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Item Somatostatin signaling via SSTR1 contributes to the quiescence of colon cancer stem cells(Biomed Central LTD, 12/7/16) Modarai,Shirin R.; Opdenaker,Lynn M.; Viswanathan,Vignesh; Fields,Jeremy Z.; Boman,Bruce M.; Shirin R. Modarai, Lynn M. Opdenaker, Vignesh Viswanathan, Jeremy Z. Fields, Bruce M. Boman; Modarai, Shirin R.; Opdenaker, Lynn Marie.; Viswanathan, Vignesh; Boman, Bruce MillringBackground: Neuroendocrine cells (NECs) reside adjacent to colonic stem cells (SCs) in the crypt stem cell (SC) niche, but how NECs are involved in regulation of SCs is unclear. We investigated NECs expressing somatostatin (SST) and somatostatin receptor type 1 (SSTR1) because SST inhibits intestinal proliferation. Hypothesis: SSTR1 cells maintain SCs in a quiescent state, and aberrant SST signaling contributes to SC overpopulation in colorectal cancer (CRC). Methods: The proportion of SCs to NECs cells was quantified, by flow cytometry, in CRC cell lines and primary normal/tumor tissues based on cellular ALDH and SSTR1 levels, respectively. Doubling time and sphere-formation was used to evaluate cell proliferation and stemness. CRC cell lines were treated with exogenous SST and SST inhibitor cyclosomatostatin (cycloSST) and analyzed for changes in SCs and growth rate. Paracrine signaling between NECs and SCs was ascertained using transwell cultures of ALDH+ and SSTR1+ cells. Results: In CRC cell lines, the proportion of ALDH+ cells inversely correlates with proportion of SSTR1+ cells and with rate of proliferation and sphere-formation. While primary normal tissue shows SST and SSTR1 expression, CRC shows only SSTR1 expression. Moreover, ALDH+ cells did not show SST or SSTR1 expression. Exogenous SST suppressed proliferation but not ALDH+ population size or viability. Inhibition of SSTR1 signaling, via cycloSST treatment, decreased cell proliferation, ALDH+ cell population size and sphere-formation. When co-cultured with SSTR1+ cells, sphere-formation and cell proliferation of ALDH+ cells was inhibited. Conclusion: That each CRC cell line has a unique ALDH+/SSTR1+ ratio which correlates with its growth dynamics, suggests feedback mechanisms exist between SCs and NECs that contribute to regulation of SCs. The growth suppression by both SST and cycloSST treatments suggests that SST signaling modulates this feedback mechanism. The ability of SSTR1+ cells to decrease sphere formation and proliferation of ALDH+ cells in transwell cultures indicates that the ALDH subpopulation is regulated by SSTR1 via a paracrine mechanism. Since ALDH+ cells lack SST and SSTR1 expression, we conjecture that SST signaling controls the rate of NEC maturation as SCs mature along the NEC lineage, which contributes to quiescence of SCs and inhibition of proliferation.Item Inhibition of T-Type Voltage Sensitive Calcium Channel Reduces Load-Induced OA in Mice and Suppresses the Catabolic Effect of Bone Mechanical Stress on Chondrocytes(PLOS (Public Library of Science), 2015-05-26) Srinivasan, Padma P.; Parajuli, Ashutosh; Price, Christopher; Wang, Liyun; Duncan, Randall L.; Kirn-Safran, Catherine B.; Padma P. Srinivasan, Ashutosh Parajuli, Christopher Price, Liyun Wang, Randall L. Duncan, Catherine B. Kirn-Safran; Srinivasan, Padma P.; Parajuli, Ashutosh; Price, Christopher; Wang, Liyun; Duncan, Randall L.; Kirn-Safran, Catherine B.Voltage-sensitive calcium channels (VSCC) regulate cellular calcium influx, one of the earliest responses to mechanical stimulation in osteoblasts. Here, we postulate that T-type VSCCs play an essential role in bone mechanical response to load and participate in events leading to the pathology of load-induced OA. Repetitive mechanical insult was used to induce OA in Cav3.2 T-VSCC null and wild-type control mouse knees. Osteoblasts (MC3T3- E1) and chondrocytes were treated with a selective T-VSCC inhibitor and subjected to fluid shear stress to determine how blocking of T-VSCCs alters the expression profile of each cell type upon mechanical stimulation. Conditioned-media (CM) obtained from static and sheared MC3T3-E1 was used to assess the effect of osteoblast-derived factors on the chondrocyte phenotype. T-VSCC null knees exhibited significantly lower focal articular cartilage damage than age-matched controls. In vitro inhibition of T-VSCC significantly reduced the expression of both early and late mechanoresponsive genes in osteoblasts but had no effect on gene expression in chondrocytes. Furthermore, treatment of chondrocytes with CM obtained from sheared osteoblasts induced expression of markers of hypertrophy in chondrocytes and this was nearly abolished when osteoblasts were pre-treated with the T-VSCC-specific inhibitor. These results indicate that T-VSCC plays a role in signaling events associated with induction of OA and is essential to the release of osteoblast-derived factors that promote an early OA phenotype in chondrocytes. Further, these findings suggest that local inhibition of T-VSCC may serve as a therapy for blocking load-induced bone formation that results in cartilage degenerationItem 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 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 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 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 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 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 Racial and Ethnic Heterogeneity in the Association Between Total Cholesterol and Pediatric Obesity(MDPI, 2015-12-23) Holmes, Laurens Jr.; LaHurd, Alex; Wasson, Emily; McClarin, Lavisha; Dabney, Kirk; Laurens Holmes Jr., Alex LaHurd, Emily Wasson, Lavisha McClarin and Kirk Dabney; LaHurd, Alex; Holmes, Laurens Jr.Total cholesterol (TC) directly correlates with overweight/obesity, but it remains unclear if this association varies by race and ethnicity. We assessed the association as well as the racial/ethnic heterogeneity in this relationship. Data on 63,863 children were assessed using electronic medical records between 2010 and 2011. A cross-sectional design was utilized with log-binomial regression model and chi-squared statistic to examine the data. Overall, abnormal total cholesterol (ATC) was 7.5% (4812). Significant racial variability in ATC was observed: Black/African American (AA) (7.4%), White (7.0%), Asian (5.1%) and some other race (SOR) children (11.3%), 2 (5) = 141.5, p < 0.0001. Black/AA (34.7%) and SOR children (41.2%) were predominantly overweight/obese, unlike the Asian children, (25.8%), 2 (5) = 324.6, p < 0.0001. The BMI percentile was highest among SOR (69.0 28.6) and Black/AA children (65.2 29.1), but lowest among Asian children (55.7 31.5). A significant racial variability was also observed in weight, with the highest mean among Black/AA children (36.8kg 23.0) and the lowest among Asian children (28.7kg 16.8), f = 7.2, p < 0.001. Relative to normal TC, children with ATC were 2.6 times as likely to have abnormal BMI, relative risk (RR) =2.60, 99% CI, 2.54–2.68). Compared to non-Hispanic (RR = 2.62, 99% CI, 2.54–2.69), the risk was lower among Hispanics (RR = 2.34, 99%, 2.21–2.48). Among children with ATC, risk for abnormal BMI was highest among Asians, adjusted RR = 2.91, 99% CI, 2.34–3.62), intermediate among AA (ARR = 2.68, 99% CI, 2.59–2.77), but lowest among Whites (ARR = 2.40, 99% CI, 2.39–2.64), and SOR (ARR = 2.33, 99% CI, 2.19–2.50). In a large sample of children, total cholesterol directly correlates with BMI, with an observed racial and ethnic heterogeneity.Item Mechanisms of extracellular S0 globule production and degradation in Chlorobaculum tepidum via dynamic cell–globule interactions(Microbiology Society, 2016-01-07) Marnocha, C. L.; Levy, A. T.; Powell, D. H.; Hanson, T. E.; Chan, C. S.; ; Marnocha, C. L.; Levy, A. T.; Powell, D. H.; Hanson, T. E.; Chan, C. S.The Chlorobiales are anoxygenic phototrophs that produce solid, extracellular elemental sulfur globules as an intermediate step in the oxidation of sulfide to sulfate. These organisms must export sulfur while preventing cell encrustation during S0 globule formation; during globule degradation they must find and mobilize the sulfur for intracellular oxidation to sulfate. To understand how the Chlorobiales address these challenges, we characterized the spatial relationships and physical dynamics of Chlorobaculum tepidum cells and S0 globules by light and electron microscopy. Cba. tepidum commonly formed globules at a distance from cells. Soluble polysulfides detected during globule production may allow for remote nucleation of globules. Polysulfides were also detected during globule degradation, probably produced as an intermediate of sulfur oxidation by attached cells. Polysulfides could feed unattached cells, which made up over 80% of the population and had comparable growth rates to attached cells. Given that S0 is formed remotely from cells, there is a question as to how cells are able to move toward S0 in order to attach. Time-lapse microscopy shows that Cba. tepidum is in fact capable of twitching motility, a finding supported by the presence of genes encoding type IV pili. Our results show how Cba. tepidum is able to avoid mineral encrustation and benefit from globule degradation even when not attached. In the environment, Cba. tepidum may also benefit from soluble sulfur species produced by other sulfur-oxidizing or sulfur-reducing bacteria as these organisms interact with its biogenic S0 globules.Item Mechanisms of extracellular S0 globule production and degradation in Chlorobaculum tepidum via dynamic cell–globule interactions(Microbiology Society, 2016-01-07) Marnocha, C. L.; Levy, A. T.; Powell, D. H.; Hanson, T. E.; Chan, C. S.; C. L. Marnocha, A. T. Levy, D. H. Powell, T. E. Hanson and C. S. Chan; Marnocha, C. L.; Levy, A. T.; Powell, D. H.; Hanson, T. E.; Chan, C. S.The Chlorobiales are anoxygenic phototrophs that produce solid, extracellular elemental sulfur globules as an intermediate step in the oxidation of sulfide to sulfate. These organisms must export sulfur while preventing cell encrustation during S0 globule formation; during globule degradation they must find and mobilize the sulfur for intracellular oxidation to sulfate. To understand how the Chlorobiales address these challenges, we characterized the spatial relationships and physical dynamics of Chlorobaculum tepidum cells and S0 globules by light and electron microscopy. Cba. tepidum commonly formed globules at a distance from cells. Soluble polysulfides detected during globule production may allow for remote nucleation of globules. Polysulfides were also detected during globule degradation, probably produced as an intermediate of sulfur oxidation by attached cells. Polysulfides could feed unattached cells, which made up over 80% of the population and had comparable growth rates to attached cells. Given that S0 is formed remotely from cells, there is a question as to how cells are able to move toward S0 in order to attach. Time-lapse microscopy shows that Cba. tepidum is in fact capable of twitching motility, a finding supported by the presence of genes encoding type IV pili. Our results show how Cba. tepidum is able to avoid mineral encrustation and benefit from globule degradation even when not attached. In the environment, Cba. tepidum may also benefit from soluble sulfur species produced by other sulfur-oxidizing or sulfur-reducing bacteria as these organisms interact with its biogenic S0 globules.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 Prohibitin involvement in the generation of mitochondrial superoxide at complex I in human sperm(Foundation for Cellular and Molecular Medicine, 2016-08-25) Chai, Ran-Ran; Chen, Guo-Wu; Shi, Hui-Juan; O, Wai-Sum; Martin-DeLeon, Patricia A.; Chen, Hong; Ran-Ran Chai, Guo-Wu Chen, Hui-Juan Shi, Wai-Sum O, Patricia A. Martin-DeLeon, Hong Chen; Martin-DeLeon, Patricia A.Prohibitin (PHB), a major mitochondrial membrane protein, has been shown earlier in our laboratoryto regulate sperm motility via an alteration in mitochondrial membrane potential (MMP) in infertile men with poor sperm quality. To test if PHB expression is associated with sperm mitochondrial superoxide (mROS) levels, here we examined sperm mROS levels, high MMP and lipid peroxidation in infertile men with poor sperm motility (asthenospermia, A) and/or low sperm concentrations (oligoasthenospermia, OA). The diaphorase-type activity of sperm mitochondrial complex I (MCI) and PHB expression were also determined. We demonstrate that mROS and lipid peroxidation levels are significantly higher in sperm from A and OA subjects than in normospermic subjects, whereas high MMP and PHB expression are significantly lower. A positive correlation between mROS and lipid peroxidation and a negative correlation of mROS with PHB expression, high MMP, and sperm motility were found in these subjects. The finding of similar diaphorase-type activity levels of sperm MCI in the three groups studied suggests that the catalytic subunits of MCI in the matrix arm may produce mROS on its own. There may be a dysfunction of electron transport at MCI associated with decreased expression of PHB in sperm with poor quality. We conclude that mROS level is increased and associated with decreased PHB expression, and it may regulate sperm motility via increases in low MMP and lipid peroxidation. This is the first report on the involvement of PHB in human sperm motility loss associated with increased generation of mROS at MCI.Item Prohibitin involvement in the generation of mitochondrial superoxide at complex I in human sperm(Wiley-Blackwell, 2016-08-25) Chai, Ran-Ran; Chen, Guo-Wu; Shi, Hui-Juan; Martin-DeLeon, Patricia A; Chen, Hong; Wai-Sum, O; Ran-Ran Chai; Guo-Wu Chen; Hui-Juan Shi; Wai-Sum O; Patricia A. Martin-DeLeon; Hong Chen; Martin-DeLeon, Patricia AProhibitin (PHB), a major mitochondrial membrane protein, has been shown earlier in our laboratoryto regulate sperm motility via an alteration in mitochondrial membrane potential (MMP) in infertile men with poor sperm quality. To test if PHB expression is associated with sperm mitochondrial superoxide (mROS) levels, here we examined sperm mROS levels, high MMP and lipid peroxidation in infertile men with poor sperm motility (asthenospermia, A) and/or low sperm concentrations (oligoasthenospermia, OA). The diaphorase-type activity of sperm mitochondrial complex I (MCI) and PHB expression were also determined. We demonstrate that mROS and lipid peroxidation levels are significantly higher in sperm from A and OA subjects than in normospermic subjects, whereas high MMP and PHB expression are significantly lower. A positive correlation between mROS and lipid peroxidation and a negative correlation of mROS with PHB expression, high MMP, and sperm motility were found in these subjects. The finding of similar diaphorase-type activity levels of sperm MCI in the three groups studied suggests that the catalytic subunits of MCI in the matrix arm may produce mROS on its own. There may be a dysfunction of electron transport at MCI associated with decreased expression of PHB in sperm with poor quality. We conclude that mROS level is increased and associated with decreased PHB expression, and it may regulate sperm motility via increases in low MMP and lipid peroxidation. This is the first report on the involvement of PHB in human sperm motility loss associated with increased generation of mROS at MCI.Item Quorum sensing regulators are required for metabolic fitness in Vibrio parahaemolyticus(American Society for Microbiology, 2017-01-09) Kalburge, Sai Siddarth; Carpenter, Megan R.; Rozovsky, Sharon; Boyd, E. Fidelma; Sai Siddarth Kalburge, Megan R. Carpenter, Sharon Rozovsky and E. Fidelma Boyd; Kalburge, Sai Siddarth; Carpenter, Megan R.; Rozovsky, Sharon; Boyd, E. FidelmaQuorum sensing (QS) is a process by which bacteria alter gene expression in response to cell density changes. In Vibrio species, at low cell density, the sigma 54-dependent response regulator LuxO, is active, and regulates the two QS master regulators AphA, which is induced and OpaR, which is repressed. At high cell density the opposite occurs, LuxO is inactive, therefore OpaR is induced and AphA is repressed. In V. parahaemolyticus, a significant enteric pathogen of humans, the role of these regulators in pathogenesis is less known. We examined deletion mutants of luxO, opaR and aphA for in vivo fitness using an adult mouse model. We found that the luxO and aphA mutants were defective in colonization compared to wild-type. The opaR mutant did not show any defect in vivo. Colonization was restored to wild-type levels in a luxO/opaR double mutant and was also increased in an opaR/aphA double mutant. These data suggest that AphA is important and that overexpression of opaR is detrimental to in vivo fitness. RNA-seq analysis of the wild-type and luxO mutant grown in mouse intestinal mucus showed that 60% of the genes that were downregulated in the luxO mutant were involved in amino acid and sugar transport and metabolism. These data suggest that the luxO mutant has a metabolic disadvantage, which was confirmed by growth pattern analysis using phenotype microarrays. Bioinformatics analysis revealed OpaR binding sites in the regulatory region of 55 carbon transporter and metabolism genes. Biochemical analysis of five representatives of these regulatory regions demonstrated direct binding of OpaR in all five tested. These data demonstrate the role of OpaR in carbon utilization and metabolic fitness, an overlooked role in the QS regulon.Item Quorum sensing regulators are required for metabolic fitness in Vibrio parahaemolyticus(American Society for Microbiology Journals, 2017-01-09) Kalburge, Sai Siddarth; Carpenter, Megan R.; Rozovsky, Sharon; Boyd, E. Fidelma; Sai Siddarth Kalburge, Megan R. Carpenter, Sharon Rozovsky, E. Fidelma Boyd; Kalburge, Sai Siddarth; Carpenter, Megan R.; Rozovsky, Sharon; Boyd, E. FidelmaQuorum sensing (QS) is a process by which bacteria alter gene expression in response to cell density changes. In Vibrio species, at low cell density, the sigma 54-dependent response regulator LuxO, is active, and regulates the two QS master regulators AphA, which is induced and OpaR, which is repressed. At high cell density the opposite occurs, LuxO is inactive, therefore OpaR is induced and AphA is repressed. In V. parahaemolyticus, a significant enteric pathogen of humans, the role of these regulators in pathogenesis is less known. We examined deletion mutants of luxO, opaR and aphA for in vivo fitness using an adult mouse model. We found that the luxO and aphA mutants were defective in colonization compared to wild-type. The opaR mutant did not show any defect in vivo. Colonization was restored to wild-type levels in a luxO/opaR double mutant and was also increased in an opaR/aphA double mutant. These data suggest that AphA is important and that overexpression of opaR is detrimental to in vivo fitness. RNA-seq analysis of the wild-type and luxO mutant grown in mouse intestinal mucus showed that 60% of the genes that were downregulated in the luxO mutant were involved in amino acid and sugar transport and metabolism. These data suggest that the luxO mutant has a metabolic disadvantage, which was confirmed by growth pattern analysis using phenotype microarrays. Bioinformatics analysis revealed OpaR binding sites in the regulatory region of 55 carbon transporter and metabolism genes. Biochemical analysis of five representatives of these regulatory regions demonstrated direct binding of OpaR in all five tested. These data demonstrate the role of OpaR in carbon utilization and metabolic fitness, an overlooked role in the QS regulon.Item Nicotinic acid inhibits glioma invasion by facilitating Snail1 degradation(Nature Publishing Group, 2017-03-03) Li, Jiejing; Qu, Jiagui; Shi, Yu; Perfetto, Mark; Ping, Zhuxian; Christian, Laura; Niu, Hua; Mei, Shuting; Zhang, Qin; Yang, Xiangcai; Wei, Shuo; Jiejing Li, Jiagui Qu, Yu Shi, Mark Perfetto, Zhuxian Ping, Laura Christian, Hua Niu, Shuting Mei, Qin Zhang, Xiangcai Yang & Shuo Wei; Wei, ShuoMalignant glioma is a formidable disease that commonly leads to death, mainly due to the invasion of tumor cells into neighboring tissues. Therefore, inhibition of tumor cell invasion may provide an effective therapy for malignant glioma. Here we report that nicotinic acid (NA), an essential vitamin, inhibits glioma cell invasion in vitro and in vivo. Treatment of the U251 glioma cells with NA in vitro results in reduced invasion, which is accompanied by a loss of mesenchymal phenotype and an increase in cell-cell adhesion. At the molecular level, transcription of the adherens junction protein E-cadherin is upregulated, leading to accumulation of E-cadherin protein at the cell-cell boundary. This can be attributed to NA’s ability to facilitate the ubiquitination and degradation of Snail1, a transcription factor that represses E-cadherin expression. Similarly, NA transiently inhibits neural crest migration in Xenopus embryos in a Snail1-dependent manner, indicating that the mechanism of action for NA in cell migration is evolutionarily conserved. We further show that NA injection blocks the infiltration of tumor cells into the adjacent brain tissues and improves animal survival in a rat model of glioma. These results suggest that NA treatment may be developed into a potential therapy for malignant glioma.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 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.Item Iron Oxidation by a Fused Cytochrome-Porin Common to Diverse Iron-Oxidizing Bacteria(mBio, 2021-07-27) Keffer, Jessica L.; McAllister, Sean M.; Garber, Arkadiy I.; Hallahan, Beverly J.; Sutherland, Molly C.; Rozovsky, Sharon; Chan, Clara S.Iron (Fe) oxidation is one of Earth’s major biogeochemical processes, key to weathering, soil formation, water quality, and corrosion. However, our understanding of microbial contribution is limited by incomplete knowledge of microbial iron oxidation mechanisms, particularly in neutrophilic iron oxidizers. The genomes of many diverse iron oxidizers encode a homolog to an outer membrane cytochrome (Cyc2) shown to oxidize iron in two acidophiles. Phylogenetic analyses show Cyc2 sequences from neutrophiles cluster together, suggesting a common function, though this function has not been verified in these organisms. Therefore, we investigated the iron oxidase function of heterologously expressed Cyc2 from a neutrophilic iron oxidizer Mariprofundus ferrooxydans PV-1. Cyc2PV-1 is capable of oxidizing iron, and its redox potential is 208 ± 20 mV, consistent with the ability to accept electrons from Fe2+ at neutral pH. These results support the hypothesis that Cyc2 functions as an iron oxidase in neutrophilic iron-oxidizing organisms. The results of sequence analysis and modeling reveal that the entire Cyc2 family shares a unique fused cytochrome-porin structure, with a defining consensus motif in the cytochrome region. On the basis of results from structural analyses, we predict that the monoheme cytochrome Cyc2 specifically oxidizes dissolved Fe2+, in contrast to multiheme iron oxidases, which may oxidize solid Fe(II). With our results, there is now functional validation for diverse representatives of Cyc2 sequences. We present a comprehensive Cyc2 phylogenetic tree and offer a roadmap for identifying cyc2/Cyc2 homologs and interpreting their function. The occurrence of cyc2 in many genomes beyond known iron oxidizers presents the possibility that microbial iron oxidation may be a widespread metabolism.