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Humanized mice have been used to study human immunodeficiency virus type 1 (HIV-1) transmission, pathogenesis, and treatment. The ability of pediatric thymus tissue implanted either in the leg (Leg PedThy) or under the renal capsule (Renal PedThy) with allogeneic CD34+ hematopoietic cells (HSCs) in NSG mice was evaluated for reconstitution of human immune cells and for rectal transmission of HIV-1. These mice were compared to traditional BLT mice implanted with fetal liver and thymus under the renal capsule and mice injected only with HSCs. Renal PedThy mice had similar immune reconstitution in the blood, spleen and intestine as BLT mice, while Leg PedThy mice had transient detection of immune cells, particularly CD4+ T cells and macrophages, the target cells for HIV-1 infection. Rectal transmission and replication of HIV-1 was efficient in BLT mice but lower and more variable in Renal PedThy mice. HIV-1 was poorly transmitted in HSC mice and not transmitted in Leg PedThy mice, which correlated with the frequencies of target cells in the spleen and intestine. Humanization of NSG mice with pediatric thymus was successful when implanted under the kidney capsule, but led to less efficient HIV-1 rectal transmission and replication compared to BLT mice., (© 2023. Springer Nature Limited.)

SARS-CoV-2 spike harbors glycans which function as ligands for lectins. Therefore, it should be possible to exploit lectins to target SARS-CoV-2 and inhibit cellular entry by binding glycans on the spike protein. Burkholderia oklahomensis agglutinin (BOA) is an antiviral lectin that interacts with viral glycoproteins via N-linked high mannose glycans. Here, we show that BOA binds to the spike protein and is a potent inhibitor of SARS-CoV-2 viral entry at nanomolar concentrations. Using a variety of biophysical approaches, we demonstrate that the interaction is avidity driven and that BOA cross-links the spike protein into soluble aggregates. Furthermore, using virus neutralization assays, we demonstrate that BOA effectively inhibits all tested variants of concern as well as SARS-CoV 2003, establishing that multivalent glycan-targeting molecules have the potential to act as pan-coronavirus inhibitors.

Visualizing the transmission and dissemination of human immunodeficiency virus type 1 (HIV-1) in real time in humanized mouse models is a robust tool to investigate viral replication during treatments and in tissue reservoirs. However, the stability and expression of HIV-1 reporter genes are obstacles for long-term serial imaging in vivo . Two replication-competent CCR5-tropic HIV-1 reporter constructs were created that encode either nanoluciferase (nLuc) or a near - infrared fluorescent protein (iRFP) upstream of nef . HIV-1 reporter virus replication and reporter gene expression was measured in cell culture and in humanized mice. While reporter gene expression in vivo correlated initially with plasma viremia, expression decreased after 4 to 5 weeks despite high plasma viremia. The reporter genes were codon optimized to remove cytosine/guanine (CG) dinucleotides, and new CO-nLuc and CO-iRFP viruses were reconstructed. Removal of CG dinucleotides in HIV-1 reporter viruses improved replication in vitro and reporter expression in vivo and ex vivo . Both codon-optimized reporter viruses could be visualized during coinfection and in vivo reporter gene expression during treatment failure preceded detection of plasma viremia. While the dynamic range of CO-iRFP HIV-1 was lower than that of CO-nLuc HIV-1, both viruses could have utility in studying and visualizing HIV-1 infection in humanized mice. IMPORTANCE Animal models are important for studying HIV-1 pathogenesis and treatments. We developed two viruses each encoding a reporter gene that can be expressed in cells after infection. This study shows that HIV-1 infection can be visualized by noninvasive, whole-body imaging in mice with human immune cells over time by reporter expression. We improved reporter expression to reflect HIV-1 replication and showed that two viral variants can be tracked over time in the same animal and can predict failure of antiretroviral therapy to suppress virus.

As a long-acting formulation of the nonnucleoside reverse transcriptase inhibitor rilpivirine (RPV LA) has been proposed for use as preexposure prophylaxis (PrEP) and the prevalence of transmitted RPV-resistant viruses can be relatively high, we evaluated the efficacy of RPV LA to inhibit vaginal transmission of RPV-resistant HIV-1 in humanized mice. Vaginal challenges of wild-type (WT), Y181C, and Y181V HIV-1 were performed in mice left untreated or after RPV PrEP. Plasma viremia was measured for 7 to 10 weeks, and single-genome sequencing was performed on plasma HIV-1 RNA in mice infected during PrEP. RPV LA significantly prevented vaginal transmission of WT HIV-1 and Y181C HIV-1, which is 3-fold resistant to RPV. However, it did not prevent transmission of Y181V HIV-1, which has 30-fold RPV resistance in the viruses used for this study. RPV LA did delay WT HIV-1 dissemination in infected animals until genital and plasma RPV concentrations waned. Animals that became infected despite RPV LA PrEP did not acquire new RPV-resistant mutations above frequencies in untreated mice or untreated people living with HIV-1, and the mutations detected conferred low-level resistance. These data suggest that high, sustained concentrations of RPV were required to inhibit vaginal transmission of HIV-1 with little or no resistance to RPV but could not inhibit virus with high resistance. HIV-1 did not develop high-level or high-frequency RPV resistance in the majority of mice infected after RPV LA treatment. However, the impact of low-frequency RPV resistance on virologic outcome during subsequent antiretroviral therapy still is unclear. IMPORTANCE The antiretroviral drug rilpivirine was developed into a long-acting formulation (RPV LA) to improve adherence for preexposure prophylaxis (PrEP) to prevent HIV-1 transmission. A concern is that RPV LA will not inhibit transmission of drug-resistant HIV-1 and may select for drug-resistant virus. In female humanized mice, we found that RPV LA inhibited vaginal transmission of WT or 3-fold RPV-resistant HIV-1 but not virus with 30-fold RPV resistance. In animals that became infected despite RPV LA PrEP, WT HIV-1 dissemination was delayed until genital and plasma RPV concentrations waned. RPV resistance was detected at similar low frequencies in untreated and PrEP-treated mice that became infected. These results indicate the importance of maintaining RPV at a sustained threshold after virus exposure to prevent dissemination of HIV-1 after vaginal infection and low-frequency resistance mutations conferred low-level resistance, suggesting that RPV resistance is difficult to develop after HIV-1 infection during RPV LA PrEP., (Copyright © 2020 Melody et al.)

Type I and III interferons (IFNs) activate similar downstream signaling cascades, but unlike type I IFNs, type III IFNs (IFNλ) do not elicit strong inflammatory responses in vivo. Here, we examined the molecular mechanisms underlying this disparity. Type I and III IFNs displayed kinetic differences in expression of IFN-stimulated genes and proinflammatory responses, with type I IFNs preferentially stimulating expression of the transcription factor IRF1. Type III IFNs failed to induce IRF1 expression because of low IFNλ receptor abundance and insufficient STAT1 activation on epithelial cells and thus did not activate the IRF1 proinflammatory gene program. Rather, IFNλ stimulation preferentially induced factors implicated in tissue repair. Our findings suggest that IFN receptor compartmentalization and abundance confer a spatiotemporal division of labor where type III IFNs control viral spread at the site of the infection while restricting tissue damage; the transient induction of inflammatory responses by type I IFNs recruits immune effectors to promote protective immunity., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Multiple studies have associated the transcription factor IRF1 with tumor-suppressive activities. Here, we report an opposite tumor cell-intrinsic function of IRF1 in promoting tumor growth. IRF1-deficient tumor cells showed reduced tumor growth in MC38 and CT26 colon carcinoma and B16 melanoma mouse models. This reduction in tumor growth was dependent on host CD8 + T cells. Detailed profiling of tumor-infiltrating leukocytes did not show changes in the various T-cell and myeloid cell populations. However, CD8 + T cells that had infiltrated IRF1-deficieint tumors in vivo exhibited enhanced cytotoxicity. IRF1-deficient tumor cells lost the ability to upregulate PD-L1 expression in vitro and in vivo and were more susceptible to T-cell-mediated killing. Induced expression of PD-L1 in IRF1-deficient tumor cells restored tumor growth. These results indicate differential activity of IRF1 in tumor escape., (©2019 American Association for Cancer Research.)

We demonstrate a facile liquid phase exfoliation method by only using perchloric acid to synthesize graphene quantum dots (GQDs) having excitation independent strong emission with a quantum yield of about 14%. The proposed simplified synthesis strategy can help in overcoming the limitations of existing aqueous routes which produce GQDs with excitation dependent emission and of low quantum efficiency. Photoluminescence (PL) properties of GQDs have been studied in detail to understand the origin of emission. As-synthesized GQDs show excitation independent photoluminesce (PL) which suggests that the synthesized materials do not have any significant defects. Spectral analysis suggests that the PL emission of the well-defined GQDs originates mainly from the peripheral functional groups conjugated with carbon backbone planes. We also demonstrate a relatively longer PL lifetime (average lifetime of about 10 ns) of the synthesized GQDs determined by time correlated single photon counting (TCSPC) measurement and this high lifetime suggests that the synthesized GQDs may be suitable for biomacromolecular probing. In addition, as-synthesized GQDs interestingly show delayed fluorescence and steady state anisotropy, which make the material an appropriate candidate for application in sensing and bioimaging of cells and organisms.

Interleukin 10tm1Cgn (IL 10tm) mice have been utilized as a model of chronic inflammation and declining health span because of their propensity to develop chronic activation in NFkB pathways, skeletal muscle and cardiac changes, and mitochondrial dysfunction. We hypothesized that older IL 10tm frail mice would have alterations similar to frail, older humans in measured parameters of glucose metabolism, oxygen consumption (VO2), respiratory quotient (RQ), spontaneous locomotor activity, body composition and plasma adipokine levels. To test this hypothesis, we investigated these metabolic parameters in cohorts of 3, 10, and 20 month old IL 10tm female mice and age and gender matched C57Bl/6 mice. Insulin sensitivity, glucose homeostasis, locomotor activity and RQ were not significantly altered between the two strains of mice. Interestingly, old IL 10tm mice had significantly decreased VO2 when normalized by lean mass, but not when normalized by fat mass or the lean/fat mass ratio. NMR based body composition analysis and dissection weights show that fat mass is decreased with age in IL 10tm mice compared to controls. Further, plasma adiponectin and leptin were also decreased in IL 10tm.These findings suggest that frailty observed in this mouse model of chronic inflammation may in part be driven by alterations in fat mass, hormone secretion and energy metabolism.

In view of their promising bio-applicability, we have synthesized water-soluble bovine serum albumin (BSA)-encapsulated ZnSe quantum dots (QDs) with visible emission with longer average luminescence lifetimes of approximately 125 ns at ambient conditions. BSA-ZnSe QDs are shown to be efficient selective copper ion probes in the presence of physiologically important metal ions through luminescence quenching with a high Stern-Volmer constant (3.3×10 5  m -1 ). The mechanism of sensing has been explained in terms of electron transfer processes and the apparent rate of electron transfer (K et ) from ZnSe QDs to Cu 2+ has been calculated to be 2.8×10 8  s -1 . It is demonstrated that the negative conduction band potential plays a major role in the feasibility of the electron transfer process, which is reflected in the higher efficacy of ZnSe QDs in sensing copper(II) ions over other group II-VI quantum dots, namely, CdSe, ZnS, or CdS. The results observed with cysteine-capped QDs are almost identical to those with BSA-encapsulated QDs and this presumably negates the possible reason of Cu II ion induced quenching ascribed to its binding with surface groups or replacement of metal sites as proposed by several groups previously., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In view of the enhanced generation of folate receptors in cancerous cells and diseases linked to the deficiency of folic acid, such as anemia, mental devolution, congenital malformation, etc., the development of a simple method for the ultra-sensitive determination of folic acid remains a long-standing issue for practical applications in medicine and biotechnology. Thus, the proposed luminescence based strategy involving multifunctional poly(amidoamine) (PAMAM) dendrimer encapsulated quantum dots (QDs) as a probe provides a simple, fast and efficient method for the selective determination of folic acid at the nano-molar level. Absorption and Fourier transform infra-red (FTIR) spectroscopy provide evidence of the binding of folic acid with dendrimer amine groups. The emission quenching of dendrimer encapsulated CdS QDs follows a linear Stern-Volmer plot with an exceedingly high value of the Stern-Volmer constant (K SV = 8.4 × 10 6 M -1 ) facilitating a higher detection efficiency. Similar quenching analysis with dendrimer-ZnS QDs showed a slightly lower Stern-Volmer constant (K SV = 2.29 × 10 6 M -1 ). The lower probing efficiency of the protein or amino acid capping of QDs has been explained through zeta potential measurements. The solvent polarity dependence suggests a charge transfer process responsible for the emission quenching of CdS QDs, which is static in nature as revealed by lifetime measurements. The determination of folic acid at this low level is not affected by possible interfering molecules, such as vitamin C, vitamin B 12 and uric acid. Calorimetric measurements showed that the exothermic binding of folic acid with a dendrimer follows enthalpy-entropy compensation. The detailed mechanistic aspect of interactions of folic acid with the QD probe helps in a better understanding of the detection process, which in turn can assist in developing a dendrimer based material for image analysis and drug delivery in folate receptor rich cells.

Graphene, and its water soluble derivative graphene oxide, has shown great promise in various biomedical applications, such as cancer therapeutics, drug delivery, etc. and in industrial applications such as enzyme immobilization, etc. Thus, modulation of the activities of different classes of enzymes by graphene materials is an important aspect in the formulation of different biological applications. We have demonstrated here how flavin adenine dinucleotide (FAD) moieties protect the binding site from conformational change in the presence of an inhibitor, graphene oxide, and also explore differences in the mode of interactions between flavo and non-flavo enzymes. It was shown that there was a much greater loss of activity with the non-flavo enzyme, l-lactate dehydrogenase (LDH), of ∼74% compared to that with the flavo-enzyme, glucose oxidase (GOX), of ∼45%, in the presence of equal concentrations of GO. Furthermore, GO acts as an enzyme inhibitor and the mode of inhibition is uncompetitive for GOX and competitive for LDH. Circular dichromism measurements showed a 21% decrease in the α helix of GOX and a 31% decrease in the α helix of LDH in the presence of a given concentration of GO (0.5 mg mL -1 ). There was a slight change in the average emission lifetime of tryptophan in GOX in the presence of GO from 3.2 to 2.6 ns. In contrast, there was no change in the average emission lifetime of tryptophan in LDH in the presence of GO. The extents of fluorescence quenching for GOX and LDH were 39% and 70% upon addition of a certain amount of GO. The present study provides insight into the development of sensors through the immobilization of enzymes and the possible formulation of a multifunctional protein and graphene composite system for various biomedical applications such as bio-sensing, gene and drug delivery, etc.

Importance: In one-third of older men with anemia, no recognized cause can be found., Objective: To determine if testosterone treatment of men 65 years or older with unequivocally low testosterone levels and unexplained anemia would increase their hemoglobin concentration., Design, Setting, and Participants: A double-blinded, placebo-controlled trial with treatment allocation by minimization using 788 men 65 years or older who have average testosterone levels of less than 275 ng/dL. Of 788 participants, 126 were anemic (hemoglobin ≤12.7 g/dL), 62 of whom had no known cause. The trial was conducted in 12 academic medical centers in the United States from June 2010 to June 2014., Interventions: Testosterone gel, the dose adjusted to maintain the testosterone levels normal for young men, or placebo gel for 12 months., Main Outcomes and Measures: The percent of men with unexplained anemia whose hemoglobin levels increased by 1.0 g/dL or more in response to testosterone compared with placebo. The statistical analysis was intent-to-treat by a logistic mixed effects model adjusted for balancing factors., Results: The men had a mean age of 74.8 years and body mass index (BMI) (calculated as weight in kilograms divided by height in meters squared) of 30.7; 84.9% were white. Testosterone treatment resulted in a greater percentage of men with unexplained anemia whose month 12 hemoglobin levels had increased by 1.0 g/dL or more over baseline (54%) than did placebo (15%) (adjusted OR, 31.5; 95% CI, 3.7-277.8; P = .002) and a greater percentage of men who at month 12 were no longer anemic (58.3%) compared with placebo (22.2%) (adjusted OR, 17.0; 95% CI, 2.8-104.0; P = .002). Testosterone treatment also resulted in a greater percentage of men with anemia of known cause whose month 12 hemoglobin levels had increased by 1.0 g/dL or more (52%) than did placebo (19%) (adjusted OR, 8.2; 95% CI, 2.1-31.9; P = .003). Testosterone treatment resulted in a hemoglobin concentration of more than 17.5 g/dL in 6 men who had not been anemic at baseline., Conclusions and Relevance: Among older men with low testosterone levels, testosterone treatment significantly increased the hemoglobin levels of those with unexplained anemia as well as those with anemia from known causes. These increases may be of clinical value, as suggested by the magnitude of the changes and the correction of anemia in most men, but the overall health benefits remain to be established. Measurement of testosterone levels might be considered in men 65 years or older who have unexplained anemia and symptoms of low testosterone levels., Trial Registration: clinicaltrials.gov Identifier: NCT00799617.

The mechanism of surface-enhanced Raman spectroscopy (SERS) is not very clear in view of the magnitude of the contribution of electromagnetic factor as well as the chemical mechanism. This report presents the extent of adsorption at different temperatures in terms of signal enhancements in SERS employing silver nanoparticles (AgNPs) of various shapes as substrate and dye molecules, crystal violet or Rhodamine 6G, as model Raman probes. Initially, the SERS signal increases with increasing temperature until a maximum intensity is reached, before it gradually decreases with increasing temperature. This trend is independent of the shape of the Raman substrates and probes. However, the temperature at which maximum intensity is obtained may depend upon the nature of the Raman probe. The energetics involved in the chemisorption process between dye molecules and AgNPs were determined through isothermal titration calorimetry and their implications for the observed SERS signals were assessed. The maximum heat change occurred at the temperature at which the maximum signal enhancement in SERS was obtained and the enhanced interaction at optimum temperature was confirmed by absorption spectroscopy., (© 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The present report demonstrates how a sulphonation process, a key route for synthesizing water soluble graphene, can influence the optical behavior of precursor graphene oxide, intermediate reaction products and sulphonated graphene. We observed that there is constant emission maximum at 500 nm for graphene oxide in the excitation range of 320-450 nm. During sulphonation, sulphonated reduced graphene oxide (rGO-SO3H) is initially formed which has an emission at 358 nm. However, the reduction of oxygen containing groups in rGO-SO3H with hydrazine hydrate leading to the formation of SG caused a shift in the emission to 430 nm, which has been attributed to the extended delocalization of π-electrons involving the phenyl sulphonate group. In the present investigation, we have identified many existing anomalies in the important spectral features of these materials, such as violation of Kasha's rule on fluorescence and pH dependence emission. Furthermore, it has also been shown that proper care is necessary to be taken in monitoring the fluorescence of sulphonated graphene in view of possible interference from the components produced during sulphonation.

Treatment of iron deficiency anemia in malaria endemic areas is complicated as iron supplementation increases malaria risk while malaria decreases iron absorption. Here we measured the influence of hepcidin expression and non-heme iron during iron supplementation on hepatic Plasmodium berghei numbers in anemic and non-anemic mice. Despite elevated hepatic non-heme iron on the high iron diet, elevated hepcidin expression is associated with less parasite bioavailable iron and lower hepatic parasite loads in anemic, iron deficient mice after both two and six weeks of supplementation. A marginal trend to lower parasite hepatic numbers was seen in non-anemic, iron replete mice. In a transgenic model of severe anemia, mice with a deletion in Sec15l1, which reportedly have normal liver iron and normal hepcidin expression, there were no changes in liver parasite numbers or blood stage numbers or outcome in the lethal Plasmodium yoelii model. In summary during iron supplementation the lower hepatic malaria numbers are regulated more by hepcidin than the absolute level of non-heme hepatic iron., (Copyright © 2015 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Evolutionary characteristics of HIV-1 have mostly studied focusing its structural genes, Gag, Pol and Env. However, regarding the process of HIV-1's evolution, few studies emphasize on genetic changes in regulatory proteins. Here we investigate the evolutionary dynamics of HIV-1, targeting one of its important regulatory proteins, Tat. We performed a phylogenetic analysis and employed a Bayesian coalescent-based approach using the BEAST package to investigate the evolutionary changes in Tat over time in the process of HIV-1 evolution. HIV-1 sequences of subtypes B and C from different parts of the world were obtained from the Los Alamos database. The mean estimated nucleotide substitution rates for Tat in HIV-1 subtypes B and C were 1.53 x 10(-3) (95% highest probability density- HPD Interval: 1.09 x10-3 to 2.08 x 10(-3)) and 2.14 x 10(-3) (95% HPD Interval: 1.35 x 10(-3) to 2.91 x 10(-3)) per site per year, respectively, which is relatively low compared to structural proteins. The median times of the most recent common ancestors (tMRCA) were estimated to be around 1933 (95% HPD, 1907-1952) and 1956 (95% HPD, 1934-1970) for subtypes B and C, respectively. Our analysis shows that subtype C appeared in the global population two decades after the introduction of subtype B. A Gaussian Markov random field (GMRF) skyride coalescent analysis demonstrates that the early expansion rate of subtype B was quite high, rapidly progressing during the 1960s and 1970s to the early 1990s, after which the rate increased up to the 2010s. In contrast, HIV-1 subtype C exhibited a relatively slow occurrence rate until the late 1980s when there was a sharp increase up to the end of 1990s; thereafter, the rate of occurrence gradually slowed. Our study highlights the importance of examining the internal/regulatory genes of HIV-1 to understand its complete evolutionary dynamics. The study results will therefore contribute to better understanding of HIV-1 evolution.

HIV-1 Tat is a regulatory protein that plays a pivotal role in viral transcription and replication. Our study aims to investigate the genetic variation of Tat exon 1 in all subtypes of HIV-1: A, B, C, D, F, G, H, J, and K. We performed phylogenetic, mutation, and selection pressure analyses on a total of 1,179 sequences of different subtypes of HIV-1 Tat obtained from the Los Alamos National Laboratory (LANL). The mean nucleotide divergences (%) among the analyzed sequences of subtypes A, B, C, D, F, G, H, J, and K were 88, 89, 90, 88, 86, 89, 88, 97, and 97, respectively. We revealed that subtype B evolved relatively faster than other subtypes. The second and fifth domains were found comparatively more variable among all subtypes. Site-by-site tests of positive selection revealed that several positions in all subtypes were under significant positive selection. Positively selected sites were found in the acidic domain at positions 3, 4, and 19, in the cysteine-rich domains at positions 24, 29, 32, and 36, in the core domain at position 40, and in the basic domain for the rest of the positions for all subtypes. Positions 58 and 68 in the basic domain were positively selected in subtypes A, B, C and B, C, F, respectively. We also observed high variability within positively selected sites in amino acid positions. Our study findings on HIV-1 Tat genetic variability may contribute to a better understanding of HIV-1 evolution as well as to the development of effective Tat-targeted therapeutics and vaccines.

Background: Hepcidin, a key iron regulatory protein, is elevated in patients with chronic kidney disease (CKD). Its role in the development and progression of the anemia of CKD in children remains poorly defined., Methods: Cross-sectional and longitudinal study in children aged 1-16 years with stage 2-4 CKD in the Chronic Kidney Disease in Children (CKiD) cohort (n = 133) with hepcidin measured at baseline and hemoglobin (HGB) measured annually at follow-up. Anemia was defined as HGB <5th percentile for age/sex OR treatment with an erythropoiesis-stimulating agent (ESA)., Results: Hepcidin levels correlated negatively with glomerular filtration rate (GFR; r = -0.22, p = 0.01) and positively with ferritin (r = 0.67, p < 0.001). At the lower end of the GFR spectrum at baseline (10th percentile, 27.5 mL/min/1.73 m(2)), higher hepcidin was associated with a 0.87 g/dL decrease in HGB during follow-up (95 % CI -1.69, -0.05 g/dL, p = 0.038). At higher GFR percentiles there was no significant association between baseline hepcidin and HGB during follow-up. Among 90 non-anemic subjects at baseline, 23.3 % developed incident anemia. In subjects with GFR  ≤  the median, a higher hepcidin level was associated with an increased risk of incident anemia (at the 10th percentile GFR, HR 3.471, 95 % CI 1.228, 9.810, p = 0.019; at the 25th percentile GFR, HR 2.641, 95 % CI 1.213, 5.750, p = 0.014; at the 50th percentile GFR, HR 1.953, 95 % CI 1.011, 3.772, p = 0.046). Among subjects with GFR at the 75th percentile or above, incrementally higher baseline hepcidin was not associated with increased anemia risk., Conclusions: Higher hepcidin levels are associated with a decreased HGB and an increased risk of incident anemia, and this association is most significant among subjects with lower GFR.

HIV-1Tat (trans-acting activator of transcription) plays essential roles in the replication through viral mRNA and genome transcription from the HIV-1 LTR promoter. However, Tat undergoes continuous amino acid substitutions. As a consequence, the virus escapes from host immunity indicating that genetic diversity of Tat protein in major HIV-1 subtypes is required to be continuously monitored. We analyzed available full-length HIV-1 sequences of subtypes B (n=493) and C (n=280) strains circulating worldwide. We observed 81% and 84% nucleotide sequence identities of HIV-1 Tat for subtypes B and C, respectively. Based on phylogenetic and mutation analyses, global diversity of subtype B was apparently higher compared to that of subtype C. Positively selected sites, such as positions Ser68 and Ser70 in both subtypes, were located in the Tat-transactivation responsive RNA (TAR) interaction domain. We also found positively selected sites in exon 2, such as positions Ser75, Pro77, Asp80, Pro81 and Ser87 for both subtypes. Our study provides useful information on the full-length HIV-1 Tat sequences in globally circulating strains.

The present study demonstrates how reducing agents play an important role in synthesis of silver nanoparticles (AgNPs) in colloidal phase. It is apparent from the observed results that borohydride, one of the most widely used reductants, induces reduction leading to the formation of spherical particles with narrowest size distribution. In contrast, ascorbic or citrate mediated reduction leads to formation of anisotropic silver nanoparticles, indicating the role of anionic carboxylate in template driving process. In view of recent green chemistry approach for synthesizing silver nanoparticles involving glucose as reductant and starch as capping groups, we have followed in detail the dependence of glucose-induced reduction process on different synthesis parameters, such as concentration, temperature and time of reactions. The phase of the synthesized particles was found to be face centred cubic (fcc), which was independent of the reductants employed. Further, we have endeavored to look into the Surface enhanced Raman spectroscopy (SERS) of crystal violet and rhodamine 6G in the presence of AgNPs substrate synthesized by using the reducing agents in question without involving any other structural modulating additive, such as ionic salt, etc. Here, the observed results provide a guideline on the selection of reducing agents and appropriate conditions for application specific synthesis of silver nanoparticles.

Over expression of hepcidin antimicrobial peptide is a common feature of iron-restricted anemia in humans. We investigated the erythroid response to either erythropoietin or RAP-011, a "murinized" ortholog of sotatercept, in C57BL/6 mice and in hepcidin antimicrobial peptide 1 over expressing mice. Sotatercept, a soluble, activin receptor type IIA ligand trap, is currently being evaluated for the treatment of anemias associated with chronic renal disease, myelodysplastic syndrome, β-thalassemia, and Diamond Blackfan anemia and acts by inhibiting signaling downstream of activin and other Transforming Growth Factor-β superfamily members. We found that erythropoietin and RAP-011 increased hemoglobin concentration in C57BL/6 mice and in hepcidin antimicrobial peptide 1 over expressing mice. While erythropoietin treatment depleted splenic iron stores in C57BL/6 mice, RAP-011 treatment did not deplete splenic iron stores in mice of either genotype. Bone marrow erythroid progenitors from erythropoietin-treated mice exhibited iron-restricted erythropoiesis, as indicated by increased median fluorescence intensity of transferrin receptor immunostaining by flow cytometry. In contrast, RAP-011-treated mice did not exhibit the same degree of iron-restricted erythropoiesis. In conclusion, we have demonstrated that RAP-011 can improve hemoglobin concentration in hepcidin antimicrobial peptide 1 transgenic mice. Our data support the hypothesis that RAP-011 has unique biologic effects which prevent or circumvent depletion of mouse splenic iron stores. RAP-011 may, therefore, be an appropriate therapeutic for trials in human anemias characterized by increased expression of hepcidin antimicrobial peptide and iron-restricted erythropoiesis., (© 2014 Wiley Periodicals, Inc.)

Anemia is common in older persons and is associated with substantial morbidity and mortality. One third of anemic older adults have unexplained anemia of the elderly (UAE). We carried out a randomized, wait list control trial in outpatients with UAE and serum ferritin levels between 20 and 200 ng/mL. Intravenous iron sucrose was given as a 200-mg weekly dose for 5 weeks either immediately after enrollment (immediate intervention group) or following a 12-week wait list period (wait list control group). The primary outcome measure was changed in 6-minute walk test (6MWT) distances from baseline to 12 weeks between the two groups. Hematologic, physical, cognitive, and quality of life parameters were also assessed. The study was terminated early after 19 subjects enrolled. The distance walked in the 6MWT increased a mean 8.05±55.48 m in the immediate intervention group and decreased a mean 11.45±49.46 m in the wait list control group (p=0.443). The hemoglobin increased a mean 0.39±0.46 g/dL in the immediate intervention group and declined a mean 0.39±0.85 g/dL in the wait list control group (p=0.026). Thus, a subgroup of adults with UAE may respond to intravenous iron. Enrollment of subjects into this type of study remains challenging., (Published by Elsevier Inc.)

Sarcopenia is an age-related decline in skeletal muscle mass and function that is multifactorial in etiology. Age-related changes in the renin-angiotensin system (RAS), increased oxidative stress, and chronic inflammation likely all contribute to its development. Losartan, an angiotensin II type I receptor blocker (ARB) decreases RAS activity and likely influences oxidative stress and inflammation. Given this, we hypothesized that losartan would improve activity levels and parameters related to inflammation and oxidative stress in older mice. We sought to test this hypothesis by comparing functional and molecular parameters between 18-month-old C57BL/6 mice treated with 50-70 mg/kg/day of losartan over a 4 month-period and age- and gender-matched mice receiving placebo. Losartan treatment significantly improved several activity measurements during treatment period compared to placebo controlled group, including increased time on treadmill, traveling activity, standing activity, and decreased grid contacts (p-values<0.05, 0.001, 0.01; and 0.04 respectively). Grip strength did not improve in treatment group relative to control group over time. Serum IL-6 level in the treated group was significantly lower than that in the control group at the end of treatment (30.3±12.9 vs. 173.0±59.5pg/ml, p<0.04), and mRNA expression of antioxidant enzymes catalase (3.9±0.9 vs. 1.0±0.4) and glutathione peroxidase (4.7±1.1 vs. 1.0±0.4) was significantly higher (p-value: 0.02, and 0.03 respectively) in quadriceps muscle after 4 months of treatment in treated and control groups. These results support the hypothesis that chronic losartan treatment improves skeletal muscle related activity measures in older mice, and that it is associated with more favorable relevant biological profiles in the treatment group. Additional studies are needed to 1) further quantify this functional improvement, 2) further identify mechanisms that influence this improvement, and 3) provide additional rationale for translating these findings into older adults., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background The prevalence of low testosterone levels in men increases with age, as does the prevalence of decreased mobility, sexual function, self-perceived vitality, cognitive abilities, bone mineral density, and glucose tolerance, and of increased anemia and coronary artery disease. Similar changes occur in men who have low serum testosterone concentrations due to known pituitary or testicular disease, and testosterone treatment improves the abnormalities. Prior studies of the effect of testosterone treatment in elderly men, however, have produced equivocal results. Purpose To describe a coordinated set of clinical trials designed to avoid the pitfalls of prior studies and to determine definitively whether testosterone treatment of elderly men with low testosterone is efficacious in improving symptoms and objective measures of age-associated conditions. Methods We present the scientific and clinical rationale for the decisions made in the design of this set of trials. Results We designed The Testosterone Trials as a coordinated set of seven trials to determine if testosterone treatment of elderly men with low serum testosterone concentrations and symptoms and objective evidence of impaired mobility and/or diminished libido and/or reduced vitality would be efficacious in improving mobility (Physical Function Trial), sexual function (Sexual Function Trial), fatigue (Vitality Trial), cognitive function (Cognitive Function Trial), hemoglobin (Anemia Trial), bone density (Bone Trial), and coronary artery plaque volume (Cardiovascular Trial). The scientific advantages of this coordination were common eligibility criteria, common approaches to treatment and monitoring, and the ability to pool safety data. The logistical advantages were a single steering committee, data coordinating center and data and safety monitoring board, the same clinical trial sites, and the possibility of men participating in multiple trials. The major consideration in participant selection was setting the eligibility criterion for serum testosterone low enough to ensure that the men were unequivocally testosterone deficient, but not so low as to preclude sufficient enrollment or eventual generalizability of the results. The major considerations in choosing primary outcomes for each trial were identifying those of the highest clinical importance and identifying the minimum clinically important differences between treatment arms for sample size estimation. Potential limitations Setting the serum testosterone concentration sufficiently low to ensure that most men would be unequivocally testosterone deficient, as well as many other entry criteria, resulted in screening approximately 30 men in person to randomize one participant. Conclusion Designing The Testosterone Trials as a coordinated set of seven trials afforded many important scientific and logistical advantages but required an intensive recruitment and screening effort.

Increased hepcidin antimicrobial peptide correlates with hypoferremia and anemia in various disease states, but its requirement for anemia of inflammation has not been adequately demonstrated. Anemia of inflammation is usually described as normocytic and normochromic, while diseases associated with over expression of hepcidin, alone, are often microcytic and hypochromic. These differences in erythrocyte parameters suggest anemia in many inflammatory states may not be fully explained by hepcidin-mediated iron sequestration. We used turpentine-induced sterile abscesses to model chronic inflammation in mice with targeted disruption of Hepcidin 1 [Hepc1 (-/-)] or its positive regulator, Interleukin-6 [IL-6 (-/-)], to determine whether these genes are required for features characteristic of anemia of inflammation. Although hemoglobin levels did not decline in Hepc1 (-/-) mice with sterile abscesses, erythrocyte numbers were significantly reduced compared to untreated Hepc1 (-/-) mice. In contrast, both hemoglobin concentration and erythrocyte number declined significantly in wild type and IL-6 (-/-) mice with sterile abscesses. Both Hepc1 (-/-) and IL-6 (-/-) mice had increased erythrocyte mean cell volume and mean cell hemoglobin following sterile abscesses, while wild types had no change. Thus, IL-6 (-/-) mice with sterile abscesses exhibit an intermediate phenotype between wild type and Hepc1 (-/-). Our results demonstrate the requirement of Hepc1 for the development of anemia in this rodent model. Simultaneously, our results demonstrate hepcidin-independent effects of inflammation on the suppression of erythropoiesis. Our results suggest chronic anemia associated with inflammation may benefit from interventions protecting erythrocyte number in addition to anti-hepcidin interventions aimed at enhancing iron availability., (Copyright © 2014 Wiley Periodicals, Inc.)

Unlabelled: Increased detection of enterovirus 68 (EV68) among patients with acute respiratory infections has been reported from different parts of the world in the late 2000s since its first detection in pediatric patients with lower-respiratory-tract infections in 1962. However, the underlying molecular mechanisms for this trend are still unknown. We therefore aimed to study the antigenicity and receptor binding properties of EV68 detected in recent years in comparison to the prototype strain of EV68, the Fermon strain. We first performed neutralization (NT) and hemagglutination inhibition (HI) tests using antisera generated for EV68 strains detected in recent years. We found that the Fermon strain had lower HI and NT titers than recently detected EV68 strains. The HI and NT titers were also significantly different between strains of different genetic lineages among recently detected EV68 strains. We further studied receptor binding specificities of EV68 strains for sialyloligosaccharides using glycan array analysis. In glycan array analysis, all tested EV68 strains showed affinity for α2-6-linked sialic acids (α2-6 SAs) compared to α2-3 SAs. Our study demonstrates that emergence of strains with different antigenicity is the possible reason for the increased detection of EV68 in recent years. Additionally, we found that EV68 preferably binds to α2-6 SAs, which suggests that EV68 might have affinity for the upper respiratory tract., Importance: Numbers of cases of enterovirus 68 (EV68) infection in different parts of the world increased significantly in the late 2000s. We studied the antigenicity and receptor binding properties of recently detected EV68 strains in comparison to the prototype strain of EV68, Fermon. The hemagglutination inhibition (HI) and neutralization (NT) titers were significantly different between strains of different genetic lineages among recently detected EV68 strains. We further studied receptor binding specificities of EV68 strains for sialyloligosaccharides using glycan array analysis, which showed affinity for α2-6-linked sialic acids (α2-6 SAs) compared to α2-3 SAs. Our study suggested that the emergence of strains with different antigenicities was the possible reason for the increased detections of EV68 in recent years. Additionally, we revealed that EV68 preferably binds to α2-6 SAs. This is the first report describing the properties of EV68 receptor binding to the specific types of sialic acids.

Anemia of inflammation or chronic disease is a highly prevalent form of anemia. The inflammatory cytokine interleukin-6 (IL-6) negatively correlates with hemoglobin concentration in many disease states. The IL-6-hepcidin antimicrobial peptide axis promotes iron-restricted anemia; however the full role of IL-6 in anemia of inflammation is not well-defined. We previously reported that chronic inflammation had a negative impact on maturation of erythroid progenitors in a mouse model. We hypothesized that IL-6 may be responsible for impaired erythropoiesis, independent of iron restriction. To test the hypothesis we utilized the human erythroleukemia TF-1 cell line to model erythroid maturation and exposed them to varying doses of IL-6 over six days. At 10 ng/ml, IL-6 significantly repressed erythropoietin-dependent TF-1 erythroid maturation. While IL-6 did not decrease the expression of genes associated with hemoglobin synthesis, we observed impaired hemoglobin synthesis as demonstrated by decreased benzidine staining. We also observed that IL-6 down regulated expression of the gene SLC4a1 which is expressed late in erythropoiesis. Those findings suggested that IL-6-dependent inhibition of hemoglobin synthesis might occur. We investigated the impact of IL-6 on mitochondria. IL-6 decreased the mitochondrial membrane potential at all treatment doses, and significantly decreased mitochondrial mass at the highest dose. Our studies indicate that IL-6 may impair mitochondrial function in maturing erythroid cells resulting in impaired hemoglobin production and erythroid maturation. Our findings may indicate a novel pathway of action for IL-6 in the anemia of inflammation, and draw attention to the potential for new therapeutic targets that affect late erythroid development., (© 2013.)

Enterovirus 68 (EV68) infection occasionally manifests with fatal outcomes. However, detection of EV68 in serum and its clinical outcomes are yet to be determined. In this study, we retrospectively tested stored serum samples collected from pediatric pneumonia patients whose nasopharyngeal specimens were positive for EV68. Of total 28 nasopharyngeal sample-positive patients, EV68 was detected in serum samples among 12 (43%) patients aged between 1 and 4 years. Our results suggest that EV68 can cause viremia by which the virus may exhibit systemic manifestations., (© 2013 The Authors. Influenza and Other Respiratory Viruses Published by John Wiley & Sons Ltd.)

Objective: To examine the association between 25-hydroxyvitamin D [25(OH)D] deficiency and anemia in a cohort of otherwise-healthy children and to determine whether race modifies the association between 25(OH)D status and hemoglobin (Hgb)., Study Design: Cross-sectional study of 10,410 children and adolescents ages 1-21 years from the 2001-2006 National Health and Nutrition Examination Survey. Anemia was defined as Hgb less than the 5th percentile for age and sex based on National Health and Nutrition Examination Survey III (1988-1994) data., Results: Lower 25(OH)D levels were associated with increased risk for anemia; <30 ng/mL, adjusted OR 1.93, 95% CI 1.21-3.08, P = .006, and <20 ng/mL, OR 1.47, 95% CI 1.14-1.89, P = .004. In linear regression, small but significant increases in Hgb were noted in the upper quartiles of 25(OH)D compared with the lowest quartile (<20 ng/mL) in the full cohort. Results of race-stratified linear regression by 25(OH)D quartile in white children were similar to those observed in the full cohort, but in black children, an increase in Hgb in the upper 25(OH)D quartiles was only apparent compared with the lowest black race-specific quartile (<12 ng/mL)., Conclusion: 25(OH)D deficiency is associated with increased risk of anemia in healthy US children, but the 25(OH)D threshold levels for lower Hgb are lower in black children in comparison with white children., (Copyright © 2014 Mosby, Inc. All rights reserved.)

Semiconductor nanomaterials have found numerous applications in optoelectronic device fabrication and in platforms for drug delivery and hyperthermia cancer treatment, and in various other biomedical fields because of their high photochemical stability and size-tunable photoluminescence (PL). However, little attention has been paid to exploring the energetics of formation of these semiconductor nanoparticles. We demonstrate that formation of nanocrystals with biofunctionalization supported by widely used groups, BSA and cysteine, is an exothermic spontaneous process driven by enthalpy. The whole energetics of the reaction shows that formation of smaller particles is favored with lower synthesis temperature. Further, it is shown that the thermodynamics of nanoparticle formation is strongly influenced by the conformation of the protein matrix. We also demonstrate that protein supported formation of nanocrystals is thermodynamically more favorable compared to that involving smaller organic thiol groups. The favorable enthalpy of formation compensates unfavorable entropy, resulting in favorable Gibbs free energy. Thus, this study can open up new avenues for establishing a thermodynamic basis for the design of nanosystems with new and tunable properties.

A classic Girl Scout song says, "Make new friends/but keep the old/One is silver/and the other gold." This review focuses on the past decade of discovery in the field of iron homeostasis, which has identified "new friends" or key modifiers of the critical systemic iron regulator, hepcidin antimicrobial peptide. The foundation for these discoveries has been the identification of mutated genes in well-characterized cohorts of patients with inherited hemochromatosis from across the globe. Transgenic mouse models of iron overload and iron-restricted anemia have also contributed to understanding molecular pathophysiology in ways that could never be accomplished in human subjects alone. The majority of these newly discovered molecules coordinate signaling through the bone morphogenetic protein pathway of ligands, receptors and coreceptors, intracellular signaling and transcription. The discovery of these proteins and their interactions with "old friends," such as the 1st known hereditary hemochromatosis gene product, HFE and transferrin receptor, has opened the field of iron homeostasis to include regulatory networks involving signal transduction pathways, in particular, the mitogen-activated protein kinase and Smad pathways. These newly discovered partnerships have also made way for opportunities to develop novel therapeutics for the treatment of iron regulatory disorders, including hemochromatosis.

Anemia is common in older adults and associated with adverse health outcomes in epidemiological studies. A thorough understanding of the complex pathophysiological mechanisms driving anemia in the elderly is lacking; but inflammation, iron restriction, and impaired erythroid maturation are thought to influence the phenotype. We hypothesized that interleukin-6 contributes to this anemia, given its pro-inflammatory activities, its ability to induce hepcidin antimicrobial peptide, and its negative impact on several tissues in older adults. We tested this hypothesis by comparing changes in indices of inflammation, iron metabolism and erythropoiesis in aged C57BL/6 mice to aged mice with targeted deletions of interleukin-6 or hepcidin antimicrobial peptide. Circulating neutrophil and monocyte numbers and inflammatory cytokines increased with age. Decline in hemoglobin concentration and red blood cell number indicated that C57BL/6, interleukin-6 knockout mice, and hepcidin antimicrobial peptide knockout mice all demonstrated impaired erythropoiesis by 24 months. However, the interleukin-6 knock out genotype and the hepcidin antimicrobial peptide knock out genotype resulted in improved erythropoiesis in aged mice. Increased erythropoietic activity in the spleen suggested that the erythroid compartment was stressed in aged C57BL/6 mice compared to aged interleukin-6 knockout mice. Our data suggest C57BL/6 mice are an appropriate mammalian model for the study of anemia with age. Furthermore, although interleukin-6 and hepcidin antimicrobial peptide are not required, they can participate in the development of anemia in aging mice, and could be targeted, pre-clinically, with existing interventions to determine the feasibility of such agents for the treatment of anemia in older adults.

Background: Detection of Enterovirus 68 (EV68) has recently been increased. However, underlying evolutionary mechanism of this increasing trend is not fully understood., Methods: Nasopharyngeal swabs were collected from 5,240 patients with acute respiratory infections in the Philippines from June 2009 to December 2011. EV68 was detected by polymerase chain reaction (PCR) targeting for 5' untranslated region (5'UTR), viral protein 1 (VP1), and VP4/VP2. Phylogenetic trees were generated using the obtained sequences., Results: Of the 5,240 tested samples, 12 EV68 positive cases were detected between August and December in 2011 (detection rate, 0.23%). The detection rate was higher among inpatients than outpatients (p<0.0001). Among VP1 sequences detected from 7 patients in 2011, 5 in lineage 2 were diverged from those detected in the Philippines in 2008, however, 2 in lineage 3 were not diverged from strains detected in the Philippines in 2008 but closely associated with strains detected in the United States. Combined with our previous report, EV68 occurrences were observed twice in the Philippines within the last four years., Conclusions: EV68 detections might be occurring in cyclic patterns, and viruses might have been maintained in the community while some strains might have been newly introduced.

The mechanisms underlying the cellular entry of the HIV-1 Tat protein transduction domain (TatP) and the molecular information necessary to improve the transduction efficiency of TatP remain unclear due to the technical limitations for direct visualization of TatP's behavior in cells. Using confocal microscopy, total internal reflection fluorescence microscopy, and four-dimensional microscopy, we developed a single-molecule tracking assay for TatP labeled with quantum dots (QDs) to examine the kinetics of TatP initially and immediately before, at the beginning of, and immediately after entry into living cells. We report that even when the number of multivalent TatP (mTatP)-QDs bound to a cell was low, each single mTatP-QD first locally induced the cell's lateral transport machinery to move the mTatP-QD toward the center of the cell body upon cross-linking of heparan sulfate proteoglycans. The centripetal and lateral movements were linked to the integrity and flow of actomyosin and microtubules. Individual mTatP underwent lipid raft-mediated temporal confinement, followed by complete immobilization, which ultimately led to endocytotic internalization. However, bivalent TatP did not sufficiently promote either cell surface movement or internalization. Together, these findings provide clues regarding the mechanisms of TatP cell entry and indicate that increasing the valence of TatP on nanoparticles allows them to behave as cargo delivery nanomachines.

Background: Human respiratory syncytial virus (HRSV) is the leading cause of acute lower respiratory tract infection in infants and young children. However, molecular characteristic of HRSV is still unknown in the Philippines., Objective: To describe the molecular epidemiology of circulating HRSV detected in the Philippines., Study Design: From May 2008 to April 2012, nasopharyngeal swabs were collected from infants and children aged between 7 days and 14 years who were hospitalized with severe pneumonia. HRSV was detected by nested PCR targeting M2 gene, and C-terminus of the G gene was sequenced for phylogenetic analysis., Result: Out of total 2150 samples, 19.3% (n = 415) were positive for HRSV, and 65.0% of them (n = 270) were identified as HRSV-A and 35.0% (n = 145) as HRSV-B. There were two major HRSV outbreaks: between June 2008 and February 2009, and between June and March 2012. Majority of HRSV strains detected during the former outbreak were HRSV-A (97.5%, 203/208) whereas during the later outbreak, both HRSV-A (54/158, 34.2%) and HRSV-B (104/158, 65.8%) were detected. All HRSV-A strains were classified as genotype NA1 and all HRSV-B as genotype BA, which had 60-nucleotide duplication in secondary hypervariable region of the G gene. Among HRSV-B positive samples, there were 2 distinct clusters with unique amino acid changes and low homology in compared to other strains in BA, suggesting emergence of new variant of HRSV-B., Conclusion: The study provides an overview of the genetic variation in circulating HRSV viruses in the Philippines along with identification of possibly a novel variant of HRSV-B., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Testosterone administration increases hemoglobin levels and has been used to treat anemia of chronic disease. Erythrocytosis is the most frequent adverse event associated with testosterone therapy of hypogonadal men, especially older men. However, the mechanisms by which testosterone increases hemoglobin remain unknown. Testosterone administration in male and female mice was associated with a greater increase in hemoglobin and hematocrit, reticulocyte count, reticulocyte hemoglobin concentration, and serum iron and transferrin saturation than placebo. Testosterone downregulated hepatic hepcidin mRNA expression, upregulated renal erythropoietin mRNA expression, and increased erythropoietin levels. Testosterone-induced suppression of hepcidin expression was independent of its effects on erythropoietin or hypoxia-sensing mechanisms. Transgenic mice with liver-specific constitutive hepcidin over-expression failed to exhibit the expected increase in hemoglobin in response to testosterone administration. Testosterone upregulated splenic ferroportin expression and reduced iron retention in spleen. After intravenous administration of transferrin-bound (58) Fe, the amount of (58) Fe incorporated into red blood cells was significantly greater in testosterone-treated mice than in placebo-treated mice. Serum from testosterone-treated mice stimulated hemoglobin synthesis in K562 erythroleukemia cells more than that from vehicle-treated mice. Testosterone administration promoted the association of androgen receptor (AR) with Smad1 and Smad4 to reduce their binding to bone morphogenetic protein (BMP)-response elements in hepcidin promoter in the liver. Ectopic expression of AR in hepatocytes suppressed hepcidin transcription; this effect was blocked dose-dependently by AR antagonist flutamide. Testosterone did not affect hepcidin mRNA stability. In conclusion, testosterone inhibits hepcidin transcription through its interaction with BMP/Smad signaling. Testosterone administration is associated with increased iron incorporation into red blood cells., (© 2013 The Authors Aging Cell © 2013 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland.)

Background: We and others have shown previously that over-expression of hepcidin antimicrobial peptide, independently of inflammation, induces several features of anemia of inflammation and chronic disease, including hypoferremia, sequestration of iron stores and iron-restricted erythropoiesis. Because the iron-restricted erythropoiesis evident in hepcidin transgenic mice differs from the normocytic, normochromic anemia most often observed in anemia of inflammation, we tested the hypothesis that chronic inflammation may contribute additional features to anemia of inflammation which continue to impair erythropoiesis following the acute phase of inflammation in which hepcidin is active., Design and Methods: We compared erythropoiesis and iron handling in mice with turpentine-induced sterile abscesses with erythropoiesis and iron handling in hepcidin transgenic mice. We compared erythrocyte indices, expression of genes in the hepcidin regulatory pathway, tissue iron distribution, expression of heme and iron transport genes in splenic macrophages, the phenotype of erythroid maturation and chloromethyl dichlorodihydrofluorescein diacetate, acetyl ester fluorescence., Results: Mice with sterile abscesses exhibited an intense, acute inflammatory phase followed by a mild to moderate chronic inflammatory phase. We found that erythrocytes in mice with sterile abscesses were normocytic and normochromic in contrast to those in hepcidin transgenic mice. We also observed that although hypoferremia resolved in the late phases of inflammation, erythropoiesis remained suppressed, with evidence of inefficient maturation of erythroid precursors in the bone marrow of mice with sterile abscesses. Finally, we observed increased oxidative stress in erythroid progenitors and circulating erythrocytes of mice with sterile abscesses which was not evident in hepcidin transgenic mice., Conclusions: Our results suggest that chronic inflammation inhibits late stages of erythroid production in the turpentine-induced sterile abscess model and induces features of impaired erythropoiesis which are distinct from those in hepcidin transgenic mice.

Inflammation alters hematopoiesis, often by decreasing erythropoiesis and enhancing myeloid output. The mechanisms behind these changes and how the BM stroma contributes to this process are active areas of research. In this study, we examine these questions in the setting of murine Toxoplasma gondii infection. Our data reveal that infection alters early myeloerythroid differentiation, blocking erythroid development beyond the Pre MegE stage, while expanding the GMP population. IL-6 was found to be a critical mediator of these differences, independent of hepcidin-induced iron restriction. Comparing the BM with the spleen showed that the hematopoietic response was driven by the local microenvironment, and BM chimeras demonstrated that radioresistant cells were the relevant source of IL-6 in vivo. Finally, direct ex vivo sorting revealed that VCAM(+)CD146(lo) BM stromal fibroblasts significantly increase IL-6 secretion after infection. These data suggest that BMSCs regulate the hematopoietic changes during inflammation via IL-6.

Objective: To assess whether selenium and carboxymethyl-lysine (CML), two biomarkers of oxidative stress, are independent predictors of anemia in older community-dwelling adults., Methods: Plasma levels of selenium, CML, folate, vitamin B12, and testosterone and markers of iron status and inflammation were measured at baseline in 1036 adults at least 65 y old in the Invecchiare in Chianti Study, a population-based cohort study of aging in Tuscany, Italy, and examined in relation to prevalent anemia and incident anemia over 6 y of follow-up., Results: At enrollment, 11.6% of participants were anemic. Of 472 participants who were non-anemic at enrollment, 72 (15.3%) developed anemia within 6 y of follow-up. At enrollment, plasma CML in the highest quartile (>425 ng/mL) and plasma selenium in the lowest quartile (<66.6 μg/L) predicted incident anemia (hazard ratio 1.67, 95% confidence interval 1.07-2.59, P = 0.02; hazard ratio 1.55, 95% confidence interval 1.01-2.38, P = 0.05, respectively) in a multivariate Cox proportional hazards model that adjusted for age, education, body mass index, cognition, inflammation, red blood cell distribution width, ferritin, vitamin B12, testosterone, and chronic diseases., Conclusion: Elevated plasma CML and low plasma selenium are long-term independent predictors of anemia in older community-dwelling adults. These findings support the idea that oxidative stress contributes to the development of anemia., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Developed countries, such as the United Kingdom, are experiencing a change in demographics resulting in the largest proportion of adults over 65 years of age that our health systems have ever experienced. As such, haematologists must be prepared to evaluate and treat anaemia in a more complicated patient population, but sufficient evidence-based guidelines are lacking. Critical next steps that must be taken to ensure the best care of this population include the determination of appropriate haemoglobin concentrations for older adults in light of age, gender, race, and comorbidities; the development of interventional trials that address physical performance outcomes in addition to haemoglobin targets; and translational studies which address the molecular pathogenesis of anaemia in older adults with the most advanced scientific approaches., (© 2011 Blackwell Publishing Ltd.)

Anemia of inflammation develops in settings of chronic inflammatory, infectious, or neoplastic disease. In this highly prevalent form of anemia, inflammatory cytokines, including IL-6, stimulate hepatic expression of hepcidin, which negatively regulates iron bioavailability by inactivating ferroportin. Hepcidin is transcriptionally regulated by IL-6 and bone morphogenetic protein (BMP) signaling. We hypothesized that inhibiting BMP signaling can reduce hepcidin expression and ameliorate hypoferremia and anemia associated with inflammation. In human hepatoma cells, IL-6-induced hepcidin expression, an effect that was inhibited by treatment with a BMP type I receptor inhibitor, LDN-193189, or BMP ligand antagonists noggin and ALK3-Fc. In zebrafish, the induction of hepcidin expression by transgenic expression of IL-6 was also reduced by LDN-193189. In mice, treatment with IL-6 or turpentine increased hepcidin expression and reduced serum iron, effects that were inhibited by LDN-193189 or ALK3-Fc. Chronic turpentine treatment led to microcytic anemia, which was prevented by concurrent administration of LDN-193189 or attenuated when LDN-193189 was administered after anemia was established. Our studies support the concept that BMP and IL-6 act together to regulate iron homeostasis and suggest that inhibition of BMP signaling may be an effective strategy for the treatment of anemia of inflammation.

The mechanism by which HIV-1-Tat protein transduction domain (TatP) enters the cell remains unclear because of an insufficient understanding of the initial kinetics of peptide entry. Here, we report the successful visualization and tracking of TatP molecular kinetics on the cell surface with 7-nm spatial precision using quantum dots. Strong cell binding was only observed with a TatP valence of ≥8, whereas monovalent TatP binding was negligible. The requirement of the cell-surface heparan sulfate (HS) chains of HS proteoglycans (HSPGs) for TatP binding and intracellular transport was demonstrated by the enzymatic removal of HS and simultaneous observation of two individual particles. Multivalent TatP induces HSPG cross-linking, recruiting activated Rac1 to adjacent lipid rafts and thereby enhancing the recruitment of TatP/HSPG to actin-associated microdomains and its internalization by macropinocytosis. These findings clarify the initial binding mechanism of TatP to the cell surface and demonstrate the importance of TatP valence for strong surface binding and signal transduction. Our data also shed light on the ability of TatP to exploit the machinery of living cells, using HSPG signaling to activate Rac1 and alter TatP mobility and internalization. This work should guide the future design of TatP-based peptides as therapeutic nanocarriers with efficient transduction.

Although anemia is regarded as a relatively common occurrence in older adults, the vigor with which the medical community should intervene to correct this common problem is disputed. Epidemiologic data clearly correlate anemia with functional decline, disability, and mortality. Anemia may contribute to functional decline by restricting oxygen delivery to muscle, or to cognitive decline by restricting oxygen delivery to the brain. On the other hand, the erythron may be a separate target of the same biologic mediators that influence deterioration of physiologic systems that contribute to weakness, functional and cognitive decline, and mortality. Clinical trials aimed at treating anemia in older adults could assess whether physical performance is improved or whether mortality risk declines with improved hemoglobin, but sufficient evidence from such trials is currently lacking. With few guidelines regarding treatment of older adults and significant risk for adverse events associated with transfusion and erythroid stimulating agents, anemia often goes untreated or ignored in geriatric clinics. This article reviews the problem of anemia in older adults, with a particular emphasis on the frail elderly. The gaps in the evidence base for the treatment of anemia in older adults are reviewed and the options for advancing the field are assessed., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Excessive iron absorption is one of the main features of β-thalassemia and can lead to severe morbidity and mortality. Serial analyses of β-thalassemic mice indicate that while hemoglobin levels decrease over time, the concentration of iron in the liver, spleen, and kidneys markedly increases. Iron overload is associated with low levels of hepcidin, a peptide that regulates iron metabolism by triggering degradation of ferroportin, an iron-transport protein localized on absorptive enterocytes as well as hepatocytes and macrophages. Patients with β-thalassemia also have low hepcidin levels. These observations led us to hypothesize that more iron is absorbed in β-thalassemia than is required for erythropoiesis and that increasing the concentration of hepcidin in the body of such patients might be therapeutic, limiting iron overload. Here we demonstrate that a moderate increase in expression of hepcidin in β-thalassemic mice limits iron overload, decreases formation of insoluble membrane-bound globins and reactive oxygen species, and improves anemia. Mice with increased hepcidin expression also demonstrated an increase in the lifespan of their red cells, reversal of ineffective erythropoiesis and splenomegaly, and an increase in total hemoglobin levels. These data led us to suggest that therapeutics that could increase hepcidin levels or act as hepcidin agonists might help treat the abnormal iron absorption in individuals with β-thalassemia and related disorders.