EML4::ALK fusions in complex lymphatic malformations.

Gorham-Stout disease (GSD) and generalized lymphatic anomaly (GLA) are subtypes of complex lymphatic malformations (CLMs) with osseous involvement that cause significant complications, including pain and pathologic fractures. As with other vascular anomalies, somatic mosaic mutations in oncogenes are often present, and the mTOR inhibitor sirolimus alleviates symptoms in some, but not all, patients. We describe two patients, one with GSD and one with GLA, found to have EML4::ALK fusions. This report of a targetable, oncogenic fusion in vascular malformations expands our understanding of the genetic basis for CLMs and suggests additional targeted therapies could be effective.

De novo heterozygous variants in SLC30A7 are a candidate cause for Joubert syndrome.

Joubert syndrome (JS), a well-established ciliopathy, is characterized by the distinctive molar tooth sign on brain MRI, ataxia, and neurodevelopmental features. Other manifestations can include polydactyly, accessory frenula, renal, or liver disease. Here, we report individuals meeting criteria for JS with de novo heterozygous variants in SLC30A7 (Chr1p21.2). The first individual is a female with history of unilateral postaxial polydactyly, classic molar tooth sign on MRI, macrocephaly, ataxia, ocular motor apraxia, neurodevelopmental delay, and precocious puberty. Exome sequencing detected a de novo heterozygous missense variant in SLC30A7: NM_133496.5: c.407 T > C, (p.Val136Ala). The second individual had bilateral postaxial polydactyly, molar tooth sign, macrocephaly, developmental delay, and an extra oral frenulum. A de novo deletion-insertion variant in SLC30A7, c.490_491delinsAG (p.His164Ser) was found. Both de novo variants affect highly conserved residues. Variants were not identified in known Joubert genes for either case. SLC30A7 has not yet been associated with a human phenotype. The SLC30 family of zinc transporters, like SLC30A7, permit cellular efflux of zinc, and although it is expressed in the brain its functions remain unknown. Published data from proteomic studies support SLC30A7 interaction with TCTN3, another protein associated with JS. The potential involvement of such genes in primary cilia suggest a role in Sonic Hedgehog signaling. SLC30A7 is a candidate JS-associated gene. Future work could be directed toward further characterization of SLC30A7 variants and understanding its function.

Immunoassay for human serum erythroferrone.

Erythroferrone (ERFE) is a glycoprotein hormone secreted by erythroblasts in response to stimulation by erythropoietin (EPO). We previously demonstrated that ERFE messenger RNA expression and serum protein concentration increase in mice subjected to hemorrhage or EPO therapy, that ERFE acts on hepatocytes to suppress hepcidin, and that the resulting decrease in hepcidin augments iron delivery for intensified erythropoiesis. We also showed that ERFE contributes to pathological hepcidin suppression and iron overload in mice with nontransfused ß-thalassemia. We now report the development and technical validation of a rabbit monoclonal antibody-based sandwich immunoassay for human ERFE. We use this assay to show that blood loss or EPO administration increases serum ERFE concentrations in humans, and that patients with both nontransfused and transfused ß-thalassemia have very high serum ERFE levels, which decrease after blood transfusion. The assay should be useful for human studies of normal and disordered erythropoiesis and its effect on iron homeostasis.

Mechanisms of plasma non-transferrin bound iron generation: insights from comparing transfused diamond blackfan anaemia with sickle cell and thalassaemia patients.

In transfusional iron overload, extra-hepatic iron distribution differs, depending on the underlying condition. Relative mechanisms of plasma non-transferrin bound iron (NTBI) generation may account for these differences. Markers of iron metabolism (plasma NTBI, labile iron, hepcidin, transferrin, monocyte SLC40A1 [ferroportin]), erythropoiesis (growth differentiation factor 15, soluble transferrin receptor) and tissue hypoxia (erythropoietin) were compared in patients with Thalassaemia Major (TM), Sickle Cell Disease and Diamond-Blackfan Anaemia (DBA), with matched transfusion histories. The most striking differences between these conditions were relationships of NTBI to erythropoietic markers, leading us to propose three mechanisms of NTBI generation: iron overload (all), ineffective erythropoiesis (predominantly TM) and low transferrin-iron utilization (DBA).

Increased leucocyte apoptosis in transfused ß-thalassaemia patients.

This exploratory study assessed apoptosis in peripheral blood leucocytes (PBL) from ß-thalassaemia patients receiving chronic transfusions and chelation therapy (deferasirox or deferoxamine) at baseline, 1, 6, and 12 months. At baseline, thalassaemic PBLs presented 50% greater levels of Bax (BAX), 75% higher caspase-3/7, 48% higher caspase-8 and 88% higher caspase-9 activities and 428% more nucleosomal DNA fragmentation than control subjects. Only neutrophils correlated significantly with apoptotic markers. Previously, we showed that over the treatment year, hepatic iron declined; we now show that the ratio of Bax/Bcl-2 (BCL2), (-27·3%/year), and caspase-9 activity (-13·3%/year) declined in both treatment groups, suggesting that chelation decreases body iron and indicators of PBL apoptosis.

The effects of glutamine supplementation on markers of apoptosis and autophagy in sickle cell disease peripheral blood mononuclear cells.

OBJECTIVES: L-Glutamine was FDA-approved for sickle cell disease (SCD) in 2017, yet the mechanism(s)-of-action are poorly understood. This study investigates the potential activation of autophagy as a previously unexplored mechanism-of-benefit. DESIGN: Prospective, open-label, 8-week, phase-2 trial of oral L-glutamine (10 g TID) in patients with SCD at risk for pulmonary hypertension identified by Doppler-echocardiography by an elevated tricuspid-regurgitant-jet-velocity (TRV)≥ 2.5 m/s. Peripheral blood mononuclear cells (PBMCs) were isolated from blood samples taken from SCD patients at baseline, two, four, six and eight weeks of glutamine therapy, and from controls at baseline; BAX (pro-apoptotic marker) and LC3-II/LC3-I (autophagy marker) were measured via western blot analysis to assess apoptosis and autophagy respectively. SETTING: Comprehensive SCD Center in Oakland, California. RESULTS: Patients with SCD (n = 8) had a mean age of 44 ± 16, 50% were male; 63% Hb-SS, and mean TRV= 3.1 ± 0.7 m/s. Controls' mean age (n = 5) was 32 ± 12% and 57% were male; all were Hb-AA with a mean TRV= 1.8 ± 0.6. At baseline, SCD-PBMCs had 2-times higher levels of BAX and LC3-I versus controls (both p = 0.03). Levels of BAX expression increased by 300% after 8-weeks of glutamine supplementation (p = 0.005); LC3-I protein levels decreased while LC3-II levels increased by 70%, giving a significant increase in the LC3-II/LC3-I ratio (p = 0.02). CONCLUSION: PBMCs from glutamine-supplemented SCD patients have upregulated apoptotic and autophagy proteins. The parallel increase in BAX and the LC3-II / LC3-I ratio with glutamine supplementation suggest a possible role of autophagic cell death. The increase in apoptotic markers provide insight into a possible mechanism used by peripheral PBMCs during glutamine supplementation in patients with SCD.

Structural basis for the allosteric inhibition of UMP kinase from Gram-positive bacteria, a promising antibacterial target.

Tuberculosis claims significantly more than one million lives each year. A feasible way to face the issue of drug resistance is the development of new antibiotics. Bacterial uridine 5'-monophosphate (UMP) kinase is a promising target for novel antibiotic discovery as it is essential for bacterial survival and has no counterpart in human cells. The UMP kinase from M. tuberculosis is also a model of particular interest for allosteric regulation with two effectors, GTP (positive) and UTP (negative). In this study, using X-ray crystallography and cryo-electron microscopy, we report for the first time a detailed description of the negative effector UTP-binding site of a typical Gram-positive behaving UMP kinase. Comparison between this snapshot of low affinity for Mg-ATP with our previous 3D-structure of the GTP-bound complex of high affinity for Mg-ATP led to a better understanding of the cooperative mechanism and the allosteric regulation of UMP kinase. Thermal shift assay and circular dichroism experiments corroborate our model of an inhibition by UTP linked to higher flexibility of the Mg-ATP-binding domain. These new structural insights provide valuable knowledge for future drug discovery strategies targeting bacterial UMP kinases.

Prevalence and factors associated with chronic use of levothyroxine: A cohort study.

IMPORTANCE: Levothyroxine prescriptions are rising worldwide. However, there are few data on factors associated with chronic use. OBJECTIVE: To assess the prevalence of chronic levothyroxine use, its rank among other chronic drugs and factors associated with chronic use. To assess the proportion of users outside the therapeutic range of thyroid-stimulating hormone (TSH). DESIGN: Cohort study (CoLaus|PsyCoLaus) with recruitment from 2003 to 2006. Follow-ups occurred 5 and 10 years after baseline. PARTICIPANTS: A random sample of Lausanne (Switzerland) inhabitants aged 35-75 years. MAIN OUTCOMES: We evaluated the prevalence of chronic levothyroxine use and we then ranked it among the other most used chronic drugs. The ranking was compared to data from health insurance across the country. We assessed the association between each factor and chronic levothyroxine use in multivariable logistic regression models. The proportion of chronic levothyroxine users outside the usual TSH therapeutic range was assessed. RESULTS: 4,334 participants were included in the analysis (mean±SD age 62.8±10.4 years, 54.9% women). 166 (3.8%) participants were chronic levothyroxine users. Levothyroxine was the second most prescribed chronic drug after aspirin in the cohort (8.2%) and the third most prescribed when using Swiss-wide insurance data. In multivariable analysis, chronic levothyroxine use was associated with increasing age [odds ratio 1.03, 95% confidence interval 1.01-1.05 per 1-year increase]; female sex [11.87 (5.24-26.89)]; BMI [1.06 (1.02-1.09) per 1-kg/m2 increase]; number of concomitant drugs [1.22 (1.16-1.29) per 1-drug increase]; and family history of thyroid pathologies [2.18 (1.37-3.48)]. Among chronic levothyroxine users with thyroid hormones assessment (n = 157), 42 (27%) were outside the TSH therapeutic range (17% overtreated and 10% undertreated). CONCLUSIONS: In this population-based study, levothyroxine ranked second among chronic drugs. Age, female sex, BMI, number of drugs and family history of thyroid pathologies were associated with chronic levothyroxine use. More than one in four chronic users were over- or undertreated.

Loss of tyrosine hydroxylase, motor deficits and elevated iron in a mouse model of phospholipase A2G6-associated neurodegeneration (PLAN).

Phospholipase A2G6-associated neurodegeneration (PLAN) is a rare early-onset monogenic neurodegenerative movement disorder which targets the basal ganglia and other regions in the central and peripheral nervous system; presenting as a series of heterogenous subtypes in patients. We describe here a B6.C3-Pla2g6m1J/CxRwb mouse model of PLAN which presents with early-onset neurodegeneration at 90 days which is analogous of the disease progression that is observed in PLAN patients. Homozygous mice had a progressively worsening motor deficit, which presented as tremors starting at 65 days and progressed to severe motor dysfunction and increased falls on the wire hang test at 90 days. This motor deficit positively correlated with a reduction in tyrosine hydroxylase (TH) protein expression in dopaminergic neurons of the substantia nigra (SN) without any neuronal loss. Fluorescence imaging of Thy1-YFP revealed spheroid formation in the SN. The spheroids in homozygous mice strongly mirrors those observed in patients and were demonstrated to correlate strongly with the motor deficits as measured by the wire hang test. The appearance of spheroids preceded TH loss and increased spheroid numbers negatively correlated with TH expression. Perls/DAB staining revealed the presence of iron accumulation within the SN of mice. This mouse model captures many of the major hallmarks of PLAN including severe-early onset neurodegeneration, a motor deficit that correlates directly to TH levels, spheroid formation and iron accumulation within the basal ganglia. Thus, this mouse line is a useful tool for further research efforts to improve understanding of how these disease mechanisms give rise to the disease presentations seen in PLAN patients as well as to test novel therapies.

Targeted Therapy and Checkpoint Immunotherapy in Lung Cancer.

Lung cancer is the leading cause of cancer mortality. It is classified into different histologic subtypes, including adenocarcinoma, squamous carcinoma, and large cell carcinoma (commonly referred as non-small cell lung cancer) and small cell lung cancer. Comprehensive molecular characterization of lung cancer has expanded our understanding of the cellular origins and molecular pathways affected in each of these subtypes. Many of these genetic alterations represent potential therapeutic targets for which drugs are constantly under development. This article discusses the molecular characteristics of the main lung cancer subtypes and discusses the current guidelines and novel targeted therapies, including checkpoint immunotherapy.

A High-Throughput System for Cyclic Stretching of Precision-Cut Lung Slices During Acute Cigarette Smoke Extract Exposure.

RATIONALE: Precision-cut lung slices (PCLSs) are a valuable tool in studying tissue responses to an acute exposure; however, cyclic stretching may be necessary to recapitulate physiologic, tidal breathing conditions. OBJECTIVES: To develop a multi-well stretcher and characterize the PCLS response following acute exposure to cigarette smoke extract (CSE). METHODS: A 12-well stretching device was designed, built, and calibrated. PCLS were obtained from male Sprague-Dawley rats (N = 10) and assigned to one of three groups: 0% (unstretched), 5% peak-to-peak amplitude (low-stretch), and 5% peak-to-peak amplitude superimposed on 10% static stretch (high-stretch). Lung slices were cyclically stretched for 12 h with or without CSE in the media. Levels of Interleukin-1ß (IL-1ß), matrix metalloproteinase (MMP)-1 and its tissue inhibitor (TIMP1), and membrane type-MMP (MT1-MMP) were assessed via western blot from tissue homogenate. RESULTS: The stretcher system produced nearly identical normal Lagrangian strains (E xx and E yy , p > 0.999) with negligible shear strain (E xy < 0.0005) and low intra-well variability 0.127 ± 0.073%. CSE dose response curve was well characterized by a four-parameter logistic model (R 2 = 0.893), yielding an IC50 value of 0.018 cig/mL. Cyclic stretching for 12 h did not decrease PCLS viability. Two-way ANOVA detected a significant interaction between CSE and stretch pattern for IL-1ß (p = 0.017), MMP-1, TIMP1, and MT1-MMP (p < 0.001). CONCLUSION: This platform is capable of high-throughput testing of an acute exposure under tightly-regulated, cyclic stretching conditions. We conclude that the acute mechano-inflammatory response to CSE exhibits complex, stretch-dependence in the PCLS.

Iron metabolism and iron chelation in sickle cell disease.

This review highlights recent advances in iron metabolism that are relevant to sickle cell disease (SCD). SCD is a common hemoglobinopathy that results in chronic inflammation. Improved understanding of how iron metabolism is controlled by proteins such as hepcidin, ferroportin, hypoxia-inducible factor 1, and growth differentiation factor 15 have revealed how they are involved in the organ toxicity of SCD. SCD patients have lower levels of non-transferrin-bound iron (NTBI) relative to other hemoglobinopathies, such as thalassemia. Care for SCD now commonly uses transfusion that results in iron overload and necessitates the need for chelation. New oral chelation therapy using deferasirox (Exjade/ICL670) appears to be safe and may even lower the amount of toxic free NTBI and enhance patient compliance. Finally, we suggest that iron metabolism and trafficking is different in SCD compared to other hemoglobinopathies. The high levels of inflammatory cytokines in SCD may enhance macrophage/reticuloendothelial cell iron and/or renal cell iron retention. This makes the tissues that retain iron different in SCD, and thus the organs that fail in SCD are different from those of other hemoglobinopathies, such as the cardiomyopathy or endocrinopathies of thalassemia.

Oligodendrocyte Death in Pelizaeus-Merzbacher Disease Is Rescued by Iron Chelation.

Pelizaeus-Merzbacher disease (PMD) is an X-linked leukodystrophy caused by mutations in Proteolipid Protein 1 (PLP1), encoding a major myelin protein, resulting in profound developmental delay and early lethality. Previous work showed involvement of unfolded protein response (UPR) and endoplasmic reticulum (ER) stress pathways, but poor PLP1 genotype-phenotype associations suggest additional pathogenetic mechanisms. Using induced pluripotent stem cell (iPSC) and gene-correction, we show that patient-derived oligodendrocytes can develop to the pre-myelinating stage, but subsequently undergo cell death. Mutant oligodendrocytes demonstrated key hallmarks of ferroptosis including lipid peroxidation, abnormal iron metabolism, and hypersensitivity to free iron. Iron chelation rescued mutant oligodendrocyte apoptosis, survival, and differentiationin vitro, and post-transplantation in vivo. Finally, systemic treatment of Plp1 mutant Jimpy mice with deferiprone, a small molecule iron chelator, reduced oligodendrocyte apoptosis and enabled myelin formation. Thus, oligodendrocyte iron-induced cell death and myelination is rescued by iron chelation in PMD pre-clinical models.

Inflammation and oxidant-stress in beta-thalassemia patients treated with iron chelators deferasirox (ICL670) or deferoxamine: an ancillary study of the Novartis CICL670A0107 trial.

BACKGROUND: We assessed whether oxidant-stress and inflammation in beta-thalassemia could be controlled by the novel oral iron chelator deferasirox as effectively as by deferoxamine. DESIGN AND METHODS: Forty-nine subjects were enrolled from seven sites and studied at baseline, and after 1, 6, and 12 months of therapy. Malondialdehyde, protein carbonyls, vitamins E and C, total non-transferrin bound iron, transferrin saturation, C-reactive protein, cytokines, serum ferritin concentration and liver iron concentration were measured. RESULTS: Liver iron concentration and ferritin declined significantly in both treatment groups during the study. This paralleled a significant decline in the oxidative-stress marker malondialdehyde (deferasirox -22%/year, deferoxamine -28%/year, average decline p=0.006). The rates of decline did not differ between treatment groups. Malondialdehyde was higher in both treatment groups than in a group of 30 non-thalassemic controls (p < 0.001). The inflammatory marker high-sensitivity C-reactive protein decreased significantly only in the group receiving deferasirox (deferasirox -51%/year, deferoxamine +8.5%/year, p = 0.02). This result was confounded by a chance difference in the level of high-sensitivity C-reactive protein between the two groups at baseline, but analyses controlling for this difference suggested an equally large treatment effect. CONCLUSIONS: Iron chelation therapy with deferoxamine or with deferasirox was equally effective in decreasing iron burden and malondialdehyde. The possible differential effect of the two chelators on inflammation warrants further investigation.

Daily supplementation with iron increases lipid peroxidation in young women with low iron stores.

The aim of this study was to determine whether women with low iron stores (plasma ferritin

Protection against aflatoxin-B1-induced liver mutagenesis by Scutellaria baicalensis.

We have measured the inhibition of the mutagenicity of the mycotoxin aflatoxin-B(1) in the liver of the rat by plant material of Scutellaria baicalensis, or Huang-qin. The addition of one percent dried Huang-qin to the feed of the animals reduced the mutant frequency of a subsequent administration of aflatoxin-B1 by approximately 60 and 77%, respectively, for two different batches of the plant material. The addition of Huang-qin also increased the expression of the gene for glutathione S-transferase A5 subunit by 2.5-3.0-fold, and decreased expression of P450 cytochrome 3A2 by 1.8-2.0-fold. The greater increase of the expression of the GST gene may result in the protection shown by Huang-qin. The sensitivity of the hepatic mitochondria to swelling, a measure of the mitochondrial permeability transition, is increased significantly in animals that are on a diet containing Huang-qin. This may lead to increased sensitivity to apoptosis on treatment with toxic compounds. The two batches of Huang-qin material show differences in both chemical composition and preventive potential. This study demonstrates how a combination of generating and analysis of plant varieties together with a mammalian assay for efficacy may improve the search for better plant-based prevention of cancer initiation.

Oxidative stress and inflammation in iron-overloaded patients with beta-thalassaemia or sickle cell disease.

Blood transfusion therapy is life-saving for patients with beta-thalassaemia and sickle cell disease (SCD), but often results in severe iron overload. This pilot study examined whether the biomarkers of tissue injury or inflammation differ in these two diseases. Plasma malondialdehyde (MDA) was significantly increased 1.8-fold in thalassaemia relative to control patients. In contrast, MDA in SCD was not significantly different from controls. In multivariate analysis, the strongest predictors of elevated MDA were liver iron concentration (P < 0.001) and specific diagnosis (P = 0.019). A significant 2-fold elevation of non-transferrin bound iron (NTBI) was observed in thalassaemia relative to SCD. NTBI was not a significant predictor of high MDA in multivariate analysis. SCD patients showed a significant 2.2-fold elevation of the inflammatory marker interleukin (IL)-6 relative to controls, and a 3.6- and 1.7-fold increase in IL-5 and IL-10 relative to thalassaemia. Although alpha-tocopherol was significantly decreased by at least 32% in both thalassaemia and SCD, indicating ongoing oxidant stress and antioxidant consumption, gamma-tocopherol, a nitric oxide-selective antioxidant, was increased 36% in SCD relative to thalassaemia. These results demonstrate that thalassaemia patients have increased MDA and circulating NTBI relative to SCD patients and lower levels of some cytokines and gamma-tocopherol. This supports the hypothesis that the biology of SCD may show increased inflammation and increased levels of protective antioxidants compared with thalassaemia.

The role of heme and iron-sulfur clusters in mitochondrial biogenesis, maintenance, and decay with age.

Mitochondria decay with age from oxidative damage and loss of protective mechanisms. Resistance, repair, and replacement mechanisms are essential for mitochondrial preservation and maintenance. Iron plays an essential role in the maintenance of mitochondria, through its two major functional forms: heme and iron-sulfur clusters. Both iron-based cofactors are formed and utilized in the mitochondria and then distributed throughout the cell. This is an important function of mitochondria that is not directly related to the production of ATP. Heme and iron-sulfur clusters are important for the normal assembly and for the optimal activity of the electron transfer complexes. Loss of mitochondrial cytochrome c oxidase (complex IV), integrity of mtDNA, and function can result from abnormal homeostasis of iron. We review the physiological role of iron-sulfur clusters and heme in the integrity of the mitochondria and the generation of oxidants.

Fe(2+) induces a transient Ca(2+) release from rat liver mitochondria.

Isolated mitochondria loaded with Ca(2+) and then exposed to Fe(2+) show a transient release of Ca(2+). The magnitude of this response depends on the Ca(2+) loading and the kinetics of the response depends on the concentration of added Fe(2+). We investigated the Fe(2+)-induced Ca(2+) release mechanism by measuring mitochondrial Ca(2+) uptake in the presence of Fe(2+). The presence of Fe(2+) inhibits Ca(2+) uptake two times. Since mitochondria can cycle Ca(2+) across their inner membrane, the suppression of Ca(2+) uptake, but not release, results in an elevation of the extramitochondrial Ca(2+), thereby varying the steady state. The transient release of Ca(2+) initially observed from mitochondria appears to occur via the electroneutral 2H(+)/Ca(2+)-exchange mechanism, since it can be markedly decreased by cyclosporin A and does not involve lipid peroxidation. When Fe(2+) accumulation is completed, reuptake of released Ca(2+) into mitochondria resumes. Finally, we propose that Fe(2+) either inhibits Ca(2+) entry at the uniporter or is transported by it into the matrix.

The role of Fe2+-induced lipid peroxidation in the initiation of the mitochondrial permeability transition.

Iron and iron complexes stimulate lipid peroxidation and formation of malondialdehyde (MDA). We have studied the effects of Fe2+ and ascorbate on mitochondrial permeability transition induced by phosphate and Ca2+. Iron is necessary for detectable MDA formation, but only Ca2+ and phosphate are necessary for the induction of membrane potential loss (Deltapsi) and Ca2+ release. Keeping the iron at a constant concentration and varying the Ca2+ level changed the mitochondrial Ca2+ retention times, but not the amount of MDA formation. The antioxidant butylated hydroxytoluene at low concentrations prevented MDA formation, but not mitochondrial Ca2+ release. Preincubation of mitochondria with Fe2+ decreased Ca2+ retention time in a concentration-dependent manner and facilitated Ca2+-stimulated MDA accumulation. Thus, Ca2+ phosphate-induced mitochondrial permeability transition (MPT) can be separated mechanistically from MDA accumulation. Lipid peroxidation products do not appear to participate in the initial phase of the permeability transition, but sensitize mitochondria toward MPT.