Vanillic Acid Restores Coenzyme Q Biosynthesis and ATP Production in Human Cells Lacking COQ6.

Coenzyme Q (CoQ), a redox-active lipid, is comprised of a quinone group and a polyisoprenoid tail. It is an electron carrier in the mitochondrial respiratory chain, a cofactor of other mitochondrial dehydrogenases, and an essential antioxidant. CoQ requires a large set of enzymes for its biosynthesis; mutations in genes encoding these proteins cause primary CoQ deficiency, a clinically and genetically heterogeneous group of diseases. Patients with CoQ deficiency often respond to oral CoQ10 supplementation. Treatment is however problematic because of the low bioavailability of CoQ10 and the poor tissue delivery. In recent years, bypass therapy using analogues of the precursor of the aromatic ring of CoQ has been proposed as a promising alternative. We have previously shown using a yeast model that vanillic acid (VA) can bypass mutations of COQ6, a monooxygenase required for the hydroxylation of the C5 carbon of the ring. In this work, we have generated a human cell line lacking functional COQ6 using CRISPR/Cas9 technology. We show that these cells cannot synthesize CoQ and display severe ATP deficiency. Treatment with VA can recover CoQ biosynthesis and ATP production. Moreover, these cells display increased ROS production, which is only partially corrected by exogenous CoQ, while VA restores ROS to normal levels. Furthermore, we show that these cells accumulate 3-decaprenyl-1,4-benzoquinone, suggesting that in mammals, the decarboxylation and C1 hydroxylation reactions occur before or independently of the C5 hydroxylation. Finally, we show that COQ6 isoform c (transcript NM_182480) does not encode an active enzyme. VA can be produced in the liver by the oxidation of vanillin, a nontoxic compound commonly used as a food additive, and crosses the blood-brain barrier. These characteristics make it a promising compound for the treatment of patients with CoQ deficiency due to COQ6 mutations.

Results of a multicenter universal newborn screening program for sickle cell disease in Italy: A call to action.

BACKGROUND: Sickle cell disease (SCD) is a chronic multisystem disorder requiring comprehensive care that includes newborn screening (NBS) as the first step of care. Italy still lacks a national SCD NBS program and policy on blood disorders. Pilot single-center screening programs and a regional targeted screening have been implemented so far, but more evidence is needed in order to impact health policies. POPULATION AND METHODS: NBS was offered to parents of newborns in gynecology clinics in Padova and Monza, tertiary care university hospitals in northern Italy. High-performance liquid chromatography (HPLC) was performed as the first test on samples collected on Guthrie cards. Molecular analysis of the beta-globin gene was performed on positive samples. RESULTS: A total of 5466 newborns were enrolled; for 5439, informed consents were obtained. A similar family origin was seen in the two centers (65% Italians, 9% mixed couples, 26% immigrants). Compared with SCD NBS programs in the United States and Europe, our results show a similar incidence of SCD patients and carriers. All SCD patients had a Sub-Saharan family background; HbS carriers were 15% Caucasians (Italian, Albanians) and 10% from other areas (North Africa-India-South America); carriers of other hemoglobin variants were mainly (47%) from other areas. CONCLUSIONS: Our results demonstrate the feasibility of a multicentric NBS program for SCD, give information on HbS epidemiology in two Northern Italian Areas, and support previous European recommendation for a universal NBS program for SCD in Italy: a high incidence of patients and carriers has been detected, with a high percentage of Caucasian carriers, impossible to identify in a targeted NBS.

COX16 is required for assembly of cytochrome c oxidase in human cells and is involved in copper delivery to COX2.

Cytochrome c oxidase (COX), complex IV of the mitochondrial respiratory chain, is comprised of 14 structural subunits, several prosthetic groups and metal cofactors, among which copper. Its biosynthesis involves a number of ancillary proteins, encoded by the COX-assembly genes that are required for the stabilization and membrane insertion of the nascent polypeptides, the synthesis of the prosthetic groups, and the delivery of the metal cofactors, in particular of copper. Recently, a modular model for COX assembly has been proposed, based on the sequential incorporation of different assembly modules formed by specific subunits. We have cloned and characterized the human homologue of yeast COX16. We show that human COX16 encodes a small mitochondrial transmembrane protein that faces the intermembrane space and is highly expressed in skeletal and cardiac muscle. Its knockdown in C. elegans produces COX deficiency, and its ablation in HEK293 cells impairs COX assembly. Interestingly, COX16 knockout cells retain significant COX activity, suggesting that the function of COX16 is partially redundant. Analysis of steady-state levels of COX subunits and of assembly intermediates by Blue-Native gels shows a pattern similar to that reported in cells lacking COX18, suggesting that COX16 is required for the formation of the COX2 subassembly module. Moreover, COX16 co-immunoprecipitates with COX2. Finally, we found that copper supplementation increases COX activity and restores normal steady state levels of COX subunits in COX16 knockout cells, indicating that, even in the absence of a canonical copper binding motif, COX16 could be involved in copper delivery to COX2.

The lncRNA CASC15 regulates SOX4 expression in RUNX1-rearranged acute leukemia.

BACKGROUND: Long non-coding RNAs (lncRNAs) play a variety of cellular roles, including regulation of transcription and translation, leading to alterations in gene expression. Some lncRNAs modulate the expression of chromosomally adjacent genes. Here, we assess the roles of the lncRNA CASC15 in regulation of a chromosomally nearby gene, SOX4, and its function in RUNX1/AML translocated leukemia. RESULTS: CASC15 is a conserved lncRNA that was upregulated in pediatric B-acute lymphoblastic leukemia (B-ALL) with t (12; 21) as well as pediatric acute myeloid leukemia (AML) with t (8; 21), both of which are associated with relatively better prognosis. Enforced expression of CASC15 led to a myeloid bias in development, and overall, decreased engraftment and colony formation. At the cellular level, CASC15 regulated cellular survival, proliferation, and the expression of its chromosomally adjacent gene, SOX4. Differentially regulated genes following CASC15 knockdown were enriched for predicted transcriptional targets of the Yin and Yang-1 (YY1) transcription factor. Interestingly, we found that CASC15 enhances YY1-mediated regulation of the SOX4 promoter. CONCLUSIONS: Our findings represent the first characterization of this CASC15 in RUNX1-translocated leukemia, and point towards a mechanistic basis for its action.

Targeting Glioblastoma with the Use of Phytocompounds and Nanoparticles.

Glioblastoma multiforme (GBM) are extremely lethal and still poorly treated primary brain tumors, characterized by the presence of highly tumorigenic cancer stem cell (CSC) subpopulations, considered responsible for tumor relapse. In order to successfully eradicate GBM growth and recurrence, new anti-cancer strategies selectively targeting CSCs should be designed. CSCs might be eradicated by targeting some of their cell surface markers and transporters, inducing their differentiation, impacting their hyper-glycolytic metabolism, inhibiting CSC-related signaling pathways and/or by targeting their microenvironmental niche. In this regard, phytocompounds such as curcumin, isothiocyanates, resveratrol and epigallocatechin-3-gallate have been shown to prevent or reverse cancer-related epigenetic dysfunctions, reducing tumorigenesis, preventing metastasis and/or increasing chemotherapy and radiotherapy efficacy. However, the actual bioavailability and metabolic processing of phytocompounds is generally unknown, and the presence of the blood brain barrier often represents a limitation to glioma treatments. Nowadays, nanoparticles (NPs) can be loaded with therapeutic compounds such as phytochemicals, improving their bioavailability and their targeted delivery within the GBM tumor bulk. Moreover, NPs can be designed to increase their tropism and specificity toward CSCs by conjugating their surface with antibodies specific for CSC antigens, with ligands or with glucose analogues. Here we discuss the use of phytochemicals as anti-glioma agents and the applicability of phytochemical-loaded NPs as drug delivery systems to target GBM. Additionally, we provide some examples on how NPs can be specifically formulated to improve CSC targeting.

Copper and bezafibrate cooperate to rescue cytochrome c oxidase deficiency in cells of patients with SCO2 mutations.

BACKGROUND: Mutations in SCO2 cause cytochrome c oxidase deficiency (COX) and a fatal infantile cardioencephalomyopathy. SCO2 encodes a protein involved in COX copper metabolism; supplementation with copper salts rescues the defect in patients' cells. Bezafibrate (BZF), an approved hypolipidemic agent, ameliorates the COX deficiency in mice with mutations in COX10, another COX-assembly gene. METHODS: We have investigated the effect of BZF and copper in cells with SCO2 mutations using spectrophotometric methods to analyse respiratory chain activities and a luciferase assay to measure ATP production.. RESULTS: Individual mitochondrial enzymes displayed different responses to BZF. COX activity increased by about 40% above basal levels (both in controls and patients), with SCO2 cells reaching 75-80% COX activity compared to untreated controls. The increase in COX was paralleled by an increase in ATP production. The effect was dose-dependent: it was negligible with 100 µM BZF, and peaked at 400 µM BZF. Higher BZF concentrations were associated with a relative decline of COX activity, indicating that the therapeutic range of this drug is very narrow. Combined treatment with 100 µM CuCl2 and 200 µM BZF (which are only marginally effective when administered individually) achieved complete rescue of COX activity in SCO2 cells. CONCLUSIONS: These data are crucial to design therapeutic trials for this otherwise fatal disorder. The additive effect of copper and BZF will allow to employ lower doses of each drug and to reduce their potential toxic effects. The exact mechanism of action of BZF remains to be determined.

Comprehensive care for sickle cell disease immigrant patients: a reproducible model achieving high adherence to minimum standards of care.

BACKGROUND: Comprehensive care and advances in clinical investigations have reduced morbidity and mortality in sickle cell disease (SCD), but only a minority of children with SCD has access to comprehensive care. In Europe the majority of patients with SCD are immigrants who present barriers in accessing the health system; therefore, new evidence-based models of comprehensive care are needed to ensure that all SCD patients receive high-quality care, overcoming patient- and health system-related barriers. We wanted to verify if addressing the specific needs of immigrant patients contributes to improving adherence. PROCEDURES: Linguistic, cultural, social issues were considered in organizing comprehensive care in 2006. Hospital's records were used to determine access from 2006 to 2010 and to compare adherence before and after 2006. RESULTS: Ninety-four patients with SCD were enrolled in comprehensive care; 94% were first generation immigrants (81% African). Age at diagnosis was higher for children born abroad vs. children born in Italy (66.08 vs 25.36 months, P < 0.005). Since 2006, children were seen at least once a year, with 100% adherence to follow-up appointments. Coverage increased from 26% to 97% for flu vaccination, from 80% to 92% for pneumococcus immunization, from 27% to 100% for Transcranial Doppler (TCD) screening (P < 0.001). Emergency Department access/patient/year and inpatient admissions/patient/year decreased from 2.3 to 0.98 and from 0.30 to 0.25, respectively (P < 0.001). CONCLUSIONS: Comprehensive care can be delivered to vulnerable groups obtaining high adherence if linguistic, cultural, social issues are addressed. This model may merit assessment in other communities where immigrants represent the majority of patients.

mll ortholog containing functional domains of human MLL is expressed throughout the zebrafish lifespan and in haematopoietic tissues.

Infant leukaemia is an embryonal disease in which the underlying MLL translocations initiate in utero. Zebrafish offer unique potential to understand how MLL impacts haematopoiesis from the earliest embryonic timepoints and how translocations cause leukaemia as an embryonal process. In this study, a zebrafish mll cDNA syntenic to human MLL spanning the 5' to 3' UTRs, was cloned from embryos, and mll expression was characterized over the zebrafish lifespan. The protein encoded by the 35-exon ORF exhibited 46·4% overall identity to human MLL and 68-100% conservation in functional domains (AT-hooks, SNL, CXXC, PHD, bromodomain, FYRN, taspase1 sites, FYRC, SET). Maternally supplied transcripts were detected at 0-2 hpf. Strong ubiquitous early zygotic expression progressed to a cephalo-caudal gradient during later embryogenesis. mll was expressed in the intermediate cell mass (ICM) where primitive erythrocytes are produced and in the kidney where definitive haematopoiesis occurs in adults. mll exhibits high cross species conservation, is developmentally regulated in haematopoietic and other tissues and is expressed from the earliest embryonic timepoints throughout the zebrafish lifespan. Haematopoietic tissue expression validates using zebrafish for MLL haematopoiesis and leukaemia models.

Symmetrical alpha-bromoacryloylamido diaryldienone derivatives as a novel series of antiproliferative agents. Design, synthesis and biological evaluation.

In a continuing study of hybrid compounds containing the alpha-bromoacryloyl moiety as potential anticancer drugs, we synthesized a novel series of hybrids 4a-h, in which this moiety was linked to a 1,5-diaryl-1,4-pentadien-3-one system. Many of the conjugates prepared (4b, 4c, 4e and 4g) demonstrated pronounced, submicromolar antiproliferative activity against four cancer cell lines. Moreover, compound 4b induced apoptosis through the mitochondrial pathway and activated caspase-3 in a concentration-dependent manner.

Central role of mitochondria and p53 in PUVA-induced apoptosis in human keratinocytes cell line NCTC-2544.

Despite strong evidence concerning the high efficiency of PUVA therapy (psoralen plus UVA light), its mechanism of action has not yet been fully elucidated. In this study, we have evaluated in a cell line of human keratinocytes (NCTC-2544) the effects of two linear psoralen derivatives, 8-methoxypsoralen (8-MOP) and 5-methoxypsoralen (5-MOP), that are widely used in PUVA therapy and two angular derivatives, Angelicin (ANG) and 4,6,4'-trymetyl angelicin (TMA). All derivatives photoinduce cellular death, TMA being the most active compound. The cell cycle analysis showed that the four derivatives induce, 24 h after irradiation, a cell cycle arrest in G1 phase later followed by massive apoptosis. The G1 arrest is correlated to an increase in the expression of p21(Waf1/Cip1), a protein associated with the cell cycle block and apoptosis. Furthermore, treatment of NCTC-2544 resulted in p53 activation by 5-MOP, 8-MOP, and ANG but not TMA and its phosphorylation at serine-15. The levels of p21(Waf1/Cip1) paralleled p53 protein staining pattern suggesting that p53 activation correlated with p21(Waf1/Cip1) induction. Simultaneous to p53 activation, psoralens induced mitochondrial depolarization, cytochrome c release, mitochondrial production of reactive oxygen species, as well as caspase-3 and -9 activation. Thus these results strongly indicate the necessity of p53 activation and the induction of the apoptotic machinery downstream of mitochondria.

Differential response of linear and angular psoralens in PUVA-induced apoptosis in HL-60 cells.

Treatment of cells with UVA radiation in combination with linear psoralens induces a cell-cycle block and subsequent apoptosis, whereas angular derivatives produce apoptosis without affecting the cellular cycle.