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Item A Review of IsomiRs in Colorectal Cancer(Non-Coding RNA, 2023-06-07) Lausten, Molly A.; Boman, Bruce M.As advancements in sequencing technology rapidly continue to develop, a new classification of microRNAs has occurred with the discovery of isomiRs, which are relatively common microRNAs with sequence variations compared to their established template microRNAs. This review article seeks to compile all known information about isomiRs in colorectal cancer (CRC), which has not, to our knowledge, been gathered previously to any great extent. A brief overview is given of the history of microRNAs, their implications in colon cancer, the canonical pathway of biogenesis and isomiR classification. This is followed by a comprehensive review of the literature that is available on microRNA isoforms in CRC. The information on isomiRs presented herein shows that isomiRs hold great promise for translation into new diagnostics and therapeutics in clinical medicine.Item Adseverin, an actin-binding protein, modulates hypertrophic chondrocyte differentiation and osteoarthritis progression(Science Advances, 2023-08-04) Chan, Byron; Glogauer, Michael; Wang, Yongqiang; Wrana, Jeffrey; Chan, Kin; Beier, Frank; Bali, Supinder; Hinz, Boris; Parreno, Justin; Ashraf, Sajjad; Kandel, RitaIn osteoarthritis (OA), a disease characterized by progressive articular cartilage degradation and calcification, the articular chondrocyte phenotype changes and this correlates with actin cytoskeleton alterations suggesting that it regulates gene expression essential for proper phenotype. This study reports that OA is associated with the loss of adseverin, an actin capping and severing protein. Adseverin deletion (Adseverin−/−) in mice compromised articular chondrocyte function, by reducing F-actin and aggrecan expression and increasing apoptosis, Indian hedgehog, Runx2, MMP13, and collagen type X expression, and cell proliferation. This led to stiffer cartilage and decreased hyaline and increased calcified cartilage thickness. Together, these changes predisposed the articular cartilage to enhanced OA severity in Adseverin−/− mice who underwent surgical induction of OA. Adseverin−/− chondrocyte RNA sequencing and in vitro studies together suggests that adseverin modulates cell viability and prevents mineralization. Thus, adseverin maintains articular chondrocyte phenotype and cartilage tissue homeostasis by preventing progression to hypertrophic differentiation in vivo. Adseverin may be chondroprotective and a potential therapeutic target.Item Association of Behavioral and Clinical Risk Factors With Cataract: A Two-Sample Mendelian Randomization Study(Investigative Ophthalmology & Visual Science, 2023-07) Jiang, Chen; Melles, Ronald B.; Sangani, Poorab; Hoffmann, Thomas J.; Hysi, Pirro G.; Glymour, M. Maria; Jorgenson, Eric; Lachke, Salil A.; Choquet, HélènePurpose: To investigate the association of genetically determined primary open-angle glaucoma (POAG), myopic refractive error (RE), type 2 diabetes (T2D), blood pressure (BP), body mass index (BMI), cigarette smoking, and alcohol consumption with the risk of age-related cataract. Methods: To assess potential causal effects of clinical or behavioral factors on cataract risk, we conducted two-sample Mendelian randomization analyses. Genetic instruments, based on common genetic variants associated with risk factors at genome-wide significance (P < 5 × 10−8), were derived from published genome-wide association studies (GWAS). For age-related cataract, we used GWAS summary statistics from our previous GWAS conducted in the Genetic Epidemiology Research on Adult Health and Aging (GERA) cohort (28,092 cataract cases and 50,487 controls; all non-Hispanic whites) or in the UK Biobank (31,852 cataract cases and 428,084 controls; all European-descent individuals). We used the inverse-variance weighted (IVW) method as our primary source of Mendelian randomization estimates and conducted common sensitivity analyses. Results: We found that genetically determined POAG and mean spherical equivalent RE were significantly associated with cataract risk (IVW model: odds ratio [OR] = 1.04; 95% confidence interval [CI], 1.01–1.08; P = 0.018; per diopter more hyperopic: OR = 0.92; 95% CI, 0.89–0.93; P = 6.51 × 10−13, respectively). In contrast, genetically determined T2D, BP, BMI, cigarette smoking, or alcohol consumption were not associated with cataract risk (P > 0.05). Conclusions: Our results provide evidence that genetic risks for POAG and myopia may be causal risk factors for age-related cataract. These results are consistent with previous observational studies reporting associations of myopia with cataract risk. This information may support population cataract risk stratification and screening strategies.Item Biophysical Regulation of Chromatin Architecture Instills a Mechanical Memory in Mesenchymal Stem Cells(Nature Publishing Group, 2015-11-23) Heo, Su-Jin; Thorpe, Stephen D.; Driscoll, Tristan P.; Duncan, Randall L.; Lee, David A.; Mauck, Robert L.; Su-Jin Heo, Stephen D. Thorpe, Tristan P. Driscoll, Randall L. Duncan, David A. Lee & Robert L. Mauck; Duncan, Randall L.Mechanical cues direct the lineage commitment of mesenchymal stem cells (MSCs). In this study, we identified the operative molecular mechanisms through which dynamic tensile loading (DL) regulates changes in chromatin organization and nuclear mechanics in MSCs. Our data show that, in the absence of exogenous differentiation factors, short term DL elicits a rapid increase in chromatin condensation, mediated by acto-myosin based cellular contractility and the activity of the histone-lysine N-methyltransferase EZH2. The resulting change in chromatin condensation stiffened the MSC nucleus, making it less deformable when stretch was applied to the cell. We also identified stretch induced ATP release and purinergic calcium signaling as a central mediator of this chromatin condensation process. Further, we showed that DL, through differential stabilization of the condensed chromatin state, established a ‘mechanical memory’ in these cells. That is, increasing strain levels and number of loading events led to a greater degree of chromatin condensation that persisted for longer periods of time after the cessation of loading. These data indicate that, with mechanical perturbation, MSCs develop a mechanical memory encoded in structural changes in the nucleus which may sensitize them to future mechanical loading events and define the trajectory and persistence of their lineage specification.Item Clinicians’ Perspectives on Proactive Patient Safety Behaviors in the Perioperative Environment(Jama Network Open, 2023-04-11) Duffy, Caoimhe; Menon, Neil; Horak, David; Bass, Geoffrey D.; Talwar, Ruchika; Lorenzi, Cara; Vo, Christina Taing; Chiang, Chienhui; Ziemba, Justin B.Importance The perioperative environment is hazardous, but patients remain safe with a successful outcome during their care due to staff adaptability and resiliency. The behaviors that support this adaptability and resilience have yet to be defined or analyzed. One Safe Act (OSA), a tool and activity developed to capture self-reported proactive safety behaviors that staff use in their daily practice to promote individual and team-based safe patient care, may allow for improved definition and analysis of these behaviors. Objective To thematically analyze staff behaviors using OSA to understand what may serve as the basis for proactive safety in the perioperative environment. Design, Setting, and Participants This qualitative thematic analysis included a convenience sample of perioperative staff at a single-center, tertiary care academic medical center who participated in an OSA activity during a 6-month period in 2021. All perioperative staff were eligible for inclusion. A combined deductive approach, based on a human factor analysis and classification framework, as well as an inductive approach was used to develop themes and analyze the self-reported staff safety behaviors. Exposures Those selected to participate were asked to join an OSA activity, which was conducted in-person by a facilitator. Participants were to self-reflect about their OSA (proactive safety behavior) and record their experience as free text in an online survey tool. Main Outcome and Measures The primary outcome was the development and application of a set of themes to describe proactive safety behaviors in the perioperative environment. Results A total of 140 participants (33 nurses [23.6%] and 18 trainee physicians [12.9%]), which represented 21.3% of the 657 total perioperative department full-time staff, described 147 behaviors. A total of 8 non–mutually exclusive themes emerged with the following categories and frequency of behaviors: (1) routine-based adaptations (46 responses [31%]); (2) resource availability and assessment adaptations (31 responses [21%]); (3) communication and coordination adaptation (23 responses [16%]); (4) environmental ergonomics adaptation (17 responses [12%]); (5) situational awareness adaptation (12 responses [8%]); (6) personal or team readiness adaptation (8 responses [5%]); (7) education adaptation (5 responses [3%]); and (8) social awareness adaptation (5 responses [3%]). Conclusions and Relevance The OSA activity elicited and captured proactive safety behaviors performed by staff. A set of behavioral themes were identified that may serve as the basis for individual practices of resilience and adaptability that promote patient safety.Item Coupling Novel Probes with Molecular Localization Microscopy Reveals Cell Wall Homeostatic Mechanisms in Staphylococcus aureus(ACS Chemical Biology, 2022-11-22) Lund, Victoria A.; Gangotra, Haneesh; Zhao, Zhen; Sutton, Joshua A. F.; Wacnik, Katarzyna; DeMeester, Kristen; Liang, Hai; Santiago, Cintia; Grimes, Catherine Leimkuhler; Jones, Simon; Foster, Simon J.Bacterial cell wall peptidoglycan is essential for viability, and its synthesis is targeted by antibiotics, including penicillin. To determine how peptidoglycan homeostasis controls cell architecture, growth, and division, we have developed novel labeling approaches. These are compatible with super-resolution fluorescence microscopy to examine peptidoglycan synthesis, hydrolysis, and the localization of the enzymes required for its biosynthesis (penicillin binding proteins (PBPs)). Synthesis of a cephalosporin-based fluorescent probe revealed a pattern of PBPs at the septum during division, supporting a model of dispersed peptidoglycan synthesis. Metabolic and hydroxylamine-based probes respectively enabled the synthesis of glycan strands and associated reducing termini of the peptidoglycan to be mapped. Foci and arcs of reducing termini appear as a result of both synthesis of glycan strands and glucosaminidase activity of the major peptidoglycan hydrolase, SagB. Our studies provide molecular level details of how essential peptidoglycan dynamics are controlled during growth and division.Item Cultural Competence in Pediatrics: Health Care Provider Knowledge, Awareness, and Skills(MDPI AG, 2015-12-15) Dabney, Kirk; McClarin, Lavisha; Romano, Emily; Fitzgerald, Diane; Bayne, Lynn; Oceanic, Patricia; Nettles, Arie L.; Holmes, Laurens Jr.; Kirk Dabney, Lavisha McClarin, Emily Romano, Diane Fitzgerald, Lynn Bayne, Patricia Oceanic, Arie L. Nettles and Laurens Holmes Jr.; Holmes, Laurens Jr.The purpose of this study was to assess the effects of a cultural competence training (CCT) program on pediatric health care providers’ self-reported ability to provide culturally competent care to a diverse pediatric patient population. This quantitative, nested ecologic level study design used a repeated measure in the form of pre-test and post-test data to assess percent change in providers’ cultural awareness, experience working or learning about different cultures, and preparedness and skills in working with different cultures before and after CCT. The study was conducted between 2011 and 2012 in a pediatric hospital and associated outpatient offices. The sample consisted of pediatric health care providers from various departments, mainly physicians and nurses (n = 69). Participants completed a pre-intervention cultural competence assessment and then were subjected to a cultural competence-training program, after which they completed the assessment a second time. The baseline and post-intervention data were collected in the form of Likert scales and transformed into a quintile or quartile scale as appropriate. Data were assessed using paired t-tests or Wilcoxon’s signed-rank tests. Providers indicated a 13% increase in knowledge (53.9% vs. 66.7%, t = 3.4, p = 0.001), 8.7% increase in awareness (46.7% vs. 55.4%, t = 3.0, p = 0.002), and 8% statistically marginal increase in skills (66.4% vs. 74.5%, z = 1.8, p = 0.06). Culturally competent training in a pediatric environment significantly enhances knowledge, awareness and to some extent skills in providing care to culturally diverse patient population.Item Discovery of Power-Law Growth in the Self- Renewal of Heterogeneous Glioma Stem Cell Populations(PLOS (Public Library of Science), 2015-08-18) Sugimori, Michiya; Hayakawa, Yumiko; Boman, Bruce M.; Fields, Jeremy Z.; Awaji, Miharu; Kozano, Hiroko; Tamura, Ryoi; Yamamoto, Seiji; Ogata, Toru; Yamada, Mitsuhiko; Endo, Shunro; Kurimoto, Masanori; Kuroda, Satoshi; Michiya Sugimori, Yumiko Hayakawa, Bruce M. Boman, Jeremy Z. Fields, Miharu Awaji, Hiroko Kozano, Ryoi Tamura, Seiji Yamamoto, Toru Ogata, Mitsuhiko Yamada, Shunro Endo, Masanori Kurimoto, Satoshi Kuroda; Boman, Bruce M.BACKGROUND Accumulating evidence indicates that cancer stem cells (CSCs) drive tumorigenesis. This suggests that CSCs should make ideal therapeutic targets. However, because CSC populations in tumors appear heterogeneous, it remains unclear how CSCs might be effectively targeted. To investigate the mechanisms by which CSC populations maintain heterogeneity during self-renewal, we established a glioma sphere (GS) forming model, to generate a population in which glioma stem cells (GSCs) become enriched. We hypothesized, based on the clonal evolution concept, that with each passage in culture, heterogeneous clonal sublines of GSs are generated that progressively show increased proliferative ability. METHODOLOGY/PRINCIPAL FINDINGS To test this hypothesis, we determined whether, with each passage, glioma neurosphere culture generated from four different glioma cell lines become progressively proliferative (i.e., enriched in large spheres). Rather than monitoring self-renewal, we measured heterogeneity based on neurosphere clone sizes (#cells/clone). Log-log plots of distributions of clone sizes yielded a good fit (r>0.90) to a straight line (log(% total clones) = k*log(#cells/ clone)) indicating that the system follows a power-law (y = xk) with a specific degree exponent (k = −1.42). Repeated passaging of the total GS population showed that the same power-law was maintained over six passages (CV = −1.01 to −1.17). Surprisingly, passage of either isolated small or large subclones generated fully heterogeneous populations that retained the original power-law-dependent heterogeneity. The anti-GSC agent Temozolomide, which is well known as a standard therapy for glioblastoma multiforme (GBM), suppressed the self-renewal of clones, but it never disrupted the power-law behavior of a GS population. CONCLUSIONS/SIGNIFICANCE Although the data above did not support the stated hypothesis, they did strongly suggest a novel mechanism that underlies CSC heterogeneity. They indicate that power-law growth governs the self-renewal of heterogeneous glioma stem cell populations. That the data always fit a power-law suggests that: (i) clone sizes follow continuous, non-random, and scale-free hierarchy; (ii) precise biologic rules that reflect self-organizing emergent behaviors govern the generation of neurospheres. That the power-law behavior and the original GS heterogeneity are maintained over multiple passages indicates that these rules are invariant. These self-organizing mechanisms very likely underlie tumor heterogeneity during tumor growth. Discovery of this power-law behavior provides a mechanism that could be targeted in the development of new, more effective, anti-cancer agents. IntroductionItem Dynamic bioinspired coculture model for probing ER+ breast cancer dormancy in the bone marrow niche(Science Advances, 2023-03-08) Pradhan, Lina; Moore, DeVonte; Ovadia, Elisa M.; Swedzinski, Samantha L.; Cossette, Travis; Sikes, Robert A.; van Golen, Kenneth; Kloxin, April M.Late recurrences of breast cancer are hypothesized to arise from disseminated tumor cells (DTCs) that reactivate after dormancy and occur most frequently with estrogen receptor–positive (ER+) breast cancer cells (BCCs) in bone marrow (BM). Interactions between the BM niche and BCCs are thought to play a pivotal role in recurrence, and relevant model systems are needed for mechanistic insights and improved treatments. We examined dormant DTCs in vivo and observed DTCs near bone lining cells and exhibiting autophagy. To study underlying cell-cell interactions, we established a well-defined, bioinspired dynamic indirect coculture model of ER+ BCCs with BM niche cells, human mesenchymal stem cells (hMSCs) and fetal osteoblasts (hFOBs). hMSCs promoted BCC growth, whereas hFOBs promoted dormancy and autophagy, regulated in part by tumor necrosis factor–α and monocyte chemoattractant protein 1 receptor signaling. This dormancy was reversible by dynamically changing the microenvironment or inhibiting autophagy, presenting further opportunities for mechanistic and targeting studies to prevent late recurrence.Item Dynamic Change and Target Prediction of Axon-Specific MicroRNAs in Regenerating Sciatic Nerve(PLOS (Public Library of Science), 2015-09-02) Phay, Monichan; Kim, Hak Hee; Yoo, Soonmoon; Monichan Phay, Hak Hee Kim, Soonmoon Yoo; Phay, MonichanInjury to axons in the peripheral nervous system induces rapid and local regenerative responses to form a new growth cone, and to generate a retrogradely transporting injury signal. The evidence for essential roles of intra-axonal protein synthesis during regeneration is now compelling. MicroRNA (miRNA) has recently been recognized as a prominent player in post-transcriptional regulation of axonal protein synthesis. Here, we directly contrast temporal changes of miRNA levels in the sciatic nerve following injury, as compared to those in an uninjured nerve using deep sequencing. Small RNAs (<200 nucleotides in length) were fractionated from the proximal nerve stumps to improve the representation of differential miRNA levels. Of 141 axoplasmic miRNAs annotated, 63 rat miRNAs showed significantly differential levels at five time points following injury, compared to an uninjured nerve. The differential changes in miRNA levels responding to injury were processed for hierarchical clustering analyses, and used to predict target mRNAs by Targetscan and miRanda. By overlapping these predicted targets with 2,924 axonally localizing transcripts previously reported, the overlapping set of 214 transcripts was further analyzed by the Gene Ontology enrichment and Ingenuity Pathway Analyses. These results suggest the possibility that the potential targets for these miRNAs play key roles in numerous neurological functions involved in ER stress response, cytoskeleton dynamics, vesicle formation, and neurodegeneration and-regeneration. Finally, our results suggest that miRNAs could play a direct role in regenerative response and may be manipulated to promote regenerative ability of injured nerves. IntroductionItem Dynamic elementomics of single-cell ICP–MS-derived signals in normal and calcium pump PMCA4-deficient mouse epididymal sperm during capacitation(Metallomics, 2023-10-16) Zhang, Bao Li; Zhang, Ze Peng; Shi, Su Meng; Shi, Hui Juan; DeLeon, Patricia A.; Shum, WinnieCurrently, clinical analysis of male infertility mainly relies on parameters of semen and sperm cells. However, the high diagnostic failure rates indicate that the current assessment methods are not sufficient and a new approach to evaluating sperm function still needs to be developed. Here we explored the feasibility of single-cell inductively coupled plasma mass spectrometry (sc-ICP–MS)-derived profiles to determine the elemental characteristics in viable capacitated sperm under normal and deficient conditions. To validate the measurements, we used male sterile Pmca4-knockout (KO) mice with impaired calcium clearance, known to be dysregulated due to loss of calcium efflux capacity during sperm capacitation. Consistently, we observed significantly increased calcium intensities in Pmca4-KO sperm upon capacitation stimulation compared with control sperm from the caudaepididymides of wild-type control (WT) mice. More importantly, we explored that the characteristic signatures of calcium intensities in individual spikes derived from sc-ICP–MS was consistent with the dynamics of relative calcium levels in single sperm reported in the literature. Prominent alterations were also observed in the dynamic signatures of sc-ICP–MS-derived profiles of essential elements, particularly the redox-labile elements including copper, iron, manganese, selenium, and zinc in Pmca4-KO sperm compared to WT controls. Therefore, our study demonstrates that elementomics of sc-ICP–MS-derived signals can reveal ionic dysregulation in plasma membrane Ca2+-ATPase isoform 4 protein deficient sperm, and that sc-ICP–MS assay can be applied for functional analysis of viable sperm in functional activities, such as capacitation stimulation. We propose that cell elementomics can be used as an alternative approach to assessing sperm quality and male fertility at the single-cell level. Graphical Abstract available at: https://doi.org/10.1093/mtomcs/mfad059Item Epidemiologic, Racial and Healthographic Mapping of Delaware Pediatric Cancer: 2004–2014(MDPI AG, 2015-12-22) Holmes, Laurens Jr.; Vandenberg, Jonathan; McClarin, Lavisha; Dabney, Kirk; Laurens Holmes Jr., Jonathan Vandenberg, Lavisha McClarin and Kirk Dabney; Holmes, Laurens Jr.Childhood cancer remains the leading cause of disease-related death among children 0 to 14 years and incidence varies by race, ethnicity, sex, geographic locale, and age at onset. However, data are unavailable in some regions, indicative of a need for such information for cancer awareness, education and prevention program. We utilized retrospective epidemiologic design to assess and characterize pediatric tumors in the Nemours Electronic Medical Records, between 2004 and 2014. Tumor frequency and children population size were used to determine the period prevalence as cumulative incidence (CI) proportion, as well as chi-square and Poisson Regression. The CI for overall childhood cancer in Delaware was 234 per 100,000 children, and varied by race, black (273 per 100,000), white (189 per 100,000). Similarly, sex variability was observed in CI, boys (237 per 100,000) and girls (230 per 100,000). The most commonly diagnosed malignancies were acute lymphoblastic leukemia (ALL), Central Nervous System (CNS)/brain and renal cancer. The geographic locales with relatively higher cancer CI in the state of DE were zip codes 19804 and 19960, but this does not imply cancer clustering. Differences in overall childhood cancer distribution occurred by race, sex, geography, and age. These findings are indicative of the need for cancer-specific health education, awareness and prevention programs in reducing the observed disparities in Delaware.Item Epididymosomes: transfer of fertility‑modulating proteins to the sperm surface(Shanghai Materia Medica, Shanghai Jiao Tong University., 2015-06-26) Martin‑DeLeon, Patricia A.; Patricia A Martin‑DeLeon; Martin‑DeLeon, Patricia A.A variety of glycosylphosphatidylinositol (GPI)‑linked proteins are acquired on spermatozoa from epididymal luminal fluids (ELF) during sperm maturation. These proteins serve roles in immunoprotection and in key steps of fertilization such as capacitation, acrosomal exocytosis and sperm‑egg interactions. Their acquisition on sperm cells is mediated both by membrane vesicles (epididymosomes, EP) which were first reported to dock on the sperm surface, and by lipid carriers which facilitate the transfer of proteins associated with the membrane‑free fraction of ELF. While the nonvesicular fraction is more efficient, both pathways are dependent on hydrophobic interactions between the GPI‑anchor and the external lipid layer of the sperm surface. More recently proteomic and hypothesis‑driven studies have shown that EP from several mammals carry transmembrane (TM) proteins, including plasma membrane Ca2+‑ATPase 4 (PMCA4). Synthesized in the testis, PMCA4 is an essential protein and the major Ca2+ efflux pump in murine spermatozoa. Delivery of PMCA4 to spermatozoa from bovine and mouse EP during epididymal maturation and in vitro suggests that the docking of EP on the sperm surface precedes fusion, and experimental evidence supports a fusogenic mechanism for TM proteins. Fusion is facilitated by CD9, which generates fusion–competent sites on membranes. On the basis of knowledge of PMCA4’s interacting partners a number of TM and membrane‑associated proteins have been identified or are predicted to be present, in the epididymosomal cargo deliverable to spermatozoa. These Ca2+‑dependent proteins, undetected in proteomic studies, play essential roles in sperm motility and fertility, and their detection highlights the usefulness of the hypothesis‑driven approach.Item Erythroid differentiation in mouse erythroleukemia cells depends on Tmod3-mediated regulation of actin filament assembly into the erythroblast membrane skeleton(FASEB Journal, 2022-02-23) Ghosh, Arit; Coffin, Megan; West, Richard; Fowler, Velia M.Erythroid differentiation (ED) is a complex cellular process entailing morphologically distinct maturation stages of erythroblasts during terminal differentiation. Studies of actin filament (F-actin) assembly and organization during terminal ED have revealed essential roles for the F-actin pointed-end capping proteins, tropomodulins (Tmod1 and Tmod3). Tmods bind tropomyosins (Tpms), which enhance Tmod capping and F-actin stabilization. Tmods can also nucleate F-actin assembly, independent of Tpms. Tmod1 is present in the red blood cell (RBC) membrane skeleton, and deletion of Tmod1 in mice leads to a mild compensated anemia due to mis-regulated F-actin lengths and membrane instability. Tmod3 is not present in RBCs, and global deletion of Tmod3 leads to embryonic lethality in mice with impaired ED. To further decipher Tmod3’s function during ED, we generated a Tmod3 knockout in a mouse erythroleukemia cell line (Mel ds19). Tmod3 knockout cells appeared normal prior to ED, but showed defects during progression of ED, characterized by a marked failure to reduce cell and nuclear size, reduced viability, and increased apoptosis. Tmod3 does not assemble with Tmod1 and Tpms into the Triton X-100 insoluble membrane skeleton during ED, and loss of Tmod3 had no effect on α1,β1-spectrin and protein 4.1R assembly into the membrane skeleton. However, F-actin, Tmod1 and Tpms failed to assemble into the membrane skeleton during ED in absence of Tmod3. We propose that Tmod3 nucleation of F-actin assembly promotes incorporation of Tmod1 and Tpms into membrane skeleton F-actin, and that this is integral to morphological maturation and cell survival during erythroid terminal differentiation.Item Fascination with Fluctuation: Luria and Delbrück’s Legacy(Axioms, 2023-03-07) Robeva, Raina S.; Jungck, John R.While Luria and Delbrück’s seminal work has found its way to some college biology textbooks, it is now largely absent from those in mathematics. This is a significant omission, and we consider it a missed opportunity to present a celebrated conceptual model that provides an authentic and, in many ways, intuitive example of the quantifiable nature of stochasticity. We argue that it is an important topic that could enrich the educational literature in mathematics, from the introductory to advanced levels, opening many doors to undergraduate research. The paper has two main parts. First, we present in detail the mathematical theory behind the Luria–Delbrück model and make suggestions for further readings from the literature. We also give ideas for inclusion in various mathematics courses and for projects that can be used in regular courses, independent projects, or as starting points for student research. Second, we briefly review available hands-on activities as pedagogical ways to facilitate problem posing, problem-based learning, and investigative case-based learning and to expose students to experiments leading to Poisson distributions. These help students with even limited mathematics backgrounds understand the significance of Luria–Delbrück’s work for determining mutation rates and its impact on many fields, including cancer chemotherapy, antibiotic resistance, radiation, and environmental screening for mutagens and teratogens.Item Genome-Wide Analysis of Differentially Expressed miRNAs and Their Associated Regulatory Networks in Lenses Deficient for the Congenital Cataract-Linked Tudor Domain Containing Protein TDRD7(Frontiers in Cell and Developmental Biology, 2021-02-16) Anand, Deepti; Al Saai, Salma; Shrestha, Sanjaya K.; Barnum, Carrie E.; Chuma, Shinichiro; Lachke, Salil A.Mutations/deficiency of TDRD7, encoding a tudor domain protein involved in post-transcriptional gene expression control, causes early onset cataract in humans. While Tdrd7 is implicated in the control of key lens mRNAs, the impact of Tdrd7 deficiency on microRNAs (miRNAs) and how this contributes to transcriptome misexpression and to cataracts, is undefined. We address this critical knowledge-gap by investigating Tdrd7-targeted knockout (Tdrd7-/-) mice that exhibit fully penetrant juvenile cataracts. We performed Affymetrix miRNA 3.0 microarray analysis on Tdrd7-/- mouse lenses at postnatal day (P) 4, a stage preceding cataract formation. This analysis identifies 22 miRNAs [14 over-expressed (miR-15a, miR-19a, miR-138, miR-328, miR-339, miR-345, miR-378b, miR-384, miR-467a, miR-1224, miR-1935, miR-1946a, miR-3102, miR-3107), 8 reduced (let-7b, miR-34c, miR-298, miR-382, miR-409, miR-1198, miR-1947, miR-3092)] to be significantly misexpressed (fold-change ≥ ± 1.2, p-value < 0.05) in Tdrd7-/- lenses. To understand how these misexpressed miRNAs impact Tdrd7-/- cataract, we predicted their mRNA targets and examined their misexpression upon Tdrd7-deficiency by performing comparative transcriptomics analysis on P4 and P30 Tdrd7-/- lens. To prioritize these target mRNAs, we used various stringency filters (e.g., fold-change in Tdrd7-/- lens, iSyTE-based lens-enriched expression) and identified 98 reduced and 89 elevated mRNA targets for overexpressed and reduced miRNAs, respectively, which were classified as “top-priority” “high-priority,” and “promising” candidates. For Tdrd7-/- lens overexpressed miRNAs, this approach identified 18 top-priority reduced target mRNAs: Alad, Ankrd46, Ceacam10, Dgat2, Ednrb, H2-Eb1, Klhl22, Lin7a, Loxl1, Lpin1, Npc1, Olfm1, Ppm1e, Ppp1r1a, Rgs8, Shisa4, Snx22 and Wnk2. Majority of these targets were also altered in other gene-specific perturbation mouse models (e.g., Brg1, E2f1/E2f2/E2f3, Foxe3, Hsf4, Klf4, Mafg/Mafk, Notch) of lens defects/cataract, suggesting their importance to lens biology. Gene ontology (GO) provided further insight into their relevance to lens pathology. For example, the Tdrd7-deficient lens capsule defect may be explained by reduced mRNA targets (e.g., Col4a3, Loxl1, Timp2, Timp3) associated with “basement membrane”. GO analysis also identified new genes (e.g., Casz1, Rasgrp1) recently linked to lens biology/pathology. Together, these analyses define a new Tdrd7-downstream miRNA-mRNA network, in turn, uncovering several new mRNA targets and their associated pathways relevant to lens biology and offering molecular insights into the pathology of congenital cataract.Item Harnessing the Power of Induced Pluripotent Stem Cells and Gene Editing Technology: Therapeutic Implications in Hematological Malignancies(Cells, 2021-10-09) Sidhu, Ishnoor; Barwe, Sonali P.; Pillai, Raju K.; Gopalakrishnapillai, AnilkumarIn vitro modeling of hematological malignancies not only provides insights into the influence of genetic aberrations on cellular and molecular mechanisms involved in disease progression but also aids development and evaluation of therapeutic agents. Owing to their self-renewal and differentiation capacity, induced pluripotent stem cells (iPSCs) have emerged as a potential source of short in supply disease-specific human cells of the hematopoietic lineage. Patient-derived iPSCs can recapitulate the disease severity and spectrum of prognosis dictated by the genetic variation among patients and can be used for drug screening and studying clonal evolution. However, this approach lacks the ability to model the early phases of the disease leading to cancer. The advent of genetic editing technology has promoted the generation of precise isogenic iPSC disease models to address questions regarding the underlying genetic mechanism of disease initiation and progression. In this review, we discuss the use of iPSC disease modeling in hematological diseases, where there is lack of patient sample availability and/or difficulty of engraftment to generate animal models. Furthermore, we describe the power of combining iPSC and precise gene editing to elucidate the underlying mechanism of initiation and progression of various hematological malignancies. Finally, we discuss the power of iPSC disease modeling in developing and testing novel therapies in a high throughput setting.Item High-resolution identification and abundance profiling of cassava (Manihot esculenta Crantz) microRNAs(BioMed Central, 2016-01-28) Khatabi, Behnam; Arikit, Siwaret; Xia, Rui; Winter, Stephan; Oumar, Doungous; Mongomake, Kone; Meyers, Blake C.; Fondong, Vincent N.; Behnam Khatabi, Siwaret Arikit, Rui Xia, Stephan Winter, Doungous Oumar, Kone Mongomake, Blake C. Meyers and Vincent N. Fondong; Arikit, Siwaret; Xia, Rui; Meyers, Blake C.BACKGROUND: Small RNAs (sRNAs) are endogenous sRNAs that play regulatory roles in plant growth, development, and biotic and abiotic stress responses. In plants, one subset of sRNAs, microRNAs (miRNAs) exhibit tissue-differential expression and regulate gene expression mainly through direct cleavage of mRNA or indirectly via production of secondary phased siRNAs (phasiRNAs) that silence cognate target transcripts in trans. RESULTS: Here, we have identified cassava (Manihot esculenta Crantz) miRNAs using high resolution sequencing of sRNA libraries from leaf, stem, callus, male and female flower tissues. To analyze the data, we built a cassava genome database and, via sequence analysis and secondary structure prediction, 38 miRNAs not previously reported in cassava were identified. These new cassava miRNAs included two miRNAs not previously been reported in any plant species. The miRNAs exhibited tissue-differential accumulation as confirmed by quantitative RT-PCR and Northern blot analysis, largely reflecting levels observed in sequencing data. Some of the miRNAs identified were predicted to trigger production of secondary phased siRNAs (phasiRNAs) from 80 PHAS loci. CONCLUSIONS: Cassava is a woody perennial shrub, grown principally for its starch-rich storage roots, which are rich in calories. In this study, new miRNAs were identified and their expression was validated using qRT-PCR of RNA from five different tissues. The data obtained expand the list of annotated miRNAs and provide additional new resources for cassava improvement research.Item Host-Derived Sialic Acids Are an Important Nutrient Source Required for Optimal Bacterial Fitness In Vivo(American Society Microbiology, 4/12/16) McDonald,Nathan D.; Lubin,Jean-Bernard; Chowdhury,Nityananda; Boyd,E. Fidelma; Nathan D. McDonald, Jean-Bernard Lubin, Nityananda Chowdhury, E. Fidelma Boyd; Boyd, Ethna FidelmaA major challenge facing bacterial intestinal pathogens is competition for nutrient sources with the host microbiota. Vibrio cholerae is an intestinal pathogen that causes cholera, which affects millions each year; however, our knowledge of its nutritional requirements in the intestinal milieu is limited. In this study, we demonstrated that V. cholerae can grow efficiently on intestinal mucus and its component sialic acids and that a tripartite ATP-independent periplasmic SiaPQM strain, transporter-deficient mutant NC1777, was attenuated for colonization using a streptomycin-pretreated adult mouse model. In in vivo competition assays, NC1777 was significantly outcompeted for up to 3 days postinfection. NC1777 was also significantly outcompeted in in vitro competition assays in M9 minimal medium supplemented with intestinal mucus, indicating that sialic acid uptake is essential for fitness. Phylogenetic analyses demonstrated that the ability to utilize sialic acid was distributed among 452 bacterial species from eight phyla. The majority of species belonged to four phyla, Actinobacteria (members of Actinobacillus, Corynebacterium, Mycoplasma, and Streptomyces), Bacteroidetes (mainly Bacteroides, Capnocytophaga, and Prevotella), Firmicutes (members of Streptococcus, Staphylococcus, Clostridium, and Lactobacillus), and Proteobacteria (including Escherichia, Shigella, Salmonella, Citrobacter, Haemophilus, Klebsiella, Pasteurella, Photobacterium, Vibrio, and Yersinia species), mostly commensals and/or pathogens. Overall, our data demonstrate that the ability to take up host-derived sugars and sialic acid specifically allows V. cholerae a competitive advantage in intestinal colonization and that this is a trait that is sporadic in its occurrence and phylogenetic distribution and ancestral in some genera but horizontally acquired in others. IMPORTANCE Sialic acids are nine carbon amino sugars that are abundant on all mucous surfaces. The deadly human pathogen Vibrio cholerae contains the genes required for scavenging, transport, and catabolism of sialic acid. We determined that the V. cholerae SiaPQM transporter is essential for sialic acid transport and that this trait allows the bacterium to outcompete non-catabolizers in vivo. We also showed that the ability to take up and catabolize sialic acid is prevalent among both commensals and pathogens that colonize the oral cavity and the respiratory, intestinal, and urogenital tracts. Phylogenetic analysis determined that the sialic acid catabolism phenotype is ancestral in some genera such as Yersinia, Streptococcus, and Staphylococcus and is acquired by horizontal gene transfer in others such as Vibrio, Aeromonas, and Klebsiella. The data demonstrate that this trait has evolved multiple times in different lineages, indicating the importance of specialized metabolism to niche expansion.Item In vitro reconstitution reveals major differences between human and bacterial cytochrome c synthases(eLife, 2021-05-11) Sutherland, Molly C.; Mendez, Deanna L.; Babbitt, Shalon E.; Tillman, Dustin E.; Melnikov, Olga; Tran, Nathan L.; Prizant, Noah T.; Collier, Andrea L.; Kranz, Robert G.Cytochromes c are ubiquitous heme proteins in mitochondria and bacteria, all possessing a CXXCH (CysXxxXxxCysHis) motif with covalently attached heme. We describe the first in vitro reconstitution of cytochrome c biogenesis using purified mitochondrial (HCCS) and bacterial (CcsBA) cytochrome c synthases. We employ apocytochrome c and peptide analogs containing CXXCH as substrates, examining recognition determinants, thioether attachment, and subsequent release and folding of cytochrome c. Peptide analogs reveal very different recognition requirements between HCCS and CcsBA. For HCCS, a minimal 16-mer peptide is required, comprised of CXXCH and adjacent alpha helix 1, yet neither thiol is critical for recognition. For bacterial CcsBA, both thiols and histidine are required, but not alpha helix 1. Heme attached peptide analogs are not released from the HCCS active site; thus, folding is important in the release mechanism. Peptide analogs behave as inhibitors of cytochrome c biogenesis, paving the way for targeted control.
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