Diffusion-weighted imaging of normal appearing corticospinal tracts in patients with multiple sclerosis.

Aim To determine if there are differences in ADC values between normal appearing corticospinal tracks in patients with MS compared to ADC values in controls. Material and methods The study population comprised 62 consecutive MS patients (36 women and 26 men; mean age 36.45±8.63 years). 50 control subjects with no neurological disabilities or intracranial were included the study (32 women and 18 men; mean age 40.18±12.25 years). All ADC maps were independently evaluated by two experienced radiologists. ROI of approximately 15-18 mm2 in capsula interna and 10-12 mm2 in mesencephalon were placed bilaterally for measurement of ADC values. Three circular ROIs were placed-one each side for internal capsule-and 6 total ROIs from right and left internal capsule were averaged for each patient. Mesencephelon ADC measurements were performed similarly. Result The mean ADC values of the left internal capsule in MS patients were significiantly lower than the control group (p:0.002). No statistically significant difference was found between the MS patients and control group mean ADC values of the right internal capsule (p>0.05). The mean ADC values of the right and left mesencephalon in MS patients were significiantly lower than the control group (respectively; p:0.031, p<0.001). The mean ADC values of the left internal capsule were significiantly lower than the right internal capsule in MS patients (p<0.001). The mean ADC values of the left mesencephalon were significiantly lower than the right mesencephalon in MS patients (p<0.001). Conclusion The mean ADC values of the normal-appearing corticospinal tract in MS patients were significantly lower than the control group except for the right internal capsule.

Efficacy of Computed Tomography (CT) Attenuation Values and CT Findings in the Differentiation of Pleural Effusion.

BACKGROUND: The aim of this study was to investigate the efficacy of computed tomography (CT) findings for characterizing pleural effusions with the use of attenuation values. MATERIAL/METHODS: One hundred and twenty eight patients with pleural effusions on thoracic CT who underwent thoracentesisis within two weeks were studied. Pleural effusions were classified as exudates or transudates according to the Light's criteria. A region of interest was placed for the measurement of Hounsfield Unit (HU) values in the area of the greatest amount of effusion on each slice of the three slices used. CT features that were evaluated for distinguishing pleural exudates from transudates included pleural nodules, pleural thickening and loculation. RESULTS: Thirty three (26%) of the 128 pleural effusions were transudates and 95 (74%) were exudates. The mean HU values of the exudates (8.82±7.04) were significantly higher than those of the transudates (2.91±8.53), (p<0.001). No statistically significant difference was found between transudate and exudate patients in terms of pleural thickness, pleural nodules and loculation (p>0.05). CONCLUSIONS: HU values can help in differentiating exudative pleural effusions from transudative pleural effusions. Because of overlapping HU values, correlation with clinical findings is essential.

The relationship of breast arterial calcification detected in mammographic examinations with cardiovascular diseases, cardiovascular risk factors, parity, and breastfeeding.

BACKGROUND/AIM: We aimed to detect the incidence of breast arterial calcification (BAC) in patients that underwent mammography and to reveal the relationship of BAC with cardiovascular diseases, cardiovascular risk factors, parity, and breastfeeding. MATERIALS AND METHODS: A total of 1195 female patients were included in this study. Cases that were positive for BAC during mammography were recorded. The relationship of BAC with age, body mass index, parity, breastfeeding, menopause, smoking, alcohol consumption, oral contraceptive use, hormone replacement therapy, and histories of hypertension (HT), diabetes mellitus (DM), coronary artery disease (CAD), and cerebral vascular diseases were investigated. RESULTS: Overall, 97 of 1195 cases were positive for BAC. In univariate analysis, age, educational status, parity, breastfeeding, menopause, hyperlipidemia, and DM, HT, and CAD histories were found to be separate risk factors that had an effect on the development of BAC. The effects of age, parity, and breastfeeding history were maintained in the logistic regression analysis (P = 0.001, P = 0.001, P = 0.024, respectively; P < 0.05 was significant), while the significance of the other analyzed variables was lost (P > 0.05). CONCLUSION: We found that BAC is associated with age, parity, and breastfeeding but not with cardiovascular diseases and cardiovascular risk factors.

The relationship of ADC values of renal parenchyma with CKD stage and serum creatinine levels.

PURPOSE: To evaluate the relationship of apparent diffusion coefficient (ADC) values of renal parenchyma with chronic kidney disease (CKD) stage and serum creatinine levels. MATERIALS AND METHODS: One hundred and ten patients who had undergone magnetic resonance imaging of the upper abdomen for different reasons were retrospectively studied. A region of interest (ROI) was placed on the renal parenchyma for measurement of ADC values of both kidneys, without any preference for cortex or medulla. Three circular ROIs were placed-one each in the upper pole, interpolar region and lower pole of both kidneys. The mean ADC values were recorded for each patient and the relationship between ADC values and stage of CKD and serum creatinine levels were evaluated. RESULTS: Statistically significant difference was determined between the ADC values of the cases according to CKD stages (p < 0.001). Paired comparisons performed to determine the group that caused the difference revealed that median ADC values of healthy subjects who formed the control group was statistically significantly higher than that of the cases with stage 3, stage 4 and stage 5 CKD (p: 0.008; p: 0.008; and p: 0.002, respectively). Sensitivity and specificity were found to be 75.44% and 69.81%, respectively in detecting stage 3, stage 4 and stage 5 CKD among the cases with ADC values of 1151 and lower. CONCLUSION: ADC values can play a role in the evaluation of renal dysfunction. However, population-and protocol-based cut-off ADC values are needed to identify renal dysfunction and to distinguish between different stages of CKD.

Evidence for AKT-independent regulation of FOXO1 and FOXO3 in haematopoietic stem and progenitor cells.

Transcription factors FOXOs (1, 3, 4) are essential for the maintenance of haematopoietic stem cells. FOXOs are evolutionary conserved substrates of the AKT serine threonine protein kinase that are also phosphorylated by several kinases other than AKT. Specifically, phosphorylation by AKT is known to result in the cytosolic localization of FOXO and subsequent inhibition of FOXO transcriptional activity. In addition to phosphorylation, FOXOs are regulated by a number of other post-translational modifications including acetylation, methylation, redox modulation, and ubiquitination that altogether determine these factors' output. Cumulating evidence raises the possibility that in stem cells, including in haematopoietic stem cells, AKT may not be the dominant regulator of FOXO. To address this question in more detail, we examined gene expression, subcellular localization, and response to AKT inhibition of FOXO1 and FOXO3, the main FOXO expressed in HSPCs (haematopoietic stem and progenitor cells). Here we show that while FOXO1 and FOXO3 transcripts are expressed at similar levels, endogenous FOXO3 protein is mostly nuclear compared to the cytoplasmic localization of FOXO1 in HSPCs. Furthermore, inhibition of AKT does not enhance nuclear localization of FOXO1 nor FOXO3. Nonetheless AKT inhibition in the context of loss of NAD-dependent SIRT1 deacetylase modulates FOXO3 localization in HSPCs. Together, these data suggest that FOXO3 is more active than FOXO1 in primitive haematopoietic stem and multipotent progenitor cells. In addition, they indicate that upstream regulators other than AKT, such as SIRT1, maintain nuclear FOXO localization and activity in HSPCs.

Myeloid Dysregulation in a Human Induced Pluripotent Stem Cell Model of PTPN11-Associated Juvenile Myelomonocytic Leukemia.

Somatic PTPN11 mutations cause juvenile myelomonocytic leukemia (JMML). Germline PTPN11 defects cause Noonan syndrome (NS), and specific inherited mutations cause NS/JMML. Here, we report that hematopoietic cells differentiated from human induced pluripotent stem cells (hiPSCs) harboring NS/JMML-causing PTPN11 mutations recapitulated JMML features. hiPSC-derived NS/JMML myeloid cells exhibited increased signaling through STAT5 and upregulation of miR-223 and miR-15a. Similarly, miR-223 and miR-15a were upregulated in 11/19 JMML bone marrow mononuclear cells harboring PTPN11 mutations, but not those without PTPN11 defects. Reducing miR-223's function in NS/JMML hiPSCs normalized myelogenesis. MicroRNA target gene expression levels were reduced in hiPSC-derived myeloid cells as well as in JMML cells with PTPN11 mutations. Thus, studying an inherited human cancer syndrome with hiPSCs illuminated early oncogenesis prior to the accumulation of secondary genomic alterations, enabling us to discover microRNA dysregulation, establishing a genotype-phenotype association for JMML and providing therapeutic targets.

FOXO3-mTOR metabolic cooperation in the regulation of erythroid cell maturation and homeostasis.

Ineffective erythropoiesis is observed in many erythroid disorders including ß-thalassemia and anemia of chronic disease in which increased production of erythroblasts that fail to mature exacerbate the underlying anemias. As loss of the transcription factor FOXO3 results in erythroblast abnormalities similar to the ones observed in ineffective erythropoiesis, we investigated the underlying mechanisms of the defective Foxo3(-/-) erythroblast cell cycle and maturation. Here we show that loss of Foxo3 results in overactivation of the JAK2/AKT/mTOR signaling pathway in primary bone marrow erythroblasts partly mediated by redox modulation. We further show that hyperactivation of mTOR signaling interferes with cell cycle progression in Foxo3 mutant erythroblasts. Importantly, inhibition of mTOR signaling, in vivo or in vitro enhances significantly Foxo3 mutant erythroid cell maturation. Similarly, in vivo inhibition of mTOR remarkably improves erythroid cell maturation and anemia in a model of ß-thalassemia. Finally we show that FOXO3 and mTOR are likely part of a larger metabolic network in erythroblasts as together they control the expression of an array of metabolic genes some of which are implicated in erythroid disorders. These combined findings indicate that a metabolism-mediated regulatory network centered by FOXO3 and mTOR control the balanced production and maturation of erythroid cells. They also highlight physiological interactions between these proteins in regulating erythroblast energy. Our results indicate that alteration in the function of this network might be implicated in the pathogenesis of ineffective erythropoiesis.

FOXO1 is an essential regulator of pluripotency in human embryonic stem cells.

Pluripotency of embryonic stem cells (ESCs) is defined by their ability to differentiate into three germ layers and derivative cell types and is established by an interactive network of proteins including OCT4 (also known as POU5F1; ref. 4), NANOG (refs 5, 6), SOX2 (ref. 7) and their binding partners. The forkhead box O (FoxO) transcription factors are evolutionarily conserved regulators of longevity and stress response whose function is inhibited by AKT protein kinase. FoxO proteins are required for the maintenance of somatic and cancer stem cells; however, their function in ESCs is unknown. We show that FOXO1 is essential for the maintenance of human ESC pluripotency, and that an orthologue of FOXO1 (Foxo1) exerts a similar function in mouse ESCs. This function is probably mediated through direct control by FOXO1 of OCT4 and SOX2 gene expression through occupation and activation of their respective promoters. Finally, AKT is not the predominant regulator of FOXO1 in human ESCs. Together these results indicate that FOXO1 is a component of the circuitry of human ESC pluripotency. These findings have critical implications for stem cell biology, development, longevity and reprogramming, with potentially important ramifications for therapy.

Regulation and function of FoxO transcription factors in normal and cancer stem cells: what have we learned?

Forkhead FoxO transcription factors exert critical biological functions in response to genotoxic stress. In mammals four FoxOs proteins are known. FoxOs induce cell cycle arrest, repair damaged DNA, or initiate apoptosis by modulating genes that control these processes. In particular, FoxO proteins are critical regulators of oxidative stress by modulating the expression of several anti-oxidant enzyme genes. This function of FoxO is essential for the regulation of stem and progenitor cell pool in the hematopoietic system and possibly other stem cells. Overall functions of FoxOs are consistent with their role as tumor suppressors as has been shown in animal models. As such, FoxOs are suppressed in various cancer cells. However, recent reports strongly suggest that FoxOs are critical for the maintenance of leukemic stem cells. The diverse functions of FoxOs are orchestrated by tight regulations of expression and activity of its family members. Here we discuss the recent progress in understanding the function of FoxOs specifically in normal and cancer stem cells and what is known about the regulation of these proteins in various cell types and tissues including in the physiological setting of primary cells in vivo. These studies underscore the importance of regulation of FoxO proteins and whether these factors play distinct or redundant functions. Understanding how FoxOs are modulated is critical for devising novel therapies based on targeted restoration/or inhibition of FoxO function in cancer and in other diseased cells in which FoxOs have a key function.

ROS-mediated amplification of AKT/mTOR signalling pathway leads to myeloproliferative syndrome in Foxo3(-/-) mice.

Reactive oxygen species (ROS) participate in normal intracellular signalling and in many diseases including cancer and aging, although the associated mechanisms are not fully understood. Forkhead Box O (FoxO) 3 transcription factor regulates levels of ROS concentrations, and is essential for maintenance of hematopoietic stem cells. Here, we show that loss of Foxo3 causes a myeloproliferative syndrome with splenomegaly and increased hematopoietic progenitors (HPs) that are hypersensitive to cytokines. These mutant HPs contain increased ROS, overactive intracellular signalling through the AKT/mammalian target of rapamycin signalling pathway and relative deficiency of Lnk, a negative regulator of cytokine receptor signalling. In vivo treatment with ROS scavenger N-acetyl-cysteine corrects these biochemical abnormalities and relieves the myeloproliferation. Moreover, enforced expression of Lnk by retroviral transfer corrects the abnormal expansion of Foxo3(-/-) HPs in vivo. Our combined results show that loss of Foxo3 causes increased ROS accumulation in HPs. In turn, this inhibits Lnk expression that contributes to exaggerated cytokine responses that lead to myeloproliferation. Our findings could explain the mechanisms by which mutations that alter Foxo3 function induce malignancy. More generally, the work illustrates how deregulated ROS may contribute to malignant progression.

Early embryonic lethality in gene trap mice with disruption of the Arfgef2 gene.

The switching of ADP-ribosylation factors from the inactive form to the active form is catalyzed by ARF-GEF (ADP ribosylation factor--guanine nucleotide exchange protein) proteins containing a Sec7 domain. The murine Arfgef2 gene encoding the BIG2 protein belongs to the class of high molecular mass (>100 kDa) ARF-GEF proteins. BIG2 is believed to be associated with the trans-Golgi network and the recycling endosomes. In humans, mutations in the ARFGEF2 gene cause autosomal recessive periventricular heterotopia with microcephaly. To elucidate the function of BIG2 in mouse we studied a gene-trap mouse line with a functional disruption of the Arfgef2 gene. Heterozygous mutants did not reveal phenotypic abnormalities and were fertile. However, no homozygous embryos were obtained from breeding heterozygous females and males. To explore the reason for embryonic lethality, we analysed the pattern of expression of Arfgef2. Arfgef2 transcripts were detected in several adult tissues. Interestingly, Arfgef2 undergoes alternative splicing and the splicing pattern differs among tissues from adult animals. Moreover, the LacZ reporter gene of the gene-trap construct was used to reveal the expression of Arfgef2 during embryonic development. Here, we show that Arfgef2 mRNA is stored in the oocyte and is likely translated during the first embryonic divisions. SNP (Single Nucleotide Polymorphism) markers were used to demonstrate that the embryonic Arfgef2 gene is activated first at the 4-cell stage, suggesting an important role for embryonic development. This assumption is supported by the failure of Arfgef2-deficient oocytes fertilized with Arfgef2-deficient sperm to develop into 4-cell stage embryos. Our results indicate that murine BIG2 is essential for early embryonic development.

Stra13 regulates oxidative stress mediated skeletal muscle degeneration.

Duchenne Muscular Dystrophy (DMD), caused by loss of dystrophin is characterized by progressive muscle cell necrosis. However, the mechanisms leading to muscle degeneration in DMD are poorly understood. Here, we demonstrate that Stra13 protects muscle cells from oxidative damage, and its absence leads to muscle necrosis in response to injury in Stra13-deficient mice. Interestingly, Stra13-/- mutants express elevated levels of TNFalpha, reduced levels of heme-oxygenase-1, and display apparent signs of oxidative stress prior to muscle death. Moreover, Stra13-/- muscle cells exhibit an increased sensitivity to pro-oxidants, and conversely, Stra13 overexpression provides resistance to oxidative damage. Consistently, treatment with anti-oxidant N-acetylcysteine ameliorates muscle necrosis in Stra13-/- mice. We also demonstrate that Stra13 expression is elevated in muscles from dystrophin-deficient (mdx) mice, and mdx/Stra13-/- double mutants exhibit an early onset of muscle degeneration. Our studies underscore the importance of oxidative stress-mediated muscle degeneration in muscular dystrophy, and reveal the contribution of Stra13 in maintenance of muscle integrity.

A possible relationship between Toxoplasma gondii and schizophrenia: A seroprevalence study.

Objective. Schizophrenia is a pervasive neuropsychiatric disease of uncertain etiology. We aimed to investigate a possible association between Toxoplasma gondii infection and schizophrenia in this study. Method. We selected individuals with schizophrenia (n=88) and tested them with commercially available enzyme-linked immunosorbent assay (ELISA) for anti-Toxoplasma IgG and IgM antibodies and compared these seropositivity rate to those of controls without psychiatric disease (n=88). Results. The rate of IgG antibody in the schizophrenia patients (47.7%) was higher than the control groups (20.4%) (P<0.001). We did not find any anti-Toxoplasma IgM seropositivity in both schizophrenia patients and control group. In schizophrenic patients with and without anti-Toxoplasma IgG groups statistical analysis did not reveal any correlation between demographic variables and Toxoplasma infection. Conclusion. Our findings supported previous studies indicate that T.gondii may play a role in etiopathogenesis in some cases of schizophrenia.

Foxo3 is essential for the regulation of ataxia telangiectasia mutated and oxidative stress-mediated homeostasis of hematopoietic stem cells.

Unchecked accumulation of reactive oxygen species (ROS) compromises maintenance of hematopoietic stem cells. Regulation of ROS by the tumor suppressor protein ataxia telangiectasia mutated (ATM) is critical for preserving the hematopoietic stem cell pool. In this study we demonstrate that the Foxo3 member of the Forkhead Box O (FoxO) family of transcription factors is essential for normal ATM expression. In addition, we show that loss of Foxo3 leads to defects in hematopoietic stem cells, and these defects result from an overaccumulation of ROS. Foxo3 suppression of ROS in hematopoietic stem cells is mediated partly by regulation of ATM expression. We identify ROS-independent modulations of ATM and p16(INK4a) and ROS-mediated activation of p53/p21(CIP1/WAF1/Sdi1) tumor suppressor pathways as major contributors to Foxo3-null hematopoietic stem cells defects. Our studies demonstrate that Foxo3 represses ROS in part via regulation of ATM and that this repression is required for maintenance of the hematopoietic stem cell pool.

Foxo3 is required for the regulation of oxidative stress in erythropoiesis.

Erythroid cells accumulate hemoglobin as they mature and as a result are highly prone to oxidative damage. However, mechanisms of transcriptional control of antioxidant defense in erythroid cells have thus far been poorly characterized. We observed that animals deficient in the forkhead box O3 (Foxo3) transcription factor died rapidly when exposed to erythroid oxidative stress-induced conditions, while wild-type mice showed no decreased viability. In view of this striking finding, we investigated the potential role of Foxo3 in the regulation of ROS in erythropoiesis. Foxo3 expression, nuclear localization, and transcriptional activity were all enhanced during normal erythroid cell maturation. Foxo3-deficient erythrocytes exhibited decreased expression of ROS scavenging enzymes and had a ROS-mediated shortened lifespan and evidence of oxidative damage. Furthermore, loss of Foxo3 induced mitotic arrest in erythroid precursor cells, leading to a significant decrease in the rate of in vivo erythroid maturation. We identified ROS-mediated upregulation of p21(CIP1/WAF1/Sdi1) (also known as Cdkn1a) as a major contributor to the interference with cell cycle progression in Foxo3-deficient erythroid precursor cells. These findings establish an essential nonredundant function for Foxo3 in the regulation of oxidative stress, cell cycle, maturation, and lifespan of erythroid cells. These results may have an impact on the understanding of human disorders in which ROS play a role.