Varying iron release from transferrin and lactoferrin proteins. A laboratory experiment

Iron metabolism is an important subject of study for undergraduate students of chemistry and biochemistry. Relevant laboratory exercises are scarce in the literature but would be very helpful in assisting students grasp key concepts. The experiment described here deals with different iron release mechanisms of two protagonists in iron metabolism: serum transferrin (Tf) and lactoferrin (Lf). Despite having very similar structures and iron‐binding sites, Tf releases practically all its iron at pH 5.5 while Lf requires a significantly lower pH of 3. This difference in behavior is directly related to their respective biological functions as Tf blood‐borne iron into the cell, while Lf competes with pathogens to sequester iron in biological fluids at more acidic pHs.  During this experiment, the students will carry out iron loading and unloading on both human Lf and Tf and monitor the iron release at different pHs using UV–Vis spectroscopy. With this simple approach, the students will discover the different patterns of iron release of Tf and Lf and how this variance in behavior relates to their biological functions. Furthermore, this laboratory practice can be expanded to allow students to investigate a variety of iron proteins

Accelerated CCl4-Induced Liver Fibrosis in Hjv-/- Mice, Associated with an Oxidative Burst and Precocious Profibrogenic Gene Expression

Hereditary hemochromatosis is commonly associated with liver fibrosis. Likewise, hepatic iron overload secondary to chronic liver diseases aggravates liver injury. To uncover underlying molecular mechanisms, hemochromatotic hemojuvelin knockout (Hjv-/-) mice and wild type (wt) controls were intoxicated with CCl4. Hjv-/- mice developed earlier (by 2-4 weeks) and more acute liver damage, reflected in dramatic levels of serum transaminases and ferritin and the development of severe coagulative necrosis and fibrosis. These responses were associated with an oxidative burst and early upregulation of mRNAs encoding α1-(I)-collagen, the profibrogenic cytokines TGF-β1, endothelin-1 and PDGF and, notably, the iron-regulatory hormone hepcidin. Hence, CCl4-induced liver fibrogenesis was exacerbated and progressed precociously in Hjv−/− animals. Even though livers of naïve Hjv−/− mice were devoid of apparent pathology, they exhibited oxidative stress and immunoreactivity towards α-SMA antibodies, a marker of hepatic stellate cells activation. Furthermore, they expressed significantly higher (2–3 fold vs. wt, p<0.05) levels of α1-(I)-collagen, TGF-β1, endothelin-1 and PDGF mRNAs, indicative of early fibrogenesis. Our data suggest that hepatic iron overload in parenchymal cells promotes oxidative stress and triggers premature profibrogenic gene expression, contributing to accelerated onset and precipitous progression of liver fibrogenesis

Siderophore-based detection of Fe(iii) and microbial pathogens

Siderophores are low-molecular-weight iron chelators that are produced and exported by bacteria, fungi and plants during periods of nutrient deprivation. The structures, biosynthetic logic, and coordination chemistry of these molecules have fascinated chemists for decades. Studies of such fundamental phenomena guide the use of siderophores and siderophore conjugates in a variety of medicinal applications that include iron-chelation therapies and drug delivery. Sensing applications constitute another important facet of siderophore-based technologies. The high affinities of siderophores for both ferric ions and siderophore receptors, proteins expressed on the cell surface that are required for ferric siderophore import, indicate that these small molecules may be employed for the selective capture of metal ions, proteins, and live bacteria. This minireview summaries progress in methods that utilize native bacterial and fungal siderophore scaffolds for the detection of Fe(III) or microbial pathogens.Massachusetts Institute of Technology. Dept. of Chemistr

From the periphery to the brain: Lipocalin-2, a friend or foe?

Lipocalin-2 (LCN2) is an acute-phase protein that, by binding to iron-loaded siderophores, acts as a potent bacteriostatic agent in the iron-depletion strategy of the immune system to control pathogens. The recent identification of a mammalian siderophore also suggests a physiological role for LCN2 in iron homeostasis, specifically in iron delivery to cells via a transferrin-independent mechanism. LCN2 participates, as well, in a variety of cellular processes, including cell proliferation, cell differentiation and apoptosis, and has been mostly found up-regulated in various tissues and under inflammatory states, being its expression regulated by several inducers. In the central nervous system less is known about the processes involving LCN2, namely by which cells it is produced/secreted, and its impact on cell proliferation and death, or in neuronal plasticity and behaviour. Importantly, LCN2 recently emerged as a potential clinical biomarker in multiple sclerosis and in ageing-related cognitive decline. Still, there are conflicting views on the role of LCN2 in pathophysiological processes, with some studies pointing to its neurodeleterious effects, while others indicate neuroprotection. Herein, these various perspectives are reviewed and a comprehensive and cohesive view of the general function of LCN2, particularly in the brain, is provided.Ana Catarina Ferreira and Sandro Da Mesquita are recipients of PhD fellowships by the Fundação para a Ciência e Tecnologia (FCT, Portugal)/FEDER. Fernanda Marques is an assistant researcher IF/ 00231/2013 of the Fundação para a Ciência e Tecnologia (FCT, Portugal). This work was supported by Fundação para a Ciência e Tecnologia (FCT) and COMPETE through the project: EXPL/NEUOSD/2196/2013 (to Marques F). The authors thank Nadine Santos for the helpful comments on the manuscript

Insights on the role of the protein hemojuvelin in the maintenance of systemic iron homeostasis

Iron is an essential nutrient, involved in a wide range of biochemical activities. However, its flexible coordination chemistry and favorable redox potential may render this element potentially toxic. Thus, tight and accurate regulation of iron absorption in humans is critical to prevent systemic excess or deficiency. This complex task is accomplished by hepcidin, a liver-derived peptide hormone that binds to the iron transporter ferroportin and promotes its degradation. This results in a decrease of the iron efflux from duodenal enterocytes, macrophages and hepatocytes into the blood stream. Severalmolecules, such as HFE, TfRs, BMP6 and HJV, are being implicated in the regulation of hepcidin expression. HJV is encoded by the HFE2 gene and functions as a BMP co-receptor, activating hepcidin expression through the BMP/SMAD pathway. It is predominantly expressed in the skeletal muscles and at lower levels in the heartand the liver. Furthermore, a putative muscle-derived soluble Hjv is supposed to circulate in the plasma. Humans bearing pathogenic mutations of the HfE2 gene develop juvenile hemochromatosis, a disease characterized by profound hepcidindeficiency and early-onset severe iron overload. Mouse models with ubiquitous disruption of the expression of HJV recapitulate the main features of the disease. Previous studies have proposed an essential role for HJV in iron sensing, however, the implicated mechanisms have not been elucidated thus far. In this work we study the function of HJV within the iron metabolism regulatory mechanisms. In chapter II we investigate the role of HJV, expressed in different tissues, in the maintenance of iron homeostasis. We generate two novelmouse models with targeted disruption of the expression of Hjv in liver hepatocytes or skeletal muscle cells and we analyze their phenotype. We show that hepatic HJV suffices to regulate the expression of hepcidin and to maintainsystemic iron homeostasis whereas skeletal muscle HJV is dispensable for systemic iron metabolism. Furthermore, we do not observe any significant iron regulatory function of a putative muscle-derived soluble Hjv.In chapter III we focus our interest on the role of hepatic HJV in iron sensing to hepcidin. To this end, we analyze the molecular responses of HJV-/- and HJV+/+ mice to dietary iron manipulations. We demonstrate that HJV is not essential for iron sensing and it rather functions as an enhancer of the primary iron signal. Furthermore, we provide evidence of iron-dependent regulation of duodenal DMT1 and tissue specific function of hepcidin in the spleen and the duodenum of the studied HJV-/- mice.Le fer est un nutriment essentiel impliqué dans une vaste gamme d'activités biochimiques. Cependant, sa coordination chimique flexible et son potentiel redox favorable rendent cet élément potentiellement toxique. Ainsi, une régulation stricte et précise de l'absorption du fer chez l'homme est essentielle pour prévenir un excès ou une insuffisance systémique. Cette tâche complexe est réalisée par l'hepcidine, une hormone peptidique dérivée du foie qui s'attache au transporteur du fer ferroportine et favorise sa dégradation. Ceci mène à une diminution de l'efflux du fer des entérocytes duodénaux, des macrophages et des hépatocytes dans le flux sanguin. Plusieurs molécules, comme le HFE, le TfR2, leBMP6 et l'HJV, sont impliquées dans la régulation de l'expression de l'hepcidine. HJV est codée par le gène HFE2 et fonctionne comme un co-récepteur de BMP, en activant l'expression d'hepcidine par la voie BMP/SMAD. Elle est principalement exprimée dans les muscles squelettiques et à des niveaux inférieurs dans le coeur et le foie. En outre, un putatif HJV soluble dérivé des muscles est supposé circuler dans le plasma. Les humains portant des mutations pathogènes du gène HFE2 développent l'hémochromatose juvénile, une maladie caractérisée par une carence profonde en hepcidine et une surcharge sévère et précoce en fer. Des modèles de souris avec une perturbation omniprésente de l'expression de l'HJV récapitulent les principales caractéristiques de la maladie. Des études antérieures ont proposé un rôle essentiel pour HJV dans la détection du fer, cependant, les mécanismes impliqués n'ont pas été élucidés à ce jour. Dans ce travail, nous étudions la fonction de HJV dans les mécanismes de régulation du métabolisme du fer. Dans le chapitre II, nous étudions le rôle de HJV, exprimé dans différents tissus, dans le maintien de l'homéostasie du fer.Nous générons deux nouveaux modèles murins avec une perturbation ciblée de l'expression de HJV dans les hépatocytes ou des cellules musculaires squelettiques et nous analysons leur phénotype. Nous montrons que l'HJV hépatique est suffisante pour réguler l'expression de l'hepcidine et de maintenir l'homéostasie du fer systémique alors que l'HJV musculaire est non essentiel pour le métabolisme du fer systémique. De plus, nous n'avons pas observé une fonction significative d'une HJV soluble musculaire hypothétique liée à la régulation du fer.Dans le chapitre III, nous nous intéressons au rôle de l'HJV hépatique dans la détection des altérations du niveau de fer et la transmission subséquente de ce signal sur l'expression de l'hepcidine. À cette fin, nous analysons les réponsesmoléculaires des souris HJV-/- et HJV+/+ à des manipulations alimentaires de fer. Nous démontrons que HJV n'est pas essentielle pour la détection des variations des niveaux ferriques mais qu'elle fonctionne plutôt comme un amplificateur dusignal primaire de fer. En outre, nous fournissons des preuves d'une régulation du DMT1 dépendante du fer et une fonction de l'hepcidine spécifique au tissu, dans le duodénum et la rate des souris HJV-/ - étudiées

Non-invasive assessment of liver fibrosis: it is time for laboratory medicine.

Abstract: Chronic liver diseases (CLDs) represent a major cause of morbidity and mortality worldwide. In all etiologies of CLDs, staging of liver fibrosis is essential for both prognosis and management. Until a few years ago, liver biopsy was the only tool for the diagnosis of liver fibrosis in patients with CLDs. However, liver biopsy is an invasive and costly procedure. More recently, various serum biomarkers and laboratory tests have been proposed as surrogates of liver histology. Due to inadequate diagnostic accuracy or to lack of sufficient validation, guidelines still do not recommend them as a substitute for liver biopsy that is still considered the gold standard for the diagnosis of liver fibrosis. Notably, non-invasive serum biomarkers, when combined, may reduce by 50%-80% the number of liver biopsies needed for correctly classifying hepatic fibrosis. However, liver biopsy cannot be avoided completely, but should be used in those cases in which non-invasive methods show poor accuracy. In this view, serum biomarkers and liver biopsy represent a union between laboratory medicine and hepatology

Hépatite B : nouvelles recommandations de prise en charge

Hepatitis B virus (HBV) infection is a major public health concern associated with major clinical complications, notably chronic liver disease that can progress with time to cirrhosis or even to hepatocellular carcinoma. The management of HBV-infected patients is complex and requires the close collaboration between the general practitioner and the specialist. This review presents an overview of recently published guidelines, from the European Association for the Study of the Liver, and suggests strategies for initial management and referral of HBV-infected patients for the general practitioner

Σχεδίαση CMOS ενισχυτή βιολογικών σημάτων χαμηλής ισχύος και χαμηλού θορύβου

Summarization: In this work we will study the designing procedure of a low-power low-noise amplifier, with application in brain signals amplification. The bio-amplifier was implemented in a 90 nm CMOS process so that it is suitable for integration into multichannel recording implants. The recording electrode of the bio-amplifier is directly connected to the input of a low-noise amplifier which includes a Miller integrator in its feedback. The integrator, achieving a long time constant, gives the bio-amplifier the ability to reject unwanted low frequency signals as it sets its low cut-off frequency at 121.5 Hz. The large time constant of the integrator results from a structure of diode-connected transistors which employs a high resistance and allows the use of capacitors of reduced capacitance and area. The design of the amplifier under the constraints resulting from the low supply voltage of 90 nm technology, 1.2 V, was done with the inversion coefficient methodology (IC) that allows design in weak, moderate and strong inversion. The midband gain of the amplifier is 46.8 dB, its bandwidth is 10.84 kHz, the noise at the input 5.8 μVrms and the power dissipation 8.4 μW.Περίληψη: Σε αυτήν την εργασία θα μελετήσουμε τη διαδικασία σχεδίασης ενός ενισχυτή χαμηλής ισχύος και χαμηλού θορύβου με εφαρμογή στην ενίσχυση εγκεφαλικών σημάτων. Ο βιο-ενισχυτής υλοποιήθηκε σε CMOS τεχνολογία 90 nm έτσι ώστε να είναι κατάλληλος για ενσωμάτωση σε πολυκάναλα καταγραφικά εμφυτεύματα. Το ηλεκτρόδιο καταγραφής σημάτων του βιο-ενισχυτή συνδέεται άμεσα στην είσοδο ενός ενισχυτή χαμηλού θορύβου ο οποίος περιλαμβάνει στην ανάδρασή του έναν ολοκληρωτή Miller. Ο ολοκληρωτής, επιτυγχάνοντας μεγάλη χρονική σταθερά, προσδίδει στον βιο-ενισχυτή την ικανότητα απόρριψης ανεπιθύμητων σημάτων χαμηλών συχνοτήτων καθώς θέτει την χαμηλή συχνότητα αποκοπής του στα 121.5 Hz. Η μεγάλη χρονική σταθερά του ολοκληρωτή προκύπτει από μια δομή διοδικών τρανζίστορ που παρουσιάζει υψηλή αντίσταση και επιτρέπει την χρήση πυκνωτή μειωμένης χωρητικότητας και επιφάνειας. Η σχεδίαση του ενισχυτή υπό τους περιορισμούς που προκύπτουν από την χαμηλή τάση τροφοδοσίας της τεχνολογίας 90 nm, 1.2 V, έγινε με την μεθοδολογία του δείκτη αναστροφής (IC). Το κέρδος του ενισχυτή στη μέση ζώνη είναι 46.8 dB, το εύρος ζώνης του 10.84 kHz, ο θόρυβος στην είσοδό 5.8 μVrms και η κατανάλωση 8.4 μW