Temporin L and aurein 2.5 have identical conformations but subtly distinct membrane and antibacterial activities.

Frogs such as Rana temporaria and Litoria aurea secrete numerous closely related antimicrobial peptides (AMPs) as an effective chemical dermal defence. Damage or penetration of the bacterial plasma membrane is considered essential for AMP activity and such properties are commonly ascribed to their ability to form secondary amphipathic, α-helix conformations in membrane mimicking milieu. Nevertheless, despite the high similarity in physical properties and preference for adopting such conformations, the spectrum of activity and potency of AMPs often varies considerably. Hence distinguishing apparently similar AMPs according to their behaviour in, and effects on, model membranes will inform understanding of primary-sequence-specific antimicrobial mechanisms. Here we use a combination of molecular dynamics simulations, circular dichroism and patch-clamp to investigate the basis for differing anti-bacterial activities in representative AMPs from each species; temporin L and aurein 2.5. Despite adopting near identical, α-helix conformations in the steady-state in a variety of membrane models, these two AMPs can be distinguished both in vitro and in silico based on their dynamic interactions with model membranes, notably their differing conformational flexibility at the N-terminus, ability to form higher order aggregates and the characteristics of induced ion conductance. Taken together, these differences provide an explanation of the greater potency and broader antibacterial spectrum of activity of temporin L over aurein 2.5. Consequently, while the secondary amphipathic, α-helix conformation is a key determinant of the ability of a cationic AMP to penetrate and disrupt the bacterial plasma membrane, the exact mechanism, potency and spectrum of activity is determined by precise structural and dynamic contributions from specific residues in each AMP sequence.

Provisional standardization of hepcidin assays: creating a traceability chain with a primary reference material, candidate reference method and a commutable secondary reference material.

Background Hepcidin concentrations measured by various methods differ considerably, complicating interpretation. Here, a previously identified plasma-based candidate secondary reference material (csRM) was modified into a serum-based two-leveled sRM. We validated its functionality to increase the equivalence between methods for international standardization. Methods We applied technical procedures developed by the International Consortium for Harmonization of Clinical Laboratory Results. The sRM, consisting of lyophilized serum with cryolyoprotectant, appeared commutable among nine different measurement procedures using 16 native human serum samples in a first round robin (RR1). Harmonization potential of the sRM was simulated in RR1 and evaluated in practice in RR2 among 11 measurement procedures using three native human plasma samples. Comprehensive purity analysis of a candidate primary RM (cpRM) was performed by state of the art procedures. The sRM was value assigned with an isotope dilution mass spectrometry-based candidate reference method calibrated using the certified pRM. Results The inter-assay CV without harmonization was 42.1% and 52.8% in RR1 and RR2, respectively. In RR1, simulation of harmonization with sRM resulted in an inter-assay CV of 11.0%, whereas in RR2 calibration with the material resulted in an inter-assay CV of 19.1%. Both the sRM and pRM passed international homogeneity criteria and showed long-term stability. We assigned values to the low (0.95±0.11 nmol/L) and middle concentration (3.75±0.17 nmol/L) calibrators of the sRM. Conclusions Standardization of hepcidin is possible with our sRM, which value is assigned by a pRM. We propose the implementation of this material as an international calibrator for hepcidin.

Proanthocyanidin trimer gallate modulates lipid deposition and fatty acid desaturation in Caenorhabditis elegans.

The incidence of obesity is rising at an alarming rate. Despite its recognition as an urgent healthcare concern, obesity remains largely an unsolved medical problem. A comprehensive screen for functional dietary phytochemicals identified proanthocyanidins as putative targets to ameliorate obesity. A full-scale purification of oligomeric proanthocyanidins (OPCs) derived from grape seed extract yielded pure OPC dimer, trimer, tetramer, and their gallates (pOPCs). Forward chemical screening conducted in Caenorhabditis elegans suggested that pOPCs reduced the activity of lipase in vitro and triglyceride storage capacity in vivo Proanthocyanidin trimer gallate in particular modified lipid desaturation in C. elegans, revealed by hyperspectral coherent anti-Stokes Raman scattering microscopy. Exposure to trimer gallate resulted in the transcriptional down-regulation of nhr-49 (an ortholog of the human peroxisome proliferator-activated receptor-α), and a key regulator of fat metabolism, and 2 downstream genes: fat-5 and acs-2 A combination exposure of 2 or 3 pOPCs (dimer gallate, trimer and/or trimer gallate) suggested the absence of synergistic potential. By using the whole-organism C. elegans coupled with versatile biochemical, biophysical, and genetic tools, we provide an account of the composition and bioactivity of individual OPCs and more generally highlight the potential of traditional Chinese medicine-derived drug leads.-Nie, Y., Littleton, B., Kavanagh, T., Abbate, V., Bansal, S. S., Richards, D., Hylands, P., Sturzenbaum, S. R. Proanthocyanidin trimer gallate modulates lipid deposition and fatty acid desaturation in Caenorhabditis elegans.

Effects of intravenous iron on fibroblast growth factor 23 (FGF23) in haemodialysis patients: a randomized controlled trial.

BACKGROUND: Intravenous iron affects serum levels of intact fibroblast growth factor-23 (iFGF23) and its cleavage product c-terminal FGF23 (cFGF23) in iron-deficient people with normal renal function. We hypothesized that intravenous iron modulates iFGF23 and cFGF23 in haemodialysis patients differently according to the type of iron used. METHODS: Prevalent, stable haemodialysis patients requiring protocol-based intravenous iron therapy were randomized to a single 200 mg dose of either ferric carboxymaltose (FCM) or iron sucrose (IS). The primary outcome was change in iFGF23 and cFGF23 from pre-infusion to Day 2 post-infusion. Serum hepcidin, ferritin and phosphate were also measured. Pair-wise comparisons utilised the Wilcoxon rank sum test; linear mixed models with an interaction term for treatment and time evaluated between-group effects. RESULTS: Forty-two participants completed the study. In those randomized to FCM (n = 22), median (interquartile range) values pre-infusion and Day 2, respectively, were 843 pg/mL (313-1922) and 576 pg/mL (356-1296, p = 0.05) for iFGF23, 704RU/mL (475-1204) and 813RU/mL (267-1156, p = 0.04) for cFGF23, and 1.53 mmol/L (1.14-1.71) and 1.37 (1.05-1.67, p = 0.03) for phosphate. These parameters did not change following IS. Both serum ferritin (p < 0.001) and hepcidin (p < 0.001) increased in both groups, and the increase in hepcidin was greater in the FCM group (p = 0.03 for between-group difference). CONCLUSIONS: Contrary to iron-deficient people with normal renal function, haemodialysis patients given protocol-driven intravenous FCM demonstrated a fall in iFGF23 and a rise in cFGF23, changes not evident with IS. This suggests a differential effect of intravenous iron treatment according to both formulation and renal function. TRIAL REGISTRATION: Australian and New Zealand Clinical Trials Register ACTRN12614000548639 . Registered 22 May 2014 (retrospectively registered).

Doxorubicin enhances curcumin's cytotoxicity in human prostate cancer cells in vitro by enhancing its cellular uptake.

Doxorubicin (DOX) is a widely used drug in cancer treatment. Despite its popularity, it suffers from systemic side effects and susceptibility to drug resistance. Curcumin (CURC), on the other hand, is a drug that recently gained popularity due to its wide range of biological activities, including anti-inflammatory and anti-cancer activities. Limitations to its clinical translation include its poor water solubility and the need for administration of high doses. Combinatory anti-cancer therapy has been proposed as a common approach to overcome one or more of these challenges. In this work, we propose a combinatory DOX and CURC anti-cancer therapy of prostate cancer cells in vitro. DOX and CURC were administered in the free drug and nanocapsule form, respectively. Cell size and complexity, cytotoxicity and apoptosis were studied by flow cytometry, MTT assay and sub-G1 quantification, respectively. Cellular uptake of CURC nanocapsules (CURC NCs) was quantified by fluorescence microscopy and high-performance liquid chromatography fluorescence detection. Results showed that in vitro treatment with CURC NCs in the presence of subtherapeutic concentrations of DOX, led to significant increase in prostate cancer cells (PC3) apoptosis and death. This was likely due to significantly enhanced CURC uptake by the cells. The study presents a good rationale for pursuing combinatory CURC/DOX therapy in pre-clinical tumor animal models in the near future.

Hepcidin Response to Iron Therapy in Patients with Non-Dialysis Dependent CKD: An Analysis of the FIND-CKD Trial.

Hepcidin is the key regulator of iron homeostasis but data are limited regarding its temporal response to iron therapy, and response to intravenous versus oral iron. In the 56-week, open-label, multicenter, prospective, randomized FIND-CKD study, 626 anemic patients with non-dialysis dependent chronic kidney disease (ND-CKD) and iron deficiency not receiving an erythropoiesis stimulating agent were randomized (1:1:2) to intravenous ferric carboxymaltose (FCM), targeting higher (400-600µg/L) or lower (100-200µg/L) ferritin, or to oral iron. Serum hepcidin levels were measured centrally in a subset of 61 patients. Mean (SD) baseline hepcidin level was 4.0(3.5), 7.3(6.4) and 6.5(5.6) ng/mL in the high ferritin FCM (n = 17), low ferritin FCM (n = 16) and oral iron group (n = 28). The mean (SD) endpoint value (i.e. the last post-baseline value) was 26.0(9.1),15.7(7.7) and 16.3(11.0) ng/mL, respectively. The increase in hepcidin from baseline was significantly smaller with low ferritin FCM or oral iron vs high ferritin FCM at all time points up to week 52. Significant correlations were found between absolute hepcidin and ferritin values (r = 0.65, p<0.001) and between final post-baseline increases in both parameters (r = 0.70, p<0.001). The increase in hepcidin levels over the 12-month study generally mirrored the cumulative iron dose in each group. Hepcidin and transferrin saturation (TSAT) absolute values showed no correlation, although there was an association between final post-baseline increases (r = 0.42, p<0.001). Absolute values (r = 0.36, p = 0.004) and final post-baseline increases of hepcidin and hemoglobin (p = 0.30, p = 0.030) correlated weakly. Baseline hepcidin levels were not predictive of a hematopoietic response to iron therapy. In conclusion, hepcidin levels rose in response to either intravenous or oral iron therapy, but the speed and extent of the rise was greatest with intravenous iron targeting a higher ferritin level. However neither the baseline level nor the change in hepcidin was able to predict response to therapy in this cohort.

Toward Worldwide Hepcidin Assay Harmonization: Identification of a Commutable Secondary Reference Material.

BACKGROUND: Absolute plasma hepcidin concentrations measured by various procedures differ substantially, complicating interpretation of results and rendering reference intervals method dependent. We investigated the degree of equivalence achievable by harmonization and the identification of a commutable secondary reference material to accomplish this goal. METHODS: We applied technical procedures to achieve harmonization developed by the Consortium for Harmonization of Clinical Laboratory Results. Eleven plasma hepcidin measurement procedures (5 mass spectrometry based and 6 immunochemical based) quantified native individual plasma samples (n = 32) and native plasma pools (n = 8) to assess analytical performance and current and achievable equivalence. In addition, 8 types of candidate reference materials (3 concentrations each, n = 24) were assessed for their suitability, most notably in terms of commutability, to serve as secondary reference material. RESULTS: Absolute hepcidin values and reproducibility (intrameasurement procedure CVs 2.9%-8.7%) differed substantially between measurement procedures, but all were linear and correlated well. The current equivalence (intermeasurement procedure CV 28.6%) between the methods was mainly attributable to differences in calibration and could thus be improved by harmonization with a common calibrator. Linear regression analysis and standardized residuals showed that a candidate reference material consisting of native lyophilized plasma with cryolyoprotectant was commutable for all measurement procedures. Mathematically simulated harmonization with this calibrator resulted in a maximum achievable equivalence of 7.7%. CONCLUSIONS: The secondary reference material identified in this study has the potential to substantially improve equivalence between hepcidin measurement procedures and contributes to the establishment of a traceability chain that will ultimately allow standardization of hepcidin measurement results.

Copper(II) binding properties of hepcidin.

Hepcidin is a peptide hormone that regulates the homeostasis of iron metabolism. The N-terminal domain of hepcidin is conserved amongst a range of species and is capable of binding Cu(II) and Ni(II) through the amino terminal copper-nickel binding motif (ATCUN). It has been suggested that the binding of copper to hepcidin may have biological relevance. In this study we have investigated the binding of Cu(II) with model peptides containing the ATCUN motif, fluorescently labelled hepcidin and hepcidin using MALDI-TOF mass spectrometry. As with albumin, it was found that tetrapeptide models of hepcidin possessed a higher affinity for Cu(II) than that of native hepcidin. The log K 1 value of hepcidin for Cu(II) was determined as 7.7. Cu(II) binds to albumin more tightly than hepcidin (log K 1 = 12) and in view of the serum concentration difference of albumin and hepcidin, the bulk of kinetically labile Cu(II) present in blood will be bound to albumin. It is estimated that the concentration of Cu(II)-hepcidin will be less than one femtomolar in normal serum and thus the binding of copper to hepcidin is unlikely to play a role in iron homeostasis. As with albumin, small tri and tetra peptides are poor models for the metal binding properties of hepcidin.

Carbon nanotubes' surface chemistry determines their potency as vaccine nanocarriers in vitro and in vivo.

Carbon nanotubes (CNTs) have shown marked capabilities in enhancing antigen delivery to antigen presenting cells. However, proper understanding of how altering the physical properties of CNTs may influence antigen uptake by antigen presenting cells, such as dendritic cells (DCs), has not been established yet. We hypothesized that altering the physical properties of multi-walled CNTs (MWNTs)-antigen conjugates, e.g. length and surface charge, can affect the internalization of MWNT-antigen by DCs, hence the induced immune response potency. For this purpose, pristine MWNTs (p-MWNTs) were exposed to various chemical reactions to modify their physical properties then conjugated to ovalbumin (OVA), a model antigen. The yielded MWNTs-OVA conjugates were long MWNT-OVA (~386nm), bearing net positive charge (5.8mV), or short MWNTs-OVA (~122nm) of increasing negative charges (-23.4, -35.8 or -39mV). Compared to the short MWNTs-OVA bearing high negative charges, short MWNT-OVA with the lowest negative charge demonstrated better cellular uptake and OVA-specific immune response both in vitro and in vivo. However, long positively-charged MWNT-OVA showed limited cellular uptake and OVA specific immune response in contrast to short MWNT-OVA displaying the least negative charge. We suggest that reduction in charge negativity of MWNT-antigen conjugate enhances cellular uptake and thus the elicited immune response intensity. Nevertheless, length of MWNT-antigen conjugate might also affect the cellular uptake and immune response potency; highlighting the importance of physical properties as a consideration in designing a MWNT-based vaccine delivery system.

Translocation of LRP1 targeted carbon nanotubes of different diameters across the blood-brain barrier in vitro and in vivo.

Brain glioblastoma and neurodegenerative diseases are still largely untreated due to the inability of most drugs to cross the blood-brain barrier (BBB). Nanoparticles have emerged as promising tools for drug delivery applications to the brain; in particular carbon nanotubes (CNTs) that have shown an intrinsic ability to cross the BBB in vitro and in vivo. Angiopep-2 (ANG), a ligand for the low-density lipoprotein receptor-related protein-1 (LRP1), has also shown promising results as a targeting ligand for brain delivery using nanoparticles (NPs). Here, we investigate the ability of ANG-targeted chemically-functionalised multi-walled carbon nanotubes (f-MWNTs) to cross the BBB in vitro and in vivo. ANG was conjugated to wide and thin f-MWNTs creating w-MWNT-ANG and t-MWNT-ANG, respectively. All f-MWNTs were radiolabelled to facilitate quantitative analyses by γ-scintigraphy. ANG conjugation to f-MWNTs enhanced BBB transport of w- and t-MWNTs-ANG compared to their non-targeted equivalents using an in vitro co-cultured BBB model consisting of primary porcine brain endothelial cells (PBEC) and primary rat astrocytes. Additionally, following intravenous administration w-MWNTs-ANG showed significantly higher whole brain uptake than the non-targeted w-MWNT in vivo reaching ~2% injected dose per g of brain (%ID/g) within the first hour post-injection. Furthermore, using a syngeneic glioma model, w-MWNT-ANG showed enhanced uptake in glioma brain compared to normal brain at 24h post-injection. t-MWNTs-ANG, on the other hand, showed higher brain accumulation than w-MWNTs. However, no significant differences were observed between t-MWNT and t-MWNT-ANG indicating the importance of f-MWNTs diameter towards their brain accumulation. The inherent brain accumulation ability of f-MWNTs coupled with improved brain-targeting by ANG favours the future clinical applications of f-MWNT-ANG to deliver active therapeutics for brain glioma therapy.

Correlates of serum hepcidin levels and its association with cardiovascular disease in an elderly general population.

BACKGROUND: The expression of the key iron regulatory hormone hepcidin is regulated by iron availability, inflammation, hormones, hypoxia, and anaemia. Increased serum concentrations of hepcidin have recently been linked to atherosclerosis. We studied demographic, haematologic, biochemical, and dietary correlates of serum hepcidin levels and its associations with incident cardiovascular disease and with carotid atherosclerosis. METHODS: Serum hepcidin concentrations were measured by tandem mass spectrometry in samples taken in 2000 from 675 infection-free participants of the prospective population-based Bruneck study (age, mean±standard deviation, 66.0±10.2; 48.1% male). Blood parameters were measured by standard methods. Dietary intakes of iron and alcohol were surveyed with a food frequency questionnaire. Carotid atherosclerosis (365 cases) was assessed by ultrasound and subjects were observed for incident stroke, myocardial infarction, or sudden cardiac death (91 events) until 2010. RESULTS: Median (interquartile range) hepcidin levels were 2.27 nM (0.86, 4.15). Most hepcidin correlates were in line with hepcidin as an indicator of iron stores. Independently of ferritin, hepcidin was related directly to physical activity (p=0.024) and fibrinogen (p<0.0001), and inversely to alcohol intake (p=0.006), haemoglobin (p=0.027), and γ-glutamyltransferase (p<0.0001). Hepcidin and hepcidin-to-ferritin ratio were not associated with prevalent carotid atherosclerosis (p=0.43 and p=0.79) or with incident cardiovascular disease (p=0.62 and p=0.33). CONCLUSIONS: In this random sample of the general community, fibrinogen and γ-glutamyltransferase were the most significant hepcidin correlates independent of iron stores, and hepcidin was related to neither atherosclerosis nor cardiovascular disease.

Design and synthesis of novel pegylated iron chelators with decreased metabolic rate.

BACKGROUND: Deferiprone has proved to be a successful iron selective chelator in a range of pathologies. However, its use is limited by rapid Phase II metabolism, necessitating the administration of large doses. In an attempt to modify metabolic rate of this class of compounds, a range of pegylated 3-hydroxypyridin-4-ones has been synthesized. EXPERIMENTAL: The synthetic route in which the polyethylene glycol counterparts are introduced to a protected pyran ring involves either a Williamson etherification reaction or direct addition leading to polyethylene glycol-containing precursors. RESULTS & DISCUSSION: The introduction of the pegylated substituent was found to lead to a relatively low rate of metabolism for some of the derivatives (6a, 6b, 8a and 8b), offering a possible improvement over deferiprone.

Synthesis of double-clickable functionalised graphene oxide for biological applications.

Azide- and alkyne-double functionalised graphene oxide (Click(2) GO) was synthesised and characterised with attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), thermogravimetric analysis (TGA) and Raman spectroscopy. Fourteen-percentage increase in azide content was found, after pre-treatment of GO with meta-chloroperoxybenzoic acid (mCPBA), determined with elemental analysis. No effect on A549 cell viability was found, up to 100 µg mL(-1) and 72 h of incubation, determined with the modified lactate dehydrogenase (mLDH) assay. Two sequential copper(i) catalysed azide-alkyne cycloaddition (CuAAC) reactions were performed to conjugate the propargyl-modified blood-brain barrier targeting peptide Angiopep-2, and a bis-azide polyethylene glycol (MW = 3500), to the Click(2) GO. The final conjugate was characterised with ATR-FTIR and TGA.

Obesity does not influence hepcidin and hemojuvelin levels in hemodialysis patients.

BACKGROUND/AIMS: Clinical studies have shown increased levels of hepcidin causing functional iron deficiency in obese individuals. This study examined whether obesity contributes to increased hepcidin and hemojuvelin levels in adult hemodialysis patients. METHODS: In a case-control design, 37 obese [body mass index (BMI) >30 kg/m(2)] stable hemodialysis patients and 37 patients with normal BMI (20-25 kg/m(2)), matched for age, gender and race, who fulfilled a strict set of inclusion and exclusion criteria were included in the study. Serum hepcidin and hemojuvelin, markers of iron status and inflammation, and routine hematological and biochemical variables were measured on samples obtained prior to the midweek hemodialysis session. RESULTS: Obese and nonobese patients (BMI 35.1 ± 3.4 vs. 22.8 ± 1.4 kg/m(2); p < 0.001) were similar with regard to basic comorbidities and use of erythropoietin and iron. Levels of hemoglobin, hypochromic red cells and reticulocytes were similar in the two groups. Serum iron and transferrin saturation levels were on the low side and not different between obese and lean individuals; total iron-binding capacity showed a trend towards higher levels in obese patients (48.4 ± 8.3 vs. 44.9 ± 7.4 µmol/l; p = 0.065). Levels of serum ferritin (651 ± 302 vs. 705 ± 327 µg/l; p = 0.46), hepcidin (118.3 ± 67.7 vs. 119.3 ± 78.0 ng/ml; p = 0.95) and hemojuvelin (1.90 ± 1.11 vs. 1.94 ± 1.24 mg/l; p = 0.90) were high but similar between the two groups. Serum hepcidin showed a significant correlation only with ferritin (r = 0.287, p = 0.013). CONCLUSIONS: Hepcidin and hemojuvelin levels are already considerably elevated in dialysis patients, but obesity does not have an additional impact. Further studies should examine whether increased weight contributes towards hepcidin elevation in predialysis individuals, in whom there is a lesser burden of systemic inflammation.

Manipulating the pH response of 2,3-diaminopropionic acid rich peptides to mediate highly effective gene silencing with low-toxicity.

Cationic amphipathic pH responsive peptides possess high in vitro and in vivo nucleic acid delivery capabilities and function by forming a non-covalent complex with cargo, protecting it from nucleases, facilitating uptake via endocytosis and responding to endosomal acidification by being released from the complex and inserting into and disordering endosomal membranes. We have designed and synthesised peptides to show how Coulombic interactions between ionizable 2,3-diaminopropionic acid (Dap) side chains can be manipulated to tune the functional pH response of the peptides to afford optimal nucleic acid transfer and have modified the hydrogen bonding capabilities of the Dap side chains in order to reduce cytotoxicity. When compared with benchmark delivery compounds, the peptides are shown to have low toxicity and are highly effective at mediating gene silencing in adherent MCF-7 and A549 cell lines, primary human umbilical vein endothelial cells and both differentiated macrophage-like and suspension monocyte-like THP-1 cells.

Functional characterization of fluorescent hepcidin.

Hepcidin is a peptide hormone that regulates homeostasis in iron metabolism. It binds to the sole known cellular iron exporter ferroportin (Fpn), triggers its internalization, and thereby modulates the efflux of iron from cells. This functional property has been adopted in this study to assess the bioactivity and potency of a range of novel fluorescent hepcidin analogues. Hepcidin was selectively labeled with 6-carboxyfluorescein (CF) and 6-carboxytetramethylrhodamine (TMR) using Fmoc solid phase peptide chemistry. Internalization of Fpn by hepcidin was assessed by high-content microscopic analysis. Both K18- and M21K-labeled hepcidin with TMR and CF exhibited measurable potency when tested in cultured MDCK and T47D cells expressing human ferroportin. The bioactivity of the labeled hepcidin varies with the type of fluorophore and site of attachment of the fluorophores on the hepcidin molecule.

Conformational flexibility determines selectivity and antibacterial, antiplasmodial, and anticancer potency of cationic α-helical peptides.

We used a combination of fluorescence, circular dichroism (CD), and NMR spectroscopies in conjunction with size exclusion chromatography to help rationalize the relative antibacterial, antiplasmodial, and cytotoxic activities of a series of proline-free and proline-containing model antimicrobial peptides (AMPs) in terms of their structural properties. When compared with proline-free analogs, proline-containing peptides had greater activity against Gram-negative bacteria, two mammalian cancer cell lines, and intraerythrocytic Plasmodium falciparum, which they were capable of killing without causing hemolysis. In contrast, incorporation of proline did not have a consistent effect on peptide activity against Mycobacterium tuberculosis. In membrane-mimicking environments, structures with high α-helix content were adopted by both proline-free and proline-containing peptides. In solution, AMPs generally adopted disordered structures unless their sequences comprised more hydrophobic amino acids or until coordinating phosphate ions were added. Proline-containing peptides resisted ordering induced by either method. The roles of the angle subtended by positively charged amino acids and the positioning of the proline residues were also investigated. Careful positioning of proline residues in AMP sequences is required to enable the peptide to resist ordering and maintain optimal antibacterial activity, whereas varying the angle subtended by positively charged amino acids can attenuate hemolytic potential albeit with a modest reduction in potency. Maintaining conformational flexibility improves AMP potency and selectivity toward bacterial, plasmodial, and cancerous cells while enabling the targeting of intracellular pathogens.

Intra-individual variability of serum hepcidin-25 in haemodialysis patients using mass spectrometry and ELISA.

BACKGROUND: Measurement of serum hepcidin levels may provide a useful alternative to the current methods of determining iron status in chronic haemodialysis (HD) patients. However, the biological variability of this pivotal regulator of iron homeostasis is unclear, and the impact of inflammation, dialysis clearance and iron therapy on hepcidin variability has not been established. METHODS: Two independent studies in chronic HD patients were conducted; serum hepcidin levels were measured at the start of dialysis sessions in 20 UK patients and in 43 Dutch patients by mass spectrometry (MS). Samples from UK patients were also analysed by a competitive enzyme-linked immunosorbent assay (cELISA). Coefficient of variance (CV(1)) was calculated and potential factors affecting CV(1) were also examined. RESULTS: The median CV(1) (inter-quartile range) was 23% (17-28) for the UK MS, 26% (17-48) for the Dutch MS and 23% (17-39) for the UK cELISA. The CV(1) was similar in those patients receiving and those not receiving regular intravenous iron. The CV(1) was not associated with the degree of inflammation. Hepcidin levels were higher following an inter-dialytic period of 3 versus 2 days (P = 0.02). CONCLUSIONS: These findings suggest considerable variability of serum hepcidin levels in HD patients. Inflammation and the use of iron did not impact on the degree of variability, and hepcidin levels were higher after an inter-dialytic period of 3 versus 2 days. These findings need to be taken into account in future studies assessing the utility of serum hepcidin as a guide to the use of iron or erythropoiesis-stimulating agents therapy.

Serum hemojuvelin and hepcidin levels in chronic kidney disease.

BACKGROUND: Hemojuvelin (HJV) has recently emerged as one of a number of significant regulators of iron homeostasis and hepcidin expression. Recently, an immunoassay has been developed to measure circulating levels of soluble HJV (sHJV). The aim of this study was to measure serum hepcidin and sHJV levels in a chronic kidney disease (CKD) population. METHODS: A total of 93 patients participated in the study (31 hemodialysis, 31 non-dialysis, 31 transplant recipients), and were matched for age and gender. Serum samples were taken for measurement of hepcidin-25 and sHJV, along with standard hematological, biochemical and inflammatory markers, and univariate/multivariate analyses were performed. RESULTS: Serum sHJV levels were markedly elevated in the hemodialysis patients (2,619 ± 1,445 ng/ml) compared to the CKD (590 ± 344 ng/ml) and transplant recipients (870 ± 638 ng/ml) (p < 0.001), normal range 370-890 ng/ml. There was a strong correlation between serum ferritin and sHJV, which remained after adjustment for potential confounders (beta 0.92, p < 0.001). In the univariate analysis, sHJV levels correlated with serum hepcidin but this was not evident in the multivariate analysis. No associations were seen between sHJV and markers of inflammation or eGFR. CONCLUSIONS: sHJV is elevated in hemodialysis patients compared to non-dialysis CKD patients. There was no association between sHJV and eGFR (in the non-dialysis groups), suggesting that factors other than decreased renal clearance are responsible for the high sHJV levels. The strong association between sHJV and ferritin suggests an interdependent relationship, although further studies are required to elucidate the possible mechanism(s) for this.

Photocrosslinked bovine serum albumin hydrogels with partial retention of esterase activity.

Novel biohybrid hydrogels based on bovine serum albumin (BSA) were synthesized by one-pot photopolymerization of chemically modified protein in the presence of N,N'-methylenebisacrylamide (MBA) as cross-linking agent under mild conditions. Two batches of methacrylated albumins were prepared by treating the protein with different amounts of methacrylic anhydride (MAN) and the degree of substitution (DS) of primary amines was quantified via trinitrobenzesulfonic acid (TNBS) colorimetric assay. Hydrogels readily formed when a diluted buffered solution of the modified protein and MBA was exposed to LW-UV in the presence of 1-[4-(2-hydroxyethoxy)phenyl]-2-hydroxy-2-methyl-1-propan-1-one (Irgacure 2959) as the radical initiator. In contrast, no hydrogel was obtained in the absence of a polymerizable BSA, nor when the cross-linker, the radical initiator or UV light exposure was excluded from the reaction, suggesting the critical importance of the combined conditions for hydrogel formation. Hydrogels were characterized via scanning electron microscopy (SEM) and the swelling ratios were monitored at different pHs. The esterolytic activity of the novel biohybrid materials was quantitatively investigated via UV-vis spectroscopy by measuring the release of para-nitrophenol upon incubation with para-nitrophenyl acetate (p-NPA) substrate. The effect of the addition of acrylic acid co-monomer and of the monomer concentration in the catalytic activity and in the swelling behavior was also examined. Finally, the reusability of the materials following one round of catalysis was evaluated.