to intermediate-risk MDS using IPSS-R were upstaged to moderate high to very high-risk MDS using IPSS-M. The majority of pts (AYA: 75%; YA: 63%, p=0.5) were transfusion independent at baseline. Single-agent hypomethylating agents were the most frequently used frontline therapy, used in 17 AYA pts (34%) and 58 YA pts (48%), with more AYA pts receiving upfront allogeneic stem cell transplantation (Allo-SCT) compared to YA pts (28% vs 5%). After frontline therapy (Table 1), an additional 14 AYA (28%) and 52 YA (42%) pts received Allo-SCT. In pts with IPSS-R intermediate to very high-risk, response rates (HI+CR+mCR+mCR with HI) to frontline therapies were 46% and 54.5% in the AYA and YA groups, respectively. After median follow-up of 24 months range, 1-218, median OS was numerically longer in the AYA versus YA group (155 vs 53 months, p=0.2). AML transformation occurred in 24% and 20% of the pts in the AYA and YA groups, respectively. Median OS was significantly longer in de novo compared to t-MDS pts (56 vs 27 months, p=0.03). Median OS among Allo-SCT compared to non-SCT pts were not reached vs 155 months ( p=0.9) in the AYA group and 53 vs 54 months ( p=0.4) in the YA group. IPSS-R (very low+low vs intermediate vs high+very high) was able to distinguish differences in OS in the YA (133 vs 53 vs 17 months, p=0.0002) and AYA (155 vs not reached vs 18 months, p=0.006) pts, with the exception of the intermediate-risk group in the AYA cohort. Conclusions: In this study, d e novoMDS was seen in the majority of younger pts with MDS (both AYA and YA). Mutations in STAG2 and NRAS were more common in the AYA pts, while YA pts had MDS more enriched for TP53, DNMT3A, TET2 and splicing mutations. This study is still ongoing with a plan to compare the abovementioned groups to older pts (≥60 years) with MDS.
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Innate immunity coordinates the first line of defense against invading pathogens. This involves the recruitment and subsequent migration of nearby immune cells to the site of infection, ...allowing the pathogen to be consumed via phagocytosis to sequester the infection. These processes require the rapid disassembly, rearrangement, and repolymerization of the network of structural proteins known as the cytoskeleton. Migration and phagocytosis are one of the functional consequences of innate immune receptor triggered signaling pathways. Signals coalesce at catalytic hubs, such as kinases, which serve to amplify and regulate the signal. In the anti‐viral response, TANK binding kinase 1 (TBK1) functions as a catalytic hub. Suppressor of IKK epsilon (SIKE) acts as a high affinity, alternate TBK1 substrate, although SIKE's downstream function is not yet defined. The primary goal of this work was to assess the effect of SIKE on cytoskeletal rearrangements in migration and phagocytosis. For these studies, a CRISPR knockout cell line was derived, SIKE‐CR, from chronic myelogenous leukemia (CML) cell line KBM‐7 named HAP1. Wound healing assays of HAP1 parental cells and SIKE‐CR illustrated that the absence of SIKE significantly decreased cell migration. In the mouse macrophage RAW 264.7 cell line, SIKE expression was knocked down via lentiviral delivery of shRNA targeting SIKE. Phagocytosis was assessed in WT and SIKE knockdown cells via uptake of latex beads labeled with IgG‐FITC. Reduced SIKE expression increased phagocytic uptake. To determine SIKE interactions with cytoskeletal proteins, co‐immunoprecipitations (cIP), in vitro precipitation (IVP) reactions, and enzyme‐linked immunosorbent assays (ELISA) were completed and demonstrated SIKE:tubulin and SIKE:α‐actinin complexes. Together, these studies establish an interaction between SIKE and cytoskeletal proteins and suggest that SIKE functions in cytoskeletal rearrangement associated with migration and phagocytosis.
Support or Funding Information
Work supported by NIH grant R21AI107447 and USD SURE.
Treponema denticola is a primary etiological agent of periodontal disease. T. denticola evades complement‐mediated killing by binding to the host's factor H (FH), a negative regulator of the ...alternative complement pathway. The T. denticola FH‐binding protein has been identified and designated as factor H‐binding protein B (FhbB). Crystals of recombinant FhbB were obtained by the hanging‐drop vapor‐diffusion method using sodium citrate and 0.2 M sodium thiocyanate. FhbB crystals diffracted to 1.8 Å resolution and belonged to space group P43212 or P41212, with unit‐cell parameters a = b = 46.76, c = 167.68 Å. Two FhbB molecules per asymmetric unit gave a Matthews coefficient of 2.2 Å3 Da−1 and a solvent content of 44%. FhbB is the smallest bacterially produced FH‐binding protein identified to date. Determination of its structure will provide unique insight into the minimal structural determinants required for FH binding.
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Innate immunity is a critical, non‐specific early defense system that allows the body to respond to pathogens prior to the recruitment of specific antibody defenses. This process ...involves the recruitment and subsequent migration of nearby immune cells to the site of infection, allowing the pathogen to be consumed via phagocytosis to prevent widespread infection. Migration and phagocytosis require the rapid disassembly, rearrangement, and assembly of a network of structural proteins known as the cytoskeleton. The link between cytoskeletal rearrangements in these processes and detection of pathogen, like viral patterns by Toll‐like receptor 3, are not well defined. TLR3 initiates proinflammatory and type I interferon responses via the TANK binding kinase 1 (TBK1) pathway. Suppressor of IKKe (SIKE) was originally proposed as a suppressor of IKKe and TBK1, playing an important inhibitory role in interferon β production. However, more recent studies have indicated SIKE acts instead as a preferred substrate of TBK1. Mass spectrometry and confocal microscopy further indicate that SIKE interacts with the cytoskeletal proteins actin, tubulin, and a‐actinin. This revised understanding of SIKE suggests that SIKE acts as a bridge between the viral stimulation of TBK1 activity and the resulting cytoskeletal rearrangements. To examine the role of SIKE in cytoskeletal rearrangement, SIKE function in cellular migration and phagocytosis were assessed. A CRISPR/Cas 9 SIKE knockout was developed in the KMB7‐derived HAP1 cells. The HAP1 SIKE
−/−
showed no detectable SIKE expression compared to the parental HAP1 cells. To determine the role of SIKE in migration, parental and HAP1 SIKE
−/−
were used in cell migration assays +/− dsRNA to simulate viral infection conditions. A lentiviral‐mediated shRNA SIKE knockdown was completed in the mouse macrophage RAW264.7 cell line. Using SL1344 Salmonella typhimurium strain, gentamicin protection assays were completed to determine SIKE's role in phagocytosis. Introduction of SL1344
Salmonella typhimurium
at MOI ≤ 100 into RAW 264.7 showed successful infection of cells by confocal microscopy and gentamicin protection assay.
Support or Funding Information
This work was supported by NIH grant R21AI107447 and University of San Diego SURE program.
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Biochemistry and molecular biology curricula serve students pursuing the major, provide breadth to non‐biochemistry majors (chemistry, biology, biophysics, behavioral neuroscience, ...engineering) and a foundational knowledge of the topic to pre‐professional students in the biomedical sciences. To meet the needs of this diverse population, the University of San Diego redesigned the biochemistry major. Central to the redesign was retaining degree certification by ACS and meeting the criteria for ASBMB accreditation. ASBMB core concepts and learning objectives, as well as ACS guidelines for introductory, foundational and in‐depth coverage of the five areas of chemistry were mapped onto the curriculum of the major. Because many biochemistry majors and students taking these courses are interested in health professions, we also took into consideration core concept coverage against the MCAT requirements. From this analysis, gaps in foundational concepts or areas with only introductory concept coverage were identified and addressed by the development of new courses; a second semester biochemistry lecture and molecular biology techniques laboratory course. A research methods course was implemented in the 4
th
semester that introduces students to primary literature, ethical conduct in research and scholarship, and professional development. With this foundation, students complete a 100 h research project with faculty or via an internship as part of their 400 h experiential learning requirement. Communication skills (written and oral competency) were integrated into the 8 h biochemistry laboratory to showcase students’ course integrated research projects. To reflect the broad interests of students, flexibility for in‐depth concept coverage in an upper division elective was expanded to include biology (Microbiology, Immunology, Adv. Molecular Biology), chemistry (Biophysical Chemistry, Adv. Synthesis, Adv. Physical Methods), and physics (Biophysics) electives. As part of the major's redesign, MCAT 2015 topics in biochemistry were considered during development of the 2‐semester biochemistry course as pre‐professional students typically will complete only one semester of biochemistry lecture. Structure/function/regulation, enzyme kinetics/thermodynamics and catabolic pathways related to glucose were maintained in the first semester. In conjunction with other coursework, this concept coverage provides adequate preparation for MCAT 2015. For students who take only a select number of courses (i.e. non‐biochemistry majors), web‐hosted materials supplement this course work to provide preparatory information for standardized exams. Together, these curricular changes provide our students with the technical and transferrable skills to be successful in their chosen career path.
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The innate immune system is the first line of defense against pathogens. Suppressor of IKKepsilon (SIKE) is a downstream component of the antiviral TLR3 innate immune pathway, although ...its function in this pathway has not yet been determined. In order to derive information about SIKE function, we undertook characterization of SIKE structure to determine a structure‐function relationship. Using PHYRE2, a SIKE model of the human isoform was predicted that consisted of a V‐shaped coiled coil fold. The secondary structure of SIKE was assessed via circular dichroism. Spectra were consistent with the predicted alpha helical model. Thermal melts (15–80°C) showed a linear, rather than sigmoidal, unfolding transition consistent with computational predictions (GlobPlot, DisEMBL, PONDR), which suggested SIKE contained 35% disordered regions. Addition of up to 25% trifluoroethanol to stabilize helical structure increased the helical content of SIKE by 35%. Fluorescence polarization and size exclusion chromatography assessed the solution size of SIKE (23 kDa) with respect to globular standards. SEC revealed a concentration dependent (0.9 – 3.6 mg/mL) association of SIKE into three distinct species of 79, 185 and 1,325 kDa. Fluorescence polarization of each species compared to standards of lysozyme (14 kDa), albumin (66 kDa) and glutamate dehydrogenase (355 kDa) were consistent with the SEC results. Limited proteolysis coupled to tandem mass spectrometry as well as crosslinking assessed by SDS‐PAGE mapped accessible SIKE sequences and association between SIKE monomers, respectively. Limited proteolysis at 1, 5, and 10 min identified trypsin cleavage sites consistent with a dimeric model of SIKE. Crosslinking with bis(sulfosuccinimidyl)suberate (BS3) identified predominantly a dimer species as well as a smaller proportion of a tetrameric species. In the cleft of the V‐shaped coiled coil, 3DLigandSite proposed a zinc binding site. To confirm an interaction between SIKE and divalent cations, SIKE‐ANS titration and fluorescence‐based thermal shift assays were completed +/− Mg, Mn, Ca, and Zn. In each case, a subset of ions perturbed SIKE structure, demonstrating a divalent cation interaction. In conclusion, using various biophysical techniques, we have found that SIKE's structure has regions of disorder bookended by alpha helices; SIKE monomers associate primarily into a dimeric species; divalent metals contribute to SIKE stabilization, and the homology model presented is consistent with these biophysical characterizations.
Support or Funding Information
Work supported by NIH grant R21AI107447 and USD SURE program.
Course-based undergraduate research experiences (CUREs) are laboratory courses that integrate broadly relevant problems, discovery, use of the scientific process, collaboration, and iteration to ...provide more students with research experiences than is possible in individually mentored faculty laboratories. Members of the national Malate dehydrogenase CUREs Community (MCC) investigated the differences in student impacts between traditional laboratory courses (control), a short module CURE within traditional laboratory courses (mCURE), and CUREs lasting the entire course (cCURE). The sample included approximately 1,500 students taught by 22 faculty at 19 institutions. We investigated course structures for elements of a CURE and student outcomes including student knowledge, student learning, student attitudes, interest in future research, overall experience, future GPA, and retention in STEM. We also disaggregated the data to investigate whether underrepresented minority (URM) outcomes were different from White and Asian students. We found that the less time students spent in the CURE the less the course was reported to contain experiences indicative of a CURE. The cCURE imparted the largest impacts for experimental design, career interests, and plans to conduct future research, while the remaining outcomes were similar between the three conditions. The mCURE student outcomes were similar to control courses for most outcomes measured in this study. However, for experimental design, the mCURE was not significantly different than either the control or cCURE. Comparing URM and White/Asian student outcomes indicated no difference for condition, except for interest in future research. Notably, the URM students in the mCURE condition had significantly higher interest in conducting research in the future than White/Asian students.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Phosphoglycerate dehydrogenase (PGDH) catalyzes the first step in the serine biosynthetic pathway. In lower plants and bacteria, the PGDH reaction is regulated by the end-product of the pathway, ...serine. The regulation occurs through a V max mechanism with serine binding and inhibition occurring in a cooperative manner. The three-dimensional structure of the serine inhibited enzyme, determined by previous work, showed a tetrameric enzyme with 222 symmetry and an unusual overall toroidal appearance. To characterize the allosteric, cooperative effects of serine, we identified W139G PGDH as an enzymatically active mutant responsive to serine but not in a cooperative manner. The position of W139 near a subunit interface and the active site cleft suggested that this residue is a key player in relaying allosteric effects. The 2.09 Å crystal structure of W139G-PGDH, determined in the absence of serine, revealed major quaternary and tertiary structural changes. Contrary to the wildtype enzyme where residues encompassing residue 139 formed extensive intersubunit contacts, the corresponding residues in the mutant were conformationally flexible. Within each of the three-domain subunits, one domain has rotated ∼42° relative to the other two. The resulting quaternary structure is now in a novel conformation creating new subunit-to-subunit contacts and illustrates the unusual flexibility in this V max regulated enzyme. Although changes at the regulatory domain interface have implications in other enzymes containing a similar regulatory or ACT domain, the serine binding site in W139G PGDH is essentially unchanged from the wildtype enzyme. The structural and previous biochemical characterization of W139G PGDH suggests that the allosteric regulation of PGDH is mediated not only by changes occurring at the ACT domain interface but also by conformational changes at the interface encompassing residue W139.
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The innate immune system rapidly responds to challenges by pathogens via activation of an inflammatory response. As a convergence point for multiple inflammatory and anti‐viral ...signaling pathways, TANK binding kinase 1 (TBK1) serves as a catalytic hub to initiate host defenses. Suppressor of IKK epsilon (SIKE) is a newly identified TBK1 substrate. The goal of this project was to identify the function of SIKE within the host's anti‐viral response. Co‐immunoprecipitation – tandem MS/MS analyses of the SIKE interaction network identified several interactions with cytoskeletal components. Immunofluorescence assays of endogenous SIKE with cytoskeletal markers and colocalization analyses indicated that SIKE colocalized with tubulin, actin, and a‐actinin. Reciprocal immunoprecipitation (RcIPs) studies confirmed the SIKE:tubulin and SIKE:a‐actinin interaction that appeared enhanced following dsRNA stimulation, a mimic of viral infection. ELISA assays were used to characterize the binding affinity between SIKE and the cytoskeletal proteins under unstimulated and simulated viral‐stimulated conditions. The SIKE:actin interaction was not detected by RcIP.
In vitro
immunoprecipitation assays mapped direct interactions between SIKE:tubulin and SIKE:a‐actinin and were used to further probe for a weak SIKE:actin interaction. Assays employed purified cytoskeletal proteins and full‐length (residues 1–207), N‐terminal (residues 1–112), C – terminal (residues 113–207) or a phosphomimetic (S6E) of a 6x‐His‐tagged SIKE construct. Together these studies establish an interaction between SIKE and cytoskeletal proteins that may provide a direct link between innate immune signaling and cytoskeletal components.
Support or Funding Information
This work was supported by NIH grant R21AI107447 and the University of San Diego SURE program.
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