KLF4-SQSTM1/p62-associated prosurvival autophagy contributes to carfilzomib resistance in multiple myeloma models.

Multiple myeloma (MM) is an incurable clonal plasma cell malignancy. Because of a high rate of immunoglobulin synthesis, the endoplasmic reticulum of MM cells is subjected to elevated basal levels of stress. Consequently, proteasome inhibitors, which exacerbate this stress by inhibiting ubiquitin-proteasome-mediated protein degradation, are an important new class of chemotherapeutic agents being used to combat this disease. However, MM cells still develop resistance to proteasome inhibitors such as carfilzomib. Toward this end, we have established carfilzomib-resistant derivatives of MM cell lines. We found that resistance to carfilzomib was associated with elevated levels of prosurvival autophagy, and Kruppel-like factor 4 (KLF4) was identified as a contributing factor. Expression levels as well as nuclear localization of KLF4 protein were elevated in MM cells with acquired carfilzomib resistance. Chromatin immunoprecipitations indicated that endogenous KLF4 bound to the promoter regions of the SQSTM1 gene encoding the ubiquitin-binding adaptor protein sequestosome/p62 that links the proteasomal and autophagic protein degradation pathways. Ectopic expression of KLF4 induced upregulation of SQSTM1. On the other hand, inhibitors of autophagy sensitized MM cells to carfilzomib, even in carfilzomib-resistant derivatives having increased expression of the multidrug resistance protein P-glycoprotein. Thus, we report here a novel function for KLF4, one of the Yamanaka reprogramming factors, as being a contributor to autophagy gene expression which moderates preclinical proteasome inhibitor efficacy in MM

Increased expression of the tight junction protein TJP1/ZO-1 is associated with upregulation of TAZ-TEAD activity and an adult tissue stem cell signature in carfilzomib-resistant multiple myeloma cells and high-risk multiple myeloma patients

Tight junction protein 1 (TJP1) has recently been proposed as a biomarker to identify multiple myeloma (MM) patients most likely to respond to bortezomiband carfilzomib-based proteasome inhibitor regimens. Herein we report increased expression of TJP1 during the adaptive response mediating carfilzomib resistance in the LP-1/Cfz MM cell line. Moreover, increased TJP1 expression delineated a subset of relapsed/refractory MM patients on bortezomib-based therapy sharing an LP-1/Cfzlike phenotype characterized by activation of interacting transcriptional effectors of the Hippo signaling cascade (TAZ and TEAD1) and an adult tissue stem cell signature. siRNA-mediated knockdown of TJP1 or TAZ/TEAD1 partially sensitized LP-1/Cfz cells to carfilzomib. Connectivity Map analysis identified translation inhibitors as candidate therapeutic agents targeting this molecular phenotype. We confirmed this prediction by showing that homoharringtonine (omacetaxine mepesuccinate) — the first translation inhibitor to be approved by the U.S. Food and Drug Administration — displayed potent cytotoxic activity on LP-1/Cfz cells. Homoharringtonine treatment reduced the levels of TAZ and TEAD1 as well as the MM-protective proteins Nrf2 and MCL1. Thus, our data suggest the importance of further studies evaluating translation inhibitors in relapsed/refractory MM. On the other hand, use of TJP1 as a MM biomarker for proteasome inhibitor sensitivity requires careful consideration

An integrated bioinformatics and computational biology approach identifies new BH3-only protein candidates

FoxD4L1 is a forkhead transcription factor that expands the neural ectoderm by down-regulating genes that promote the onset of neural differentiation and up-regulating genes that maintain proliferative neural precursors in an immature state. We previously demonstrated that binding of Grg4 to an Eh-1 motif enhances the ability of FoxD4L1 to down-regulate target neural genes but does not account for all of its repressive activity. Herein we analyzed the protein sequence for additional interaction motifs and secondary structure. Eight conserved motifs were identified in the C-terminal region of fish and frog proteins. Extending the analysis to mammals identified a high scoring motif downstream of the Eh-1 domain that contains a tryptophan residue implicated in protein-protein interactions. In addition, secondary structure prediction programs predicted an α-helical structure overlapping with amphibian-specific Motif 6 inXenopus, and similarly located α-helical structures in other vertebrate FoxD proteins. We tested functionality of this site by inducing a glutamine-to-proline substitution expected to break the predicted α-helical structure; this significantly reduced FoxD4L1’s ability to repress zic3and irx1. Because this mutation does not interfere with Grg4 binding, these results demonstrate that at least two regions, the Eh-1 motif and a more C-terminal predicted α-helical/Motif 6 site, additively contribute to repression. In the N-terminal region we previously identified a 14 amino acid motif that is required for the up-regulation of target genes. Secondary structure prediction programs predicted a short β-strand separating two acidic domains. Mutant constructs show that the β-strand itself is not required for transcriptional activation. Instead, activation depends upon a glycine residue that is predicted to provide sufficient flexibility to bring the two acidic domains into close proximity. These results identify conserved predicted motifs with secondary structures that enable FoxD4L1 to carry out its essential functions as both a transcriptional repressor and activator of neural genes

Free-sugar, total-sugar, fibre and micronutrient intake within elite youth British soccer players: a nutritional transition from schoolboy to fulltime soccer player.

It is recommended that soccer players consume a high carbohydrate (CHO) diet to augment performance. However, growing evidence suggests that there is a link between high free-sugar (FS) intake (>5% total energy intake; TEI) and metabolic diseases. Furthermore, foods that are often high in sugar, such as processed foods, are typically lacking in nutrient quality. We therefore analysed total- and FS, dietary fibre and micronutrient intake of players from an English Premier League academy under(U) 18 (n=13); U15/16 (n=25); U13/14 (n=21) using a 7-day food diary. Data was compared to current UK dietary reference value (DRV) for free-sugar via a t-test. The U13/14s (1018 %) and U15/16s (1130 %) both consumed higher amounts of free-sugar in comparison to the UK DRV of 5% TEI 5% (P<0.01), conversely, the U18s did not exceed the DRV (513 %). Furthermore, FS intake of the U18s was significantly lower than the U13/14s and U15/16s (P<0.01). Dietary fibre was below the DRV (25g/d for U13/14 & U15/16s; 30g/d for U18s) for all squads (19.04.7; 19.68.3; 17.14.2 g/d, respectively), but not different between squads. Additionally, micronutrient reference intakes were generally met. In conclusion, we provide novel data on dietary sugar, fibre and micronutrient intake within elite youth soccer players. We report an apparent 'nutritional transition' from schoolboy to fulltime soccer player, with U18s showing a significantly lower intake of sugar in comparison to younger squads, and a similar intake of FS to the UK DRVs. Practitioners should target improving player education around sugar and fibre consumption

Phenotype correction of fanconi anemia group a hematopoietic stem cells using lentiviral vector

AbstractFanconi anemia (FA) is an autosomal recessive disease characterized by progressive bone marrow failure due to defective stem cell function. FA patients' cells are hypersensitive to DNA cross-linking agents such as mitomycin C (MMC), exposure to which results in cytogenetic aberrations and cell death. To date Moloney murine leukemia virus vectors have been used in clinical gene therapy. Recently, third-generation lentiviral vectors based on the HIV-1 genome have been developed for efficient gene transfer to hematopoietic stem cells. We generated a self-inactivating lentiviral vector expressing the FA group A cDNA driven by the murine stem cell virus U3 LTR promoter and used the vector to transduce side-population (SP) cells isolated from bone marrow of Fanconi anemia group A (Fanca) knockout mice. One thousand transduced SP cells reconstituted the bone marrow of sublethally irradiated Fanca recipient mice. Phenotype correction was demonstrated by stable hematopoiesis following MMC challenge. Using real-time PCR, one proviral vector DNA copy per cell was detected in all lineage-committed cells in the peripheral blood of both primary and secondary recipients. Our results suggest that the lentiviral vector transduces stem cells capable of self-renewal and long-term hematopoiesis in vivo and is potentially useful for clinical gene therapy of FA hematopoietic cells

Decreased venous thrombosis with an oral inhibitor of P selectin

BackgroundP-selectin inhibition with protein therapeutics such as antibodies or soluble ligands given intravenously can decrease thrombosis in a mouse ligation model of venous thrombosis. In this study, we hypothesized that oral inhibition of P selectin with a novel oral nonprotein inhibitor (PSI-697) would decrease thrombosis and circulating microparticle populations. This study evaluated the effects on thrombosis and circulating microparticle populations in this murine venous thrombosis model.MethodsMice underwent inferior vena cava ligation to induce thrombosis. Mice with high circulating level of P selectin, Delta Cytoplasmic Tail (^CT), mice gene-deleted for both E- and P-selectin knockout (EPKO), and wild-type C57BL/6 mice (WT) were studied without and with administration of PSI-697 in food (100 mg/kg daily) from 2 days before thrombosis until the end of the study. Animals were killed 2 and 6 days later. Evaluations included thrombus weight (TW), vein wall morphometrics, microparticle quantification by using fluorescence-activated cell sorter analysis, and vein wall enzyme-linked immunosorbent assays for interleukin (IL)-10, P selectin, and monocyte chemotactic protein 1.ResultsPSI-697 significantly decreased TW in WT and ^CT mice, with a treated vs nontreated TW of 132 ± 24 vs 228 ± 29 × 10−4 g (P = .014) and 166 ± 19 vs 281 ± 16 × 10−4 g (P = .001), respectively. At day 6, the effect was significant only in the ^CT group (P < .05). Drug therapy at day 2 significantly increased vein wall monocytes in WT mice and increased monocytes and total inflammatory cells in ^CT animals. A significant decrease in neutrophils and total inflammatory cells was seen in EPKO mice at day 2 with therapy. Therapy significantly increased platelet-derived microparticles and total microparticles in ^CT mice on day 2. Changes in treated WT and treated EPKO animals were not significant compared with respective vehicle treatments at day 2. On day 6, therapy significantly decreased total microparticles in EPKO animals. Vein wall expression of IL-10 increased in all groups with therapy at day 2 (n = 18) and was significantly increased in WT (2687.5 ± 903 pg/mL vs 636 ± 108 pg/mL total protein; P = .038) and ^CT (2078 ± 295 pg/mL vs 432 ± 62 pg/mL total protein; P = .001) mice. Therapy significantly decreased vein wall P selectin, monocyte chemotactic protein 1, and IL-10 levels at day 6.ConclusionsPSI-697 decreased thrombosis. P-selectin inhibition allowed vein wall inflammatory cell extravasation in this model of complete ligation. Circulating microparticles (platelet-derived microparticles and total microparticles) increased with P-selectin inhibition, possibly because of decreased consumption into the thrombus. In summary, the oral administration of an inhibitor to P selectin provides significant TW reduction.Clinical RelevanceDeep venous thrombosis is a significant national health problem in the general population. The average annual incidence of deep venous thrombosis is approximately 250,000 cases per year. The selectin family of adhesion molecules is thought to be largely responsible for the initial attachment and rolling of leukocytes on stimulated vascular endothelium. Recent studies have explored the possible therapeutic implications of P-selectin inhibition to modulate venous thrombosis. For example, prophylactic dosing of a recombinant P-selectin ligand decreases venous thrombosis in a dose-dependent fashion in both feline and nonhuman primate animal models. Additionally, treatment of 2-day iliac thrombi with a recombinant protein, P-selectin inhibitor, significantly improves vein reopening in nonhuman primates. It is interesting to note that P-selectin inhibition decreases thrombosis without adverse anticoagulation. On the basis of the results from these previous studies, the use of P-selectin antagonism is a logical therapeutic approach to treat venous thrombosis. All inhibitors developed to date are either proteins or small molecules with low oral bioavailability that require intravenous or subcutaneous injection. This study evaluates, for the first time, a novel orally bioavailable inhibitor of P-selectin (PSI-697)

Impact of Community-Based Larviciding on the Prevalence of Malaria Infection in Dar es Salaam, Tanzania.

The use of larval source management is not prioritized by contemporary malaria control programs in sub-Saharan Africa despite historical success. Larviciding, in particular, could be effective in urban areas where transmission is focal and accessibility to Anopheles breeding habitats is generally easier than in rural settings. The objective of this study is to assess the effectiveness of a community-based microbial larviciding intervention to reduce the prevalence of malaria infection in Dar es Salaam, United Republic of Tanzania. Larviciding was implemented in 3 out of 15 targeted wards of Dar es Salaam in 2006 after two years of baseline data collection. This intervention was subsequently scaled up to 9 wards a year later, and to all 15 targeted wards in 2008. Continuous randomized cluster sampling of malaria prevalence and socio-demographic characteristics was carried out during 6 survey rounds (2004-2008), which included both cross-sectional and longitudinal data (N = 64,537). Bayesian random effects logistic regression models were used to quantify the effect of the intervention on malaria prevalence at the individual level. Effect size estimates suggest a significant protective effect of the larviciding intervention. After adjustment for confounders, the odds of individuals living in areas treated with larviciding being infected with malaria were 21% lower (Odds Ratio = 0.79; 95% Credible Intervals: 0.66-0.93) than those who lived in areas not treated. The larviciding intervention was most effective during dry seasons and had synergistic effects with other protective measures such as use of insecticide-treated bed nets and house proofing (i.e., complete ceiling or window screens). A large-scale community-based larviciding intervention significantly reduced the prevalence of malaria infection in urban Dar es Salaam

An Initial Survey of White Dwarfs in the Sloan Digital Sky Survey

An initial assessment is made of white dwarf and hot subdwarf stars observed in the Sloan Digital Sky Survey. In a small area of sky (190 square degrees), observed much like the full survey will be, 269 white dwarfs and 56 hot subdwarfs are identified spectroscopically where only 44 white dwarfs and 5 hot subdwarfs were known previously. Most are ordinary DA (hydrogen atmosphere) and DB (helium) types. In addition, in the full survey to date, a number of WDs have been found with uncommon spectral types. Among these are blue DQ stars displaying lines of atomic carbon; red DQ stars showing molecular bands of C_2 with a wide variety of strengths; DZ stars where Ca and occasionally Mg, Na, and/or Fe lines are detected; and magnetic WDs with a wide range of magnetic field strengths in DA, DB, DQ, and (probably) DZ spectral types. Photometry alone allows identification of stars hotter than 12000 K, and the density of these stars for 15<g<20 is found to be ~2.2 deg^{-2} at Galactic latitudes 29-62 deg. Spectra are obtained for roughly half of these hot stars. The spectra show that, for 15<g<17, 40% of hot stars are WDs and the fraction of WDs rises to ~90% at g=20. The remainder are hot sdB and sdO stars.Comment: Accepted for AJ; 43 pages, including 12 figures and 5 table