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1.  PEGylation, increasing specific activity and multiple dosing as strategies to improve the risk-benefit profile of targeted radionuclide therapy with 177Lu-DOTA-bombesin analogues
EJNMMI Research  2012;2:24.
Radiolabelled bombesin (BN) conjugates are promising radiotracers for imaging and therapy of breast and prostate tumours, in which BN2/gastrin-releasing peptide receptors are overexpressed. We describe the influence of the specific activity of a 177Lu-DOTA-PEG5k-Lys-B analogue on its therapeutic efficacy and compare it with its non-PEGylated counterpart.
Derivatisation of a stabilised DOTA-BN(7–14)[Cha13,Nle14] analogue with a linear PEG molecule of 5 kDa (PEG5k) was performed by PEGylation of the ϵ-amino group of a β3hLys-βAla-βAla spacer between the BN sequence and the DOTA chelator. The non-PEGylated and the PEGylated analogues were radiolabelled with 177Lu. In vitro evaluation was performed in human prostate carcinoma PC-3 cells, and in vivo studies were carried out in nude mice bearing PC-3 tumour xenografts. Different specific activities of the PEGylated BN analogue and various dose regimens were evaluated concerning their therapeutic efficacy.
The specificity and the binding affinity of the BN analogue for BN2/GRP receptors were only slightly reduced by PEGylation. In vitro binding kinetics of the PEGylated analogue was slower since steady-state condition was reached after 4 h. PEGylation improved the stability of BN conjugate in vitro in human plasma by a factor of 5.6. The non-PEGylated BN analogue showed favourable pharmacokinetics already, i.e. fast blood clearance and renal excretion, but PEGylation improved the in vivo behaviour further. One hour after injection, the tumour uptake of the PEG5k-BN derivative was higher compared with that of the non-PEGylated analogue (3.43 ± 0.63% vs. 1.88 ± 0.4% ID/g). Moreover, the increased tumour retention resulted in a twofold higher tumour accumulation at 24 h p.i., and increased tumour-to-non-target ratios (tumour-to-kidney, 0.6 vs. 0.4; tumour-to-liver, 8.8 vs. 5.9, 24 h p.i.). In the therapy study, both 177Lu-labelled BN analogues significantly inhibited tumour growth. The therapeutic efficacy was highest for the PEGylated derivative of high specific activity administered in two fractions (2 × 20 MBq = 40 MBq) at day 0 and day 7 (73% tumour growth inhibition, 3 weeks after therapy).
PEGylation and increasing the specific activity enhance the pharmacokinetic properties of a 177Lu-labelled BN-based radiopharmaceutical and provide a protocol for targeted radionuclide therapy with a beneficial anti-tumour effectiveness and a favourable risk-profile at the same time.
PMCID: PMC3478187  PMID: 22681935
Gastrin-releasing peptide; Prostate cancer; 177Lu; Radionuclide therapy; PEGylation
2.  Hemoglobin Syracuse (alpha2beta2-143(H21)His leads to Pro), a new high-affinity variant detected by special electrophoretic methods. Observations on the auto-oxidation of normal and variant hemoglobins.
Journal of Clinical Investigation  1975;55(3):469-477.
Family members from four generations were found to have polycythemia and increased whole blood O2 affinity (P50; 11 mm Hg; normal, 27 mm Hg). No abnormal hemoglobin bands were seen after electrophoresis on starch gel at pH 8.6 or agar gel at pH 6.0. Analysis of the oxygenated hemolysate by isoelectric focusing on polyacrylamide gel revealed two closely spaced bands. When deoxygenated hemolysate was analyzed in oxygen-free gels, the two components were more widely separated. About 40% of the patient's hemoglobin focused at a more acid pH than hemoglobin A, indicating a hemoglobin variant with impaired Bohr effect. Chromatography of globin in 8 M urea revealed two beta-chain peaks, the first of which was eluted at a lower buffer molarity than normal beta chain. Fingerprints of tryptic digests of the aminoethylated chains were done on silica gel thin-layer plates. Tp 14 from the abnormal beta chain had slower electrophoretic mobility and a greater Rf value. Amino acid analyses of this peptide gave values identical with those of betaTp 14, except that it contained one proline residue and no histidine. Since the one His in betaTp 14 is in position 143, hemoglobin Syracuse in alpha2beta2-143 His leads to Pro. Native Hb Syracuse could be separated from hemoglobin A on a carboxymethylcellulose column. The inclusion of 0.1 mM EDTA in the preparative buffers proved very useful in reducing the formation of methemoglobin. Oxygen equilibria of purified hemoglobin Syracuse showed high oxygen affinity (P50 value 12% that of hemoglobin A) and lack of cooperativity between subunits (Hill's n equals 1.1). The alkaline Bohr effect was about half that of hemoglobin A. The proline substitution at betaH21 disrupts the helical configuration and probably prevents the formation of salt bonds that are important in stabilizing the deoxy structure and contribute to the alkaline Bohr effect. Since beta143 His is a binding site for 2,3-diphosphoglycerate (2,3-DPG), it is not suprising that hemoglobin Syracuse had markedly impaired reactivity with 2,3-DPG. Hemoglobin Syracuse auto-oxidized more slowly than hemoglobin A, probably reflecting a slower rate of dissociation of oxygen from fully liganded hemoglobin.
PMCID: PMC301774  PMID: 234980
3.  Subunit dissociation of certain abnormal human hemoglobins
Journal of Clinical Investigation  1969;48(1):126-138.
The extent of dissociation of various hemoglobins into subunits was estimated from their elution volumes (Ve) on G-100 Sephadex. Under the same controlled conditions carboxyhemoglobins A, A3 (A1), F, S, and C all had the same elution volumes. The carboxy and cyanmet derivatives of hemoglobin Kansas (a variant with very low oxygen affinity) had a relatively high Ve, indicating a decreased mean molecular weight and therefore an increased tendency to form dimers and even monomers. Conversely, the liganded derivatives of hemoglobin Chesapeake (a variant with high oxygen affinity) had a relatively low Ve, suggestive of an impaired degree of subunit dissociation. Deoxyhemoglobin Chesapeake had a Ve identical with that of deoxyhemoglobin A. Cat hemoglobin, known to have an unusually low oxygen affinity, was found to have a higher Ve than human, dog, rabbit, rat, or guinea pig hemoglobins.
Haptoglobin is thought to bind αβ dimers in preference to the α2β2-tetramer. The comparative haptoglobin affinities of the human hemoglobins were measured by competition between the test hemoglobin and radioactive reference hemoglobin for haptoglobin binding sites. Hemoglobins A, F, S, and C all seemed to bind equally readily, but hemoglobin Kansas and cat hemoglobin showed a higher affinity, and hemoglobin Chesapeake a lower affinity.
These results are in accord with recently proposed models which predict that hemoglobins which have an increased degree of subunit dissociation will have a low oxygen affinity, and vice versa.
PMCID: PMC322198  PMID: 5765016
Protein used in medicine, e.g. interferon, are immunogenic and quickly broken down by the body. Pegylation is a recognized way of preserving their integrity and reducing immune reactions, and works well with enzymes used to degrade amino acids, a recent focus of attention in controlling cancer growth. Of the two arginine-degrading enzymes being explored clinically, arginine deiminase is a decidedly foreign mycoplasm-derived enzyme, whereas human arginase 1 is a native liver enzyme. Both have been pegylated, the former with adjuncts of 20 kD, the latter with 5 kD PEG. Pegylation is done by several different methods, not all of which are satisfactory or desirable.
The preparation of novel polyethylene glycol (PEG) derivatives for modifying proteins is described, but directed specifically at pegylation of recombinant human arginase 1 (rhArg1). rhArg1 expressed in Escherichia coli was purified and coupled in various ways with 5 different PEG molecules to compare their protective properties and the residual enzyme activity, using hepatocellular cell lines both in vitro and in vivo.
Methoxypolyethylene glycol-succinimidyl propionate (mPEG-SPA 5,000) coupled with very high affinity under mild conditions. The resulting pegylated enzyme (rhArg1-peg5,000 mw) had up to 6 PEG chains of 5K length which not only protected it from degradation and any residual immunogenicity, but most importantly let it retain >90% of its native catalytic activity. It remained efficacious in depleting arginine in rats after a single ip injection of 1,500 U of the conjugate as the native enzyme, plasma arginine falling to >0.05 μM from ~170 μM within 20 min and lasting 6 days. The conjugate had almost the same efficacy as unpegylated rhArg1 on 2 cultured human liver cancer (HCC) cell lines. It was considerably more effective than 4 other pegylated conjugates prepared.
Valuable data on the optimization of the pegylation procedure and choice of ligand that best stabilizes the enzyme arginase 1 are presented, a protocol that should equally fit many other enzymes and proteins. It is a long lasting arginine-depleting enzyme in vivo which will greatly improve its use in anti-cancer therapy.
PMCID: PMC2679726  PMID: 19374748
5.  A Long-Acting, MonoPEGylated Human Growth Hormone Analog is a Potent Stimulator of Weight Gain and Bone Growth in Hypophysectomized Rats.
Endocrinology  2007;148(4):1590-1597.
Recombinant human Growth Hormone (GH) is used to treat growth hormone deficiency in children and adults, and wasting in AIDS patients. GH has a circulating half-life of only a few hours in humans and must be administered to patients by daily injection for maximum effectiveness. Previous studies showed that longer-acting forms of GH could be created by modification of GH with multiple 5 kDa amine-reactive polyethylene glycols (PEGs). Eight of nine lysine residues and the N-terminal amino acid were modified to varying extents by amine-PEGylation of GH. The amine-PEGylated GH product comprised a complex mixture of multiple PEGylated species that differed from one another in mass, in vitro bioactivity and in vivopotency. In vitro bioactivity of GH was reduced 100- to 1,000-fold by extensive amine-PEGylation of the protein. Here we describe a homogeneously modified, monoPEGylated GH protein that possesses near complete in vitro bioactivity, a long half-life and increased potency in vivo. The monoPEGylated GH was created by substituting cysteine for threonine-3 (T3C) of GH, followed by modification of the added cysteine residue with a single 20 kDa cysteine-reactive PEG. The PEG-T3C protein has an approximate 8-fold longer half-life than GH following sc administration to rats. Every other day or every third day administration of PEG-T3C stimulates increases in body weight and tibial epiphysis growth comparable to that produced by daily administration of GH in hypophysectomized rats. Long-acting, monoPEGylated GH analogs such as PEG-T3C are promising candidate for future testing in humans.
PMCID: PMC1892190  PMID: 17234711
growth hormone; polyethylene glycol; rat; bone; growth
Video abstract
Alpha-momorcharin (α-MMC) and momordica anti-HIV protein (MAP30) derived from Momordica charantia L. have been confirmed to possess antitumor and antivirus activities due to their RNA-N-glycosidase activity. However, strong immunogenicity and short plasma half-life limit their clinical application. To solve this problem, the two proteins were modified with (mPEG)2-Lys-NHS (20 kDa).
Methodology/principal findings
In this article, a novel purification strategy for the two main type I ribosome-inactivating proteins (RIPs), α-MMC and MAP30, was successfully developed for laboratory-scale preparation. Using this dramatic method, 200 mg of α-MMC and about 120 mg of MAP30 was obtained in only one purification process from 200 g of Momordica charantia seeds. The homogeneity and some other properties of the two proteins were assessed by gradient SDS-PAGE, electrospray ionization quadruple mass spectrometry, and N-terminal sequence analysis as well as Western blot. Two polyethylene glycol (PEG)ylated proteins were synthesized and purified. Homogeneous mono-, di-, or tri-PEGylated proteins were characterized by matrix-assisted laser desorption ionization-time of flight mass spectrometry. The analysis of antitumor and antivirus activities indicated that the serial PEGylated RIPs preserved moderate activities on JAR choriocarcinoma cells and herpes simplex virus-1. Furthermore, both PEGylated proteins showed about 60%–70% antitumor and antivirus activities, and at the same time decreased 50%–70% immunogenicity when compared with their unmodified counterparts.
α-MMC and MAP30 obtained from this novel purification strategy can meet the requirement of a large amount of samples for research. Their chemical modification can solve the problem of strong immunogenicity and meanwhile preserve moderate activities. All these findings suggest the potential application of PEGylated α-MMC and PEGylated MAP30 as antitumor and antivirus agents. According to these results, PEGylated RIPs can be constructed with nanomaterials to be a targeting drug that can further decrease immunogenicity and side effects. Through nanotechnology we can make them low-release drugs, which can further prolong their half-life period in the human body.
PMCID: PMC3396394  PMID: 22802682
ribosome-inactivating proteins; alpha-momorcharin; momordica anti-HIV protein; antitumor; antivirus; (mPEG)2-Lys-NHS (20 kDa); immunogenicity
7.  PEGylation Potentiates the Effectiveness of an Antagonistic Peptide That Targets the EphB4 Receptor with Nanomolar Affinity
PLoS ONE  2011;6(12):e28611.
The EphB4 receptor tyrosine kinase together with its preferred ligand, ephrin-B2, regulates a variety of physiological and pathological processes, including tumor progression, pathological forms of angiogenesis, cardiomyocyte differentiation and bone remodeling. We previously reported the identification of TNYL-RAW, a 15 amino acid-long peptide that binds to the ephrin-binding pocked of EphB4 with low nanomolar affinity and inhibits ephrin-B2 binding. Although ephrin-B2 interacts promiscuously with all the EphB receptors, the TNYL-RAW peptide is remarkably selective and only binds to EphB4. Therefore, this peptide is a useful tool for studying the biological functions of EphB4 and for imaging EphB4-expressing tumors. Furthermore, TNYL-RAW could be useful for treating pathologies involving EphB4-ephrin-B2 interaction. However, the peptide has a very short half-life in cell culture and in the mouse blood circulation due to proteolytic degradation and clearance by the kidneys and reticuloendothelial system. To overcome these limitations, we have modified TNYL-RAW by fusion with the Fc portion of human IgG1, complexation with streptavidin or covalent coupling to a 40 KDa branched polyethylene glycol (PEG) polymer. These modified forms of TNYL-RAW all have greatly increased stability in cell culture, while retaining high binding affinity for EphB4. Furthermore, PEGylation most effectively increases peptide half-life in vivo. Consistent with increased stability, submicromolar concentrations of PEGylated TNYL-RAW effectively impair EphB4 activation by ephrin-B2 in cultured B16 melanoma cells as well as capillary-like tube formation and capillary sprouting in co-cultures of endothelial and epicardial mesothelial cells. Therefore, PEGylated TNYL-RAW may be useful for inhibiting pathological forms of angiogenesis through a novel mechanism involving disruption of EphB4-ephrin-B2 interactions between endothelial cells and supporting perivascular mesenchymal cells. Furthermore, the PEGylated peptide is suitable for other cell culture and in vivo applications requiring prolonged EphB4 receptor targeting.
PMCID: PMC3237458  PMID: 22194865
8.  Design of Homogeneous, MonoPEGylated Erythropoietin Analogs With Preserved In Vitro Bioactivity
Experimental hematology  2006;34(6):697-704.
Erythropoietin (Epo) bioactivity is significantly reduced by modification of lysine residues with amine-reactive reagents, which are the most commonly used reagents for attaching polyethylene glycols (PEGs) to proteins to improve protein half-life in vivo. The aims of this study were to determine whether Epo bioactivity can be preserved by targeting attachment of maleimide-PEGs to engineered cysteine analogs of Epo, and to determine whether the PEGylated Epo cysteine analogs have improved pharmacokinetic properties in vivo.
Materials and Methods
Thirty-four Epo cysteine analogs were constructed by site-directed mutagenesis and expressed as secreted proteins in baculovirus-infected insect cells. Following purification, monoPEGylated derivatives of 12 cysteine analogs were prepared using 20 kDa-maleimide-PEGs. In vitro biological activities of the proteins were measured in an Epo-dependent cell proliferation assay. Plasma levels of insect cell-expressed wild type Epo (BV Epo) and a PEGylated Epo cysteine analog were quantitated by ELISA following intravenous administration to rats.
Biological activities of 17 purified Epo cysteine analogs and 10 purified PEGylated Epo cysteine analogs were comparable to that of BV Epo in the in vitro bioassay. The only PEGylated cysteine analogs that displayed consistently reduced in vitro bioactivities were substitutions for lysine residues, PEG-K45C and PEG-K154C. The PEGylated Epo cysteine analog had a slower initial distribution phase and a longer terminal half-life than BV Epo in rats, but the majority of both proteins were cleared rapidly from the circulation.
Targeted attachment of maleimide-PEGs to engineered Epo cysteine analogs permits rational design of monoPEGylated Epo analogs with minimal loss of in vitro biological activity. Insect cell-expressed EPO proteins are cleared rapidly from the circulation in rats, possibly due to improper glycosylation. Site-specific PEGylation appears to improve the pharmacokinetic properties of Epo.
PMCID: PMC1634893  PMID: 16728273
Biochemistry  2007;46(15):4554-4564.
HexaPEGylated Hb, a non-hypertensive Hb, exhibits high O2-affinity which makes it difficult to deliver desired levels of oxygen to tissue. PEGylation of very low O2-affinity Hbs is now contemplated as the strategy to generate PEGylated Hbs with intermediate levels of O2-affinity. Towards this goal, a doubly modified Hb with very low O2-affinity has been generated. The amino terminal of β-chain of HbA is modified by 2-hydroxy, 3-phospho propylation first to generate a low oxygen affinity Hb, HPPr-HbA. The oxygen affinity of this Hb is insensitive to DPG and IHP. Molecular modeling studies indicated potential interactions between the covalently linked phosphate group and Lys-82 of the trans β-chain. To further modulate the oxygen affinity of Hb, the αα-fumaryl crossbridge has been introduced into HPPr-HbA in the mid central cavity. The doubly modified HbA (αα-fumaryl-HPPr-HbA) exhibits an O2-affinity lower than that of either of the singly modified Hbs, with a partial additivity of the two modifications. The geminate recombination and the visible resonance Raman spectra of the photoproduct of αα-fumaryl-HPPr-HbA also reflect a degree of additive influence of each of these modifications. The two modifications induced a synergistic influence on the chemical reactivity of Cys-93(β). It is suggested that the doubly modified Hb has accessed the low affinity T-state that is non-responsive to effectors. The doubly modified Hb is considered as a potential candidate for generating PEGylated Hbs with an O2-affinity comparable to that of erythrocytes for developing blood substitutes.
PMCID: PMC2572862  PMID: 17381072
10.  Effects of PEGylation on the physicochemical properties and in vivo distribution of organic nanotubes
PMCID: PMC4270402  PMID: 25540582
nanostructure; dispersibility; distribution
11.  Synthesis and Characterization of PEGylated Toll Like Receptor 7 Ligands
Bioconjugate chemistry  2011;22(3):445-454.
Toll like receptor 7 (TLR7) is located in the endosomal compartment of immune cells. Signaling through TLR7, mediated by the adaptor protein MyD88, stimulates the innate immune system and shapes adaptive immune responses. Previously, we characterized TLR7 ligands conjugated to protein, lipid or polyethylene glycol (PEG). Among the TLR7 ligand conjugates, the addition of PEG chains reduced the agonistic potency. PEGs are safe in humans and widely used for improvement of pharmacokinetics in existing biologics and some low molecular weight compounds. PEGylation could be a feasible method to alter the pharmacokinetics and pharmacodynamics of TLR7 ligands. In this study, we systematically studied the influence of PEG chain length on the in vitro and in vivo properties of potent TLR7 ligands. PEGylation increased solubility of the TLR7 ligands and modulated protein binding. Adding a 6–10 length PEG to the TLR7 ligand reduced its potency toward induction of interleukin (IL)-6 by murine macrophages in vitro and IL-6 and tumor necrosis factor (TNF) in vivo. However, PEGylation with 18 or longer chain restored, and even enhanced, the agonistic activity of the drug. In human peripheral blood mononuclear cells, similar effects of PEGylation were observed for secretion of proinflammatory cytokines, IL-6, IL-12, TNF-α, IL-1β and type 1 interferon, as well for B cell proliferation. In summary, these studies demonstrate that conjugation of PEG chains to a synthetic TLR ligand can impact its potency for cytokine induction depending on the size of the PEG moiety. Thus, PEGylation may be a feasible approach to regulate the pharmacological properties of TLR7 ligands.
PMCID: PMC3063487  PMID: 21338093
polyethylene glycol; PEG; Toll like receptor 7
Journal of Clinical Investigation  1973;52(8):2060-2067.
Erythrocytosis associated with the presence of a hemoglobin with increased oxygen affinity has been reported for 10 hemoglobin variants, most of which demonstrate altered electrophoretic mobility. Several members of a family were found to have erythrocytosis, and both the whole blood and the hemoglobin exhibited increased oxygen affinity. Phosphate-free hemoglobin solutions had a normal Bohr effect and reactivity to 2,3-diphosphoglycerate. The electrophoretic properties of the hemoglobin were normal, but on peptide mapping of a tryptic digest of the isolated β-chains, a normal βT11 peptide and an abnormal βT11 with greater Rf were seen. Analysis of the abnormal peptide showed the substitution of leucine for the normal proline at β100 (helical residue G2).
The hemoglobin variant, designated Hb Brigham, serves to emphasize the necessity for detailed evaluation of the structure and function of hemoglobin in familial erythrocytosis even with electrophoretically “normal” hemoglobin.
PMCID: PMC302488  PMID: 4719677
13.  Deer mouse hemoglobin exhibits a lowered oxygen affinity owing to mobility of the E helix
PMCID: PMC3614163  PMID: 23545644
hemoglobin; deer mouse; oxygen affinity; Peromyscus maniculatus
14.  PEGylation of interleukin-10 for the mitigation of enhanced pain states
The anti-inflammatory cytokine interleukin-10 (IL-10) shows promise for the treatment of neuropathic pain, but for IL-10 to be clinically useful as a short-term therapeutic its duration needs to be improved. In this study, IL-10 was covalently modified with polyethylene glycol (PEG) with the goal of stabilizing and increasing protein levels in the CSF to improve the efficacy of IL-10 for treating neuropathic pain. Two different PEGylation methods were explored in vitro to identify suitable PEGylated IL-10 products for subsequent in vivo testing. PEGylation of IL-10 by acylation yielded a highly PEGylated product with a 35-fold in vitro biological activity reduction. PEGylation of IL-10 by reductive amination yielded products with a minimal number of PEG molecules attached and in vitro biological activity reductions of ~3-fold. In vivo collections of cerebrospinal fluid after intrathecal administration demonstrated that 20 kDa PEG attachment to IL-10 increased the concentration of IL-10 in the cerebrospinal fluid over time. Relative to unmodified IL-10, the 20 kDa PEG-IL-10 product exhibited an increased therapeutic duration and magnitude in an animal model of neuropathic pain. This suggests that PEGylation is a viable strategy for the short-term treatment or, in conjunction with other approaches, the long-term treatment of enhanced pain states.
PMCID: PMC3604983  PMID: 19768789
glia; interleukin-10; intrathecal delivery; neuropathic pain; PEGylation
15.  Mapping Polymerization and Allostery of Hemoglobin S Using Point Mutations
The journal of physical chemistry. B  2013;117(42):13058-13068.
Hemoglobin is a complex system that undergoes conformational changes in response to oxygen, allosteric effectors, mutations, and environmental changes. Here, we study allostery and polymerization of hemoglobin and its variants by application of two previously described methods: (i) AllosMod for simulating allostery dynamics given two allosterically related input structures and (ii) a machine-learning method for dynamics- and structure-based prediction of the mutation impact on allostery (Weinkam et al. J. Mol. Biol. 2013), now applicable to systems with multiple coupled binding sites such as hemoglobin. First, we predict the relative stabilities of substates and microstates of hemoglobin, which are determined primarily by entropy within our model. Next, we predict the impact of 866 annotated mutations on hemoglobin’s oxygen binding equilibrium. We then discuss a subset of 30 mutations that occur in the presence of the sickle cell mutation and whose effects on polymerization have been measured. Seven of these HbS mutations occur in three predicted druggable binding pockets that might be exploited to directly inhibit polymerization; one of these binding pockets is not apparent in the crystal structure but only in structures generated by AllosMod. For the 30 mutations, we predict that mutation-induced conformational changes within a single tetramer tend not to significantly impact polymerization; instead, these mutations more likely impact polymerization by directly perturbing a polymerization interface. Finally, our analysis of allostery allows us to hypothesize why hemoglobin evolved to have multiple subunits and a persistent low frequency sickle cell mutation.
PMCID: PMC3973026  PMID: 23957820
Energy landscape; funnel; Gō model; molecular dynamics; machine-learning
16.  Effects of Shielding Adenoviral Vectors with Polyethylene Glycol on Vector-Specific and Vaccine-Mediated Immune Responses
Human Gene Therapy  2008;19(12):1369-1382.
PMCID: PMC2922072  PMID: 18778197
17.  A Better Anti-Diabetic Recombinant Human Fibroblast Growth Factor 21 (rhFGF21) Modified with Polyethylene Glycol
PLoS ONE  2011;6(6):e20669.
As one of fibroblast growth factor (FGF) family members, FGF21 has been extensively investigated for its potential as a drug candidate to combat metabolic diseases. In the present study, recombinant human FGF21 (rhFGF21) was modified with polyethylene glycol (PEGylation) in order to increase its in vivo biostabilities and therapeutic potency. At N-terminal residue rhFGF21 was site-selectively PEGylated with mPEG20 kDa-butyraldehyde. The PEGylated rhFGF21 was purified to near homogeneity by Q Sepharose anion-exchange chromatography. The general structural and biochemical features as well as anti-diabetic effects of PEGylated rhFGF21 in a type 2 diabetic rat model were evaluated. By N-terminal sequencing and MALDI-TOF mass spectrometry, we confirmed that PEG molecule was conjugated only to the N-terminus of rhFGF21. The mono-PEGylated rhFGF21 retained the secondary structure, consistent with the native rhFGF21, but its biostabilities, including the resistance to physiological temperature and trypsinization, were significantly enhanced. The in vivo immunogenicity of PEGylated rhFGF21 was significantly decreased, and in vivo half-life time was significantly elongated. Compared to the native form, the PEGylated rhFGF21 had a similar capacity of stimulating glucose uptake in 3T3-L1 cells in vitro, but afforded a significantly long effect on reducing blood glucose and triglyceride levels in the type 2 diabetic animals. These results suggest that the PEGylated rhFGF21 is a better and more effective anti-diabetic drug candidate than the native rhFGF21 currently available. Therefore, the PEGylated rhFGF21 may be potentially applied in clinics to improve the metabolic syndrome for type 2 diabetic patients.
PMCID: PMC3108960  PMID: 21673953
18.  Preparation and Characterization of PEGylated Amylin
AAPS PharmSciTech  2013;14(3):1083-1097.
Amylin is a pancreatic hormone that plays important roles in overall metabolism and in glucose homeostasis. The therapeutic restoration of postprandial and basal amylin levels is highly desirable for patients with diabetes who need to avoid glucose excursions. Protein conjugation with polyethylene glycol (PEG) has long been known to be a convenient approach for extending the biological effects of biopharmaceuticals. We have investigated the reactivity of amylin with methoxy polyethylene glycol succinimidyl carbonate and methoxy polyethylene glycol succinimidyl propionate, which have an average molecular weight of 5 kDa. The reaction, which was conducted in both aqueous and organic (dimethyl sulfoxide) solvents, occurred within a few minutes and resulted in at least four detectable products with distinct kinetic phases. These results suggest a kinetic selectivity for PEGylation by succinimidyl derivatives; these derivatives exhibit enhanced reactivity with primary amine groups, as indicated by an evaluation of the remaining amino groups using fluorescamine. The analysis of tryptic fragments from mono- and diPEGylated amylin revealed that conjugation occurred within the 1-11 amino acid region, most likely at the two amine groups of Lys1. The reaction products were efficiently separated by C-18 reversed phase chromatography. Binding assays confirmed the ability of mono- and diPEGylated amylin to interact with the amylin co-receptor receptor activity-modifying protein 2. Subcutaneous administration in mice revealed the effectiveness of monoPEG-amylin and diPEG-amylin in reducing glycemia; both compounds exhibited prolonged action compared to unmodified amylin. These features suggest the potential use of PEGylated amylin to restore basal amylin levels.
PMCID: PMC3755165  PMID: 23818080
amylin; diabetes; islet-associated polypeptide; PEGylation; receptor activity-modifying protein
19.  Structural and functional studies on hemoglobin Bethesda (α2β2145 His), a variant associated with compensatory erythrocytosis
Journal of Clinical Investigation  1972;51(9):2299-2309.
Studies have been performed on a 12-yr-old Chinese girl with compensatory erythrocytosis due to the presence of hemoglobin Bethesda comprising about 45% of the red cell hemoglobin. Her parents and three siblings were normal. The oxygen affinity of her blood was markedly increased: under physiological conditions (pH 7.40, 37°C). P50 was 12.8 mm Hg (normal = 26.5 mm Hg). The red cell 2,3-diphosphoglycerate (2.3-DPG) level was normal. The abnormal hemoglobin could not be separated from hemoglobin A by zone electrophoresis at pH 8.6 or isoelectric focusing on polyacrylamide gel. However, after the hemoglobin was split into free α and β chains by treatment with p-hydroxymercuribenzoate (PMB) or 6 M urea, an abnormal β chain was readily demonstrated having a higher isoelectric point (more positive net charge) than normal βA. Structural analysis of the variant β chain demonstrated the substitution of histidine for tyrosine at position 145: hemoglobin Bethesda (α2β2145His). From earlier chemical and crystallographic studies, it has been postulated that this residue is a critical determinant of hemoglobin function. Hemoglobin Bethesda was separated from hemoglobin A by column chromatography. Oxygen equilibria of purified hemoglobin Bethesda revealed an extremely high oxygen affinity (exceeding that of isolated α and β chains), and markedly reduced cooperativity. The Bohr effect of hemoglobin Bethesda was 1/3 that of hemoglobin A. However, hemoglobin Bethesda showed a significant interaction with 2.3-DPG and inositol hexaphosphate.
PMCID: PMC292395  PMID: 4639015
20.  In Vitro Evaluation of the Efficacy of Liposomal and Pegylated Liposomal Hydroxyurea
Breast cancer is one of the most frequent cancer types within women population. Hydroxyurea (HU) is a chemotherapy compound for treatment of patients with cancer diagnosis, including breast cancer associated with several adverse effects. In this study, we applied nanotechnology to decreased drug side effects along with improvement of therapeutic index. Liposomation is widely used in modern pharmacological developments in order to enhance the effects of the drugs. To achieve this, in this study a mixture of phosphatidylcholine and cholesterol was made up and HU was added to the resultant mixture, was then pegylated using Polyethylene Glycol 2000 to increase resistance, applicability and solubility. The mean diameters of nanoliposomal and pegylated nanoliposomal HU were measured by Zeta sizer device and obtained about 402.5 and 338.2 nm. The efficiency of non-pegylated and pegylated liposomal HU was 70.8 and 64.2, respectively. Releasing HU in both formulations was estimated about 25.8 and 21.7 %. Also, this study investigated the cytotoxicity effect of nanoliposomal and pegylated nanoliposomal HU using MTT assay. Results of this investigation showed that the cytotoxic properties of pegylated HU was 3.6 % more than those non-pegylated form, while was 38.93 % more than ordinary from of HU. This study showed that the stability, releasing pattern and cytotoxicity of the pegylated nanoliposomal HU is better than that of nanoliposomal HU.
PMCID: PMC3903928  PMID: 24478555
Breast cancer; Hydroxyurea; Liposome; Pegylated liposome
PMCID: PMC2950395  PMID: 20957159
gene delivery; DOTAP; polyethylene glycol (PEG); biodistribution; lung cancer; xenograft tumor model
22.  Site-Selective Glycosylation of Hemoglobin with Variable Molecular Weight Oligosaccharides: A Potential Alternative to PEGylation
Poly(ethylene glycol) (PEG) conjugation (i.e. PEGylation) is a commonly used strategy to increase the circulatory half-life of therapeutic proteins and colloids, however, few viable alternatives exist to replicate its functions. Herein, we report a method for the rapid site-selective glycosylation of proteins with various sized carbohydrates, up to a molecular weight (MW) of 10,000 Da, thus, serving as a potential alternative for PEGylation. More importantly, the method developed has two unique features. First, traditional protecting group strategies that typically accompany the modification of the carbohydrate fragments are circumvented, allowing for the facile site-selective glycosylation of a desired protein with various sized glycans. Second, the methodology employed is not limited by oligosaccharide size; consequently, glycans of a similar MW to that of PEG, used in the PEGylation of therapeutic proteins, can be employed. To demonstrate the usefulness of this technology, hemoglobin (Hb) was site-selectively glycosylated with a series of carbohydrates of increasing MW (504 to ~10,000 Da). Hb was selected based on the vast wealth of biochemical and biophysical knowledge present in the literature and because of its use as a precursor in the synthesis/formulation of artificial red blood cell substitutes. Following the successful site-selective glycosylation of Hb, the impact of increasing the glycan MW on Hb’s biophysical properties was investigated in vitro.
PMCID: PMC3353419  PMID: 22489605
Glycosylation; glycoprotein synthesis; hemoglobin cysteine 93; PEGylation; carbohydrate; saccharide
23.  PEGylated Cationic Liposome – DNA Complexation in Brine is Pathway-Dependent
Biochimica et biophysica acta  2013;1838(1):398-412.
Cationic liposome-DNA (CL-DNA) complexes, are regarded as promising materials for safe and efficient delivery of genes for therapeutical applications. In order to be used in vivo, these complexes may be coated with a hydrophilic polymer (e.g. polyethylene-glycol, PEG) that provides steric stabilization towards adhesion of proteins and removal by the immune system. In this work we study the influence of the initial salt concentration (Cs) – which modulates the electrostatic interaction between oppositely charged vesicles and DNA – on the structure and stability of PEGylated CL-DNA particles. Previous small-angle X-ray scattering has shown that if non-PEGylated or PEGylated CL-DNA lamellar complexes are prepared in water, their structure is well defined with a high number of lipid membrane-DNA layers (larger than 20). Here we show that if these complexes are transferred to saline media (150 mM NaCl or DMEM, both near physiological conditions), this structure remains nearly unchanged. Conversely, if PEGylated complexes are prepared in saline media, their lamellar structure is much looser, with fewer number of layers. This pathway dependent behavior of PEGylated complex formation in brine is modulated by the liposome membrane charge density and the mole fraction of PEG 2000 in the membranes, with the average number of layers decreasing with increasing Cs and in going from 5 mol% to 10 mol% PEG-lipid. Each of these structures (high and low number of layers) is stable with time, suggesting that despite complex formation being thermodynamically favored, the complexation process in PEGylated membranes, which determines the number of layers per particle, is kinetically controlled. In the extreme case (when polymer repulsions from 10 mol% PEG-lipid are maximized and electrostatic attraction between PEGylated CLs and DNA are minimized at low membrane charge density) complex formation is suppressed at high Cs=150 mM.
PMCID: PMC3891919  PMID: 24060564
CL-DNA; Lipoplexes; DNA; cationic lipid; PEG; PEGylated; salt; steric stabilization; SAXS; gene therapy
24.  Effects of T-state and R-state stabilization on deoxyhemoglobin-nitrite reactions and stimulation of nitric oxide signaling
Recent data suggest that transitions between the relaxed (R) and tense (T) state of hemoglobin control the reduction of nitrite to nitric oxide (NO) by deoxyhemoglobin. This reaction may play a role in physiologic NO homeostasis and be a novel consideration for the development of the next generation of hemoglobin-based blood oxygen carriers (HBOCs, i.e. artificial blood substitutes). Herein we tested the effects of chemical stabilization of bovine hemoglobin in either the T- (THb) or R-state (RHb) on nitrite reduction kinetics, NO-gas formation and ability to stimulate NO-dependent signaling. These studies were performed over a range of fractional saturations that is expected to mimic biological conditions. The initial rate for nitrite-reduction decreased in the following order RHb > bHb > THb, consistent with the hypothesis that the rate constant for nitrite reduction is faster with R-state Hb and slower with T-state Hb. Moreover, RHb produced more NO-gas and inhibited mitochondrial respiration more potently than both bHb and THb. Interestingly, at low oxygen fractional saturations, THb produced more NO and stimulated nitrite-dependent vasodilation more potently than bHb despite both derivatives having similar initial rates for nitrite reduction and a more negative reduction potential in THb versus bHb. These data suggest that cross-linking of bovine hemoglobin in the T-state conformation leads to a more effective coupling of nitrite reduction to NO-formation. Our results support the model of allosteric regulation of nitrite reduction by deoxyhemoglobin and show that cross-linking hemoglobins in distinct quaternary states can generate products with increased NO yields from nitrite reduction that could be harnessed to promote NO-signaling in vivo.
PMCID: PMC3115472  PMID: 21277987
Hypoxia; blood flow; oxygen sensing; blood substitute; nitrite reduction
25.  The acetylation of hemoglobin by aspirin. In vitro and in vivo.
Journal of Clinical Investigation  1975;56(1):201-207.
PMCID: PMC436570  PMID: 237937

Results 1-25 (816156)


Australian Broadcasting Corporation

LOCATION: http://www.abc.net.au/7.30/content/2002/s668492.htm

Broadcast: 05/09/2002

Earth Summit success?: Clarke & Dawe

Reporter: Clarke and Dawe

MARK BANNERMAN: And finally, John Clarke and Bryan Dawe turn their attention to the UN Earth Summit in Johannesburg.

Bryan Dawe is the interviewer.

Dr Kemp is played by John Clarke.

INTERVIEWER: Dr Kemp, thanks for your time.

DR KEMP: Good evening.

Very nice to be talking to you.

INTERVIEWER: How's it going over there?

DR KEMP: Well, it's a very, very interesting experience for me.

It's a most fascinating place.

INTERVIEWER: How did your address go?

DR KEMP: I've got to say that was a bit character building.

Um, but, if nothing else, I think that I've certainly expressed some very interesting ideas.

INTERVIEWER: Did anyone come up to you and say that you'd expressed some interesting ideas?

DR KEMP: I haven't had a lot of contact with the other delegates since I put Australia's position on the environment.

INTERVIEWER: Yes, but, Minister, as Minister for the Environment, surely you must have met other environment ministers.

DR KEMP: I beg your pardon?

As Minister for the Environment?

INTERVIEWER: Yes, you are the Minister for the Environment, aren't you?

DR KEMP: Just bear with me, will you.

Minister for the Environment, Minister FOR the Environment you said?

INTERVIEWER: Minister for the Environment, yep.

DR KEMP: Minister FOR the Environment?

That's what I thought you said.

Yeah, it's David.

INTERVIEWER: Isn't that why you're up there?

That's why you're doing the address?

DR KEMP: I'm just doing an interview with somebody at the moment, John.

No, somebody back there.

Sorry, what's your name?

DR KEMP: With Brian Doyle.

INTERVIEWER: No, Bryan Dawe.

DR KEMP: And he's telling me, John, that I'm the Minister FOR the Environment.

Is that right?

INTERVIEWER: Well, it must be right, Minister.

That's why you're up at the summit.

DR KEMP: Look, John, I've got to tell you, I wish you'd pointed that out a little bit earlier than this.

Well, because I'm over here arguing the opposite.

INTERVIEWER: Minister, you are the Minister for the Environment.

DR KEMP: I mean, I'm actually over here saying the Kyoto protocols, John, aren't the point.

INTERVIEWER: Of course they're the point.

How can you move forward, Minister, if you don't accept the protocols?

DR KEMP: You know what I said, John, because you saw it and because I discussed it with you before leaving.

I basically got up there and told them all to go and get stuffed.

Dr Kemp?

DR KEMP: No, I don't care how good it looked, John.

It's completely out of keeping with our international thinking on the environment, which, it turns out, is what the conference is about.

INTERVIEWER: Minister, does John know that Russia and China have agreed to sign the protocols?

DR KEMP: Just hang on a minute, Barry, I'm just talking to John.

INTERVIEWER: And the fact that Australia is at the moment in the grip of the worst drought it's had in its history?

DR KEMP: That's a very good point.

Good point.

Bill makes a good point, John.

DR KEMP: A lot of the people at the conference here are saying to me how can I, coming as I do from a country with a history of droughts and which is currently in the grip of one of the worst droughts in its own history, come here and argue that we shouldn't do anything about it?

INTERVIEWER: Dr Kemp, I wonder if -

DR KEMP: Alright, I'll give it a go.

DR KEMP: Of course it's a long shot but I'll try anything at this stage, John.

DR KEMP: Just go back to your telly.

I'll give it a crack.

DR KEMP: OK, bye.

INTERVIEWER: Dr Kemp, as Minister for the Environment - .

DR KEMP: Can I just correct you there?

DR KEMP: I'm not the Australian Minister for the Environment.

INTERVIEWER: What do you mean you're not?

Well, what are you?

DR KEMP: I'm actually a wildebeest who has roamed unaccountably from his reservation and finds himself strangely in a television studio trying to conduct an interview about the environment with an interviewer in another country.

INTERVIEWER: Dr Kemp, thanks for your time.

DR KEMP: Put yourself in my position.


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For Immediate Release: February 22, 2011

When was the last time the gas tax increased in Maryland?

Maryland’s gas tax was last increased in 1992 to 23.5 cents per gallon.  The amount of the gas tax today remains 23.5 cents per gallon.

Has the buying power of the state’s gas tax decreased over time?

Yes.  One dollar in 1992 is worth 62 cents today, a reduction of 38%.  While the cost of materials and labor has increased over time, Maryland’s ability to pay for needed transportation improvements has decreased 38%.

What is being proposed?

Under existing Maryland law, gasoline is exempt from the 6% state sales tax.  The proposal now before the General Assembly calls for a phased-in application of the sales tax on gasoline over three years, 2% each year through 2015.

When would this take effect?

If passed as proposed, the bill would take effect July 1, 2012.  Implementation of the first phase (the first 2% of the 6% sales tax) also would take effect July 1, 2012.

How much will a 6% gas tax add to the price of a gallon at the pump?

Based on current average gas price of $3.49 per gallon, this translates into an increase at the pump of    6 cents in the first year, 12 cents in the second year and 18 cents per gallon in the third year at which point the full 6% sales tax will be in effect.

Will the sales tax be applied to the price of a gallon of gas including federal and state gas taxes?

No, the sales tax is only applied to the retail price of a gallon of gasoline once the state gas tax (23.5 cents) and the federal gas tax (18.4 cents) have been subtracted.

Here is an example of how the rate would be calculated at full implementation of the 6% sales tax.  The average retail price of gasoline is $3.49 per gallon based on the final quarter of 2011, according to the American Petroleum Institute.   After subtracting the 23.5 cents per gallon state gas tax and the 18.4 cents per gallon federal gas tax (total of 41.9 cents per gallon), the net retail price equals $3.07 per gallon.  Sales tax of 6% applied to $3.07 equals to 18.4 cents per gallon.

What is the impact, per vehicle of the 6% sales tax?

When the full 6% sales tax is phased in, the typical driver is projected to pay roughly $2.00 more per week, per vehicle.

If the price of gas dramatically increases during the phase-in period will the sales tax on gas dramatically increase as well?

No.  A braking mechanism will be in place to slow down the phase-in of the sales tax if a dramatic fluctuation in the price of gas occurs.  If the price of gasoline in Year One were to increase by more than 15% over the prior year, the rate in Year Two would remain at 2%.  A similar approach will be used in each successive year until the full 6% is implemented.

Will there be any way to make sure that transportation money doesn’t get used for other needs?

The legislation now being considered by the 2012 General Assembly contains a series of measures to ensure transportation revenue is used solely for transportation purposes except only in the most dire circumstances, such as when a Governor declares a state of emergency as a result of a major catastrophe.  A transfer would require prior consultation with the Treasurer regarding its financial impact on the Transportation Trust Fund, as well as a three-fifths majority vote by two legislative financial committees before being considered by the full House and Senate.   The legislation also requires an automatic repayment within five years.

Some say the sales tax will be complicated to implement.  Is that correct?

No.  The sales tax will be collected at the wholesale level just as the state gas tax is today.

Where will Maryland’s gas tax rank among other states if the increase is implemented?

In the first year of the phased implementation of the sales tax, the amount Marylander’s pay in tax related to gasoline would increase from 23.5 cents - 30th in the nation to 29.5 cents - 18th in the nation.  This amount is still below the national average of 30.5 cents per gallon.

How many other states impose a sales tax on gasoline?

According to the American Petroleum Institute, 39 states apply a sales tax or some other form of tax to their gas tax.  Specifically, eight states apply the sales tax to gasoline.  They include:

California         Indiana
Florida             Michigan
Georgia           New York
Illinois             Virginia (Northern Virginia Transportation District)

What are the current total tax rates on gasoline in surrounding states and where do those states rank?

West Virginia            33.40 cents/gal        11th highest in the nation
Pennsylvania            32.30 cents/gal        15th highest in the nation
District of Columbia  23.50 cents/gal        29th highest in the nation
Delaware                 23.00 cents/gal        32nd highest in the nation
Virginia                    19.80 cents/gal        41st highest in the nation

Has all the money borrowed from the Transportation Trust Fund for non-transportation purposes been repaid to the Transportation Trust Fund?

Yes.  Since 1984, $540 million has been transferred from the Transportation Trust Fund to the General Fund.   During that same period of time, a total of $544 million has been transferred from the General Fund to the Trust Fund as repayment or as part of the independent funding plan enacted by the General Assembly for the Intercounty Connector.

How does the amount of money generated by the 6% sales tax on gasoline compare with what was recommended by the Blue Ribbon Commission?

The Blue Ribbon Commission recommended a series of potential increases using various fees and taxes to reach its recommended level of $870 million annually, including a phased-in 15 cent increase in the state gas tax.

The current proposal is more streamlined and simpler for consumers to follow and is less of a burden on the taxpayer.  By focusing solely on the application of the sales tax on gasoline, this proposal generates $613 million annually for transportation.  While it adds an estimated 18 cents to the retail price of a gallon of gasoline, it does not subject taxpayers to a variety of other transportation-related fees that would increase the financial burden on motorists even more.  The estimated 18 cents is based on applying the 6% sales tax to the average retail price of gasoline minus the federal and state gas taxes.

What has taken place regarding transportation aid to counties, municipalities and Baltimore City?

It is important to make the distinction between the transfer of dollars between the Trust Fund and the General Fund versus what has taken place regarding local transportation aid to counties, municipalities and Baltimore City.  The State provides local transportation aid just like the state provides local aid for education, school construction or public safety.  As budgets became tighter in recent years, state leadership was faced with the difficult decision of what local aid to reduce as part of the overall effort to balance the state budget, as required by law.  Going back to as early as 2003, the difficult decision was made by leadership to reduce local transportation aid while opting to maintain the flow of other types of aid to the counties, municipalities and Baltimore City.

Does this proposal provide any ability to restore local transportation aid to counties, municipalities and Baltimore City?

Yes.  The proposal would establish a Local Transportation Infrastructure Aid Account where the counties, municipalities and Baltimore City would share a portion of the revenue generated by the application of the 6% sales tax on gasoline.  Twenty percent of the revenue generated by the sales tax would go to local governments.  This amount is in addition to the level of funding provided from existing transportation aid, known as Highway User Revenues, which will continue.  Combined, existing Highway User Revenues and the new Local Transportation Infrastructure Aid funding will restore local funding closer to the level provided in 2008 -- the year when state support for local transportation needs reached the highest level in Maryland history.

Why does the state need more money for transportation improvements?

Since 2008, except for a handful of projects made possible as a result of the infusion of federal dollars from the American Recovery and Reinvestment Act, Maryland has not been able to break ground on any major transportation improvements, all while its citizens face some of the worst traffic congestion in the nation.

Maintaining the state’s existing roads, bridges, transit, port and airport is Maryland’s top priority.  It is only after this existing system preservation need has been met that the state dedicates any dollars remaining to new projects intended to improve or expand the transportation network.  The state’s ability to do so has been dramatically reduced due to the national economic downturn and its impact on revenues that support transportation.  Maryland now has a backlog of projects needed to address congestion or to support economic development.  Simply building the top priority project as identified by the each of the 23 counties and Baltimore City would cost roughly $12 billion.
With any new funding, Maryland will continue to implement a balanced approach in the selection of priority projects that would address all transportation needs, including:  roads, bridges, transit, port and airport.  The top priority projects as identified by the 23 counties and Baltimore City demonstrate the varied needs across the State. The projects can be found here:

Why is an increase in transportation funding necessary when tolls were recently raised?

When it comes to funding, Maryland’s toll facilities are entirely separate from the rest of the state’s transportation network.   The Hatem, Nice, Key and Bay bridges, the Baltimore Harbor tunnels, the Kennedy Highway along I-95 and the ICC are owned and operated by the independent Maryland Transportation Authority (MDTA).  All of these facilities are supported solely by the toll revenue that they generate.  Tolls cover the cost of constructing, operating and maintaining these eight facilities.  The MDTA receives no funding from the state Transportation Trust Fund.  Conversely, the Transportation Trust Fund receives no revenue from the MDTA’s toll facilities.

The recent toll increase was required to pay for prior decisions made as far back as 2005 to fund needed rehabilitation of existing MDTA facilities, some of which are up to 70 years old, and simultaneously construct two major transportation projects (I-95 express toll lanes north of Baltimore and the ICC).

# # #


Latest Earthquakes - 14 of 13

Latest Earthquakes FAQs - 13 Found

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Why isn't the distance to the nearest fault provided?

Seismologists evaluate the hypocenter location and the focal mechanism of an earthquake to decide if the earthquake occurs on a named fault. Research shows that many earthquakes occur on small, un-named faults located near well known faults. For example, most of the aftershocks of the 1989 M6.9 Loma Prieta earthquake occurred on small, subsidiary faults within a few hundred meters of the mainshock rupture plane. On other fault segments like the San Andreas fault near Parkfield, most of the earthquakes occur on the San Andreas fault. It is difficult to automate this decision process, and it would be misleading to imply on the basis of only distance that an earthquake occurs on a named fault.


Slow, cold start to universe suggested

Idea provides alternative to Big Bang theory of cosmic origin

10:10am, January 31, 2014
Sponsor Message

The universe may have emerged not with a hot Big Bang but with a long, cold slog, a physicist proposes in a paper posted online January 21 at arXiv.org.

Over the last half-century, most cosmologists have come to agree that all matter initially exploded from a single point. An instant later, the hot, dense universe ballooned dramatically in an event called inflation. A slower expansion then proceeded for billions of years.

But the Big Bang model requires the universe to start from what physicists call a singularity, a point of infinite density at which physical laws break down. A theory that avoids a singularity without introducing other complications would fit better with quantum mechanics and general relativity, physicists’ best explanations of nature’s fundamental forces.

Christof Wetterich, a theoretical physicist at Heidelberg University in Germany, says he has created such a picture. In Wetterich’s theory, fundamental particles become heavier over time, while gravity weakens. This logic leads to a cosmic history in which the universe still underwent inflation but did not necessarily continue expanding. And instead of starting with a Big Bang, time before inflation could stretch into the infinite past.

Whether researchers adopt Wetterich’s theory may depend on how it fares on a subjective criterion physicists call naturalness, Afshordi says; the term means that a theory does not require strange-seeming explanations of phenomena scientists observe. Wetterich argues that, by eliminating the Big Bang, his model may provide a more natural picture of cosmic evolution.

But Afshordi doubts cosmologists will accept changing particle masses to get rid of a singularity. “Physicists happen to be conservative by nature,” he says. “We don’t take models seriously until basically we have no other option.”

Ruth Durrer, a theoretical physicist at the University of Geneva, agrees that Wetterich’s paper is interesting, but she is unsure whether it adds anything fundamentally new. Other researchers have built models with changing particle masses, she notes. “I think this paper looks more revolutionary than it is,” she adds.

Wetterich stresses that he is not trying to overthrow the Big Bang; he says both it and his slow freeze model are consistent with all known observations.

More from Science News


Gestational diabetes develops during pregnancy and usually disappears afterwards, but it increases the risk of later diabetes. The condition is managed by changing the diet and activity and sometimes adding insulin or other medications.

The study points out the need for doctors to monitor patients with gestational diabetes after they give birth to help prevent atherosclerosis — the buildup of plaque in the arteries that can lead to heart attack and stroke, said researchers.

“Pregnancy has been under-recognized as an important time period that can signal a woman’s greater risk for future heart disease. This signal is revealed by gestational diabetes, a condition of elevated blood sugar during pregnancy,” said Erica P. Gunderson, Ph.D., M.S., M.P.H., study lead author and senior research scientist in the Division of Research at Kaiser Permanente Northern California in Oakland, Calif.

Researchers measured heart disease risk factors in 898 women, ages 18-to-30-years old who did not have heart disease or diabetes before the study. They started before pregnancy and continued after they gave birth. The women were periodically tested for diabetes and metabolic conditions.

They found that 119 women, or 13 percent, developed gestational diabetes.

Researchers took measurements of carotid artery wall thickness using ultrasound imaging an average of 12 years after pregnancy. This is an early measure of early atherosclerosis.

Among the women who did not develop diabetes or the metabolic syndrome during the 20-year follow up, they found a 0.023 mm larger average carotid artery intima-media thickness in those who had gestational diabetes compared to those who didn’t. The difference was not attributed to obesity or elevated glucose before pregnancy.

At the end of the study, 13 women had cardiovascular events, one of whom had gestational diabetes.

The study controlled for age, race, number of births, pre-pregnancy body mass index, fasting blood glucose, insulin, lipids and blood pressure.

Currently, there are no uniform national recommendations for screening for heart disease risk factors in pregnancy to determine risk of later heart disease, Gunderson said.

The article was published online in the Journal of the American Heart Association.

For more information:


I am not particularly welcome in some circles because my attempts to avoid the smoke are seen as "grandstanding" or being a ridiculous jerk. My only other option is to stay home and miss out on time with family and the concerts, festivals and other outdoor activities I enjoy.

If I were on the Nobel Prize nominating committee, I would certainly vote for you in the category of "Patience and Compassion." It's sometimes difficult for me to understand how some of these people can actually manage in this world. Keep up the good work. -- M. in Montreal

DEAR M.: One reason we write this column is to help those who want an unbiased opinion about situations they cannot wrap their heads around. We know that it often takes an objective viewpoint to see the forest for the trees, because when you are in the middle of a crisis -- unhappy, confused and hurt -- it helps to have someone in your corner to steer you in a more focused direction. We hope we can provide that for our readers. And if it holds your interest at the same time, even better. Thanks for writing.


Facial perception

Facing up to faces: perception from brains to robots

Facial perception Your brain processes lots of tiny and subtle clues about faces whenever you interact with others. Could robots and computers learn to do the same thing?

Face it, faces matter. They are windows to our emotions and identities. At this exhibit you can explore how scientists are trying to understand how the brain understands faces and you can help scientists create the next generation of life-changing software and robots.

How does it work?

There are parts of the human brain that scientists believe are specifically concerned with processing faces. If we can understand this part of the brain, in particular how moving faces and emotional expressions are processed, then we can start to build computer systems that mimic this useful ability.

If computer systems like these are built, then they might be used in robots to allow them to recognise faces and facial expressions.These robots could respond to people’s social cues, allowing new and useful technologies to be built.

This exciting scientific approach of using biology to build better technology calls for a range of different types of experts like psychologists and computer scientists to work together to explore new ways to create tomorrow’s gadgets, gizmos and applications.

Video and Audio

The video below and podcast demonstrate some of the science of this exhibit. The first shows movement from the face on the left is transferred to the face on the right.


The Maya invented dentistry. But not for teeth health or cleanliness – they were all about aesthetic. Mayan dentists would drill into patients teeth and precisely place gemstones in the cavity. So precisely, in fact, that most skeletons’ teeth still hold their gemstones, a thousand years later. And Mayan dentistry was not just for the wealthy or powerful.  Citizens from all classes had their teeth filled with gemstones!


Strategy Guide - Guide for Krazy Ivan

Scroll down to read our guide named "Strategy Guide" for Krazy Ivan on PlayStation (PSX), or click the above links for more cheats.

                              Krazy Ivan FAQ 1.1
                              by Wendell Martin
                               Created 5/25/96
                               Revised 7/11/96

 Krazy Ivan is a Psygnosis game for the Sony PlayStation.  It has a look
 somewhat similar to giant robot/mech games, and resembles them in a few
 ways, but is more of a 3D shooter.  Actually, it plays rather like an
 improved (*much* improved ) version of Cyber Sled.  Viewed from this
 perspective, Krazy Ivan succeeds well.

 You are Ivan Popovich, commander of a Steel Cossack powersuit, who is
 fighting to free Earth from alien domination.  Your psychotic hatred for
 aliens makes you perfect for the job.  "Ivan" is pronounced "ee-VAHN" in
 most of the game (since that's the correct Russian pronunciation), but the
 American in a cut scene does pronounce it "EYE-van."  BTW, the Russian word
 appearing sideways on many pages of the manual and on the CD is OPASNO (in
 Latin letters) and means "dangerous."

 The game has well-done FMV introduction and cut scenes (especially if you
 like heavy Russian accents ), and the in-game graphics are beautiful.
 The controls can be reconfigured into one of several useful presets, so
 they're pretty easy to get used to, regardless of how you prefer them.  One
 odd aspect is that side-stepping/strafing is not continuous; you must
 repeatedly press the button to repeatedly side-step (unless your controller
 has a turbo option).

 There are five zones in the game, each with several "sentients" (bosses)
 and several types of drones (20 sentients and 10 drone types total).  You
 are free to engage the sentients in any order, but it's probably wisest to
 take out the toughest one first, while you're at full capacity.  Once you
 engage a sentient, you must defeat it before going on.

 Destroying sentients and drones releases prisoners, which you can pick up
 for points, and various pickups which are usually, but not always, helpful
 to pick up.  You can destroy drones in order to gather prisoners and
 pickups, but if you take too long between engaging sentients, Black Knight
 will appear, and you must defeat him before going on.  Once you've defeated
 all sentients on a level, you must destroy the zonal shield generator to
 complete the zone and get to the upgrade section.

 After each upgrade, you may save your game to a memory card before
 continuing.  You also need to save your configuration if you want to keep
 your high scores and audio and control settings, but don't save your
 configuration onto a saved game or you'll lose your progress in that game.

 The game has three difficulty levels, and it's simple to beat it on Easy.
 It's correspondingly more difficult at Medium and Hard, but is a fairly
 quick game at any level (each zone takes about 10-20 minutes to play,
 depending on how much time you spend on the drones).

 This is one of the easier games available for PlayStation, and seems to have
 no cheat codes (if I learn of any, I'll post them).  If you're having
 trouble winning, try the Easy setting (it's *really* easy).  If Easy gives
 you trouble, try circling your enemies (side-stepping while turning to
 bring your guns to bear).  Fire quickly and repeatedly, and use your
 heaviest (missile/special) weapon against the toughest enemy on the level.

 Guns Rearm (vertical bullet) - rearms all guns
 Missiles Rearm (angled missile) - rearms your missiles
 Special Rearm (five small yellow balls) - rearms your special weapon

 Energia Mode (yellow lightning bolt) - brief invincibility plus speed boost
 Zombie Mode (skull) - brief invincibility plus speed reduction
 Schizo Mode (light/dark circle) - brief invincibility plus reversed controls

 Air Strike (large yellow ball) - Arrow drones appear and attack you

 Energy Core (red sphere) - repairs your shields and provides a variable
   number of energy cores which are used to "buy" post-mission upgrades.

ZONE INFORMATION (sentient strengths are the initial levels on your HUD)

 Available in any zone

  Arrow - flying dark green drone, summoned by Air Strike pickup

  Black Knight - black powersuit sentient - strength 7

 Zone: Russia - 50% difficulty - 154 prisoners

  Trilobite - tracked black land drone that lays mines
  Bane - flying green drone with four jet engine pods
  Manta - flying red drone with flapping wings
  Transona - flying green drone with three jet engine pods

  Dedlok - gray and brown, ape-like sentient - strength 5.5
  Dwarf - gray and red bipedal walker with huge launchers - strength 5.5
  Raptor - green bipedal walker sentient, toughest in zone - strength 5.5

 Zone: Saudi Arabia/Middle East - 70% difficulty - 187 prisoners

  Manta, Trilobite
  Hovertank - green land drone with turret on top
  Demonseed - floating brown drone with two engine pods

  Prowl - orange tiger sentient, toughest in zone - strength 5
  Qualm - light blue humanoid sentient - strength 5
  Sentinel - floating brown squid sentient - strength 6.5

 Zone: France/Europe - 80% difficulty - 416 prisoners

  Bane, Hovertank, Trilobite
  Contrail - flying dark blue/gray and red drone with hang-down turret
  Wasp - flying brown wasp-like drone

  Arachan - tall, white, four-legged spider sentient - strength 8
  Behemoth - gray and red "Empire Strikes Back AT-AT" walker - strength 8
  Buzzsaw - flying green and yellow insectoid sentient - strength 5
  Carapace - flying brown insectoid sentient with jet engines - strength 6.5
  Scorch - dark green snake sentient - strength 6

 Zone: North America/USA - 90% difficulty - 362 prisoners

  Arrow, Bane, Contrail, Demonseed, Transona, Trilobite, Wasp
  Reaper - red and white land drone with helicopter blades
  Roach - brown roach-like land drone that lays mines

  Fireball - purple and yellow bipedal walker sentient - strength 7
  Granite - green, domed, quadrupedal sentient - strength 7.5
  Reflex - flying red "Empire Strikes Back probe droid" sentient - strength 5
  Thunderfoot - red and gray humanoid sentient - strength 8

 Zone: Japan - 100% difficulty - 408 prisoners
       (Runnning into the shield generator as is explodes will take you
       to the "real" last zone, facing Black Knight.)

  Arrow, Contrail, Demonseed, Reaper, Roach, Transona, Wasp

  Arclight - gray and yellow tripedal sentient - strength 9
  Claw - yellow and red crab-like sentient - strength 8
  Colossus - tan bipedal walker sentient - strength 9
  Flamefront - light blue humanoid sentient - strength 9

(Energy core "prices" are for Easy/Medium/Hard)

 Armor - 1/2/2 energy core per 10% (up to 100%)

 Guns (up to five)

   2 x 30mm Rotary Cannon - infinite ammo - initial equipment
   2 x 50mm Rotary Cannon - 300 rounds each - initial equipment
   2 x Laser Cannon - 250 rounds each - 10/12/14 energy cores
   2 x Plasma Cannon - 60 rounds each - 14/16/18 energy cores
   2 x Z Beam Cannon - 70 rounds each - 20/24/30 energy cores

 Missile Weapons (one at a time)

   2 x Wolf Rocket Launcher - 10 rockets each - initial equipment
   Hyena Missile Launcher - 10 missiles - 10/12/14 energy cores
   2 x Hyena Missile Launcher - 10 missiles each - 5/7/9 energy cores
   Cerberus Missile Launcher - 10 missiles - 12/14/6 energy cores
   2 x Cerberus Missile Launcher - 10 missiles each - 6/8/10 energy cores
   4 x Cerberus Missile Launcher - 10 missiles each - 10/12/14 energy cores

 Special Weapons (one at a time)

   Scythe Beam - 5 charges - initial equipment
   Vortex Bomb - 3 charges - 10/12/14 energy cores
   ULF Pulse - 1 charge - 12/14/16 energy cores
   EM Pulse -  1 charge - 15/17/19 energy cores
   Kraken - 1 charge - 18/20/22 energy cores
   Medusa - 1? charge - 23/25/27? energy cores

If you have corrections or additions to this FAQ, please e-mail me and I'll
implement them with credit.  The latest version of this FAQ can be found via
my home page.

  [email protected]

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Picture of Block Party 2.0
Throwing a block party is a great way to get to know your neighbors. Just ask York from Chicago.

York has lived on the same block almost his entire life -- nearly 40 years -- but, as his wife teased him, he didn't really know his neighbors. One big reason is that York lives on a block near Lake Michigan that primarily is home to condo buildings, rather than the single-family bungalows or three-story greystones and A-frame homes that characterize many Chicago neighborhoods. Without a front stoop, or even their own front doors, neighbors can easily become invisible to one another.

When York became president of his neighborhood group, he realized how much easier it is to solve community problems if you know your neighbors. He decided to throw a block party after talking to the staff at his local alderman's office to see what was entailed.

York's block party was a huge success, attracting about 200 people from his block. This Instructable recreates how he turned those strangers into neighbors.

Step 1: Organize a team of 10 to 12 volunteers

Picture of Organize a team of 10 to 12 volunteers
In early May, York posted a flyer in his neighborhood, calling for volunteers to help organize a block party. He initially attracted a group of about 7 or 8 enthusiastic individuals, and word-of-mouth grew the group to his target number of 12.

You may want to organize your volunteers into committees, by task. Here are four basic committees every block party needs:

food and drinks
set up and clean up
Megan Allen6 months ago

Seriously awesome 'ible! Not only were the instructions clear for organizing a neighborhood block party, BUT also on top of that there was some great inspiration for how to use marketing tools and techniques in everyday life. Word-of-mouth advertising, local business outreach, and surveys - all great things!

JohnJY5 years ago
 I hate the people who live near me, but this is a great Instructable.


Basic Preserved Lemons

Preserved lemons are easy to make -- all you need are lemons and salt. Regular table salt, kosher salt or rock salt can be used, but I find rock salt best as it dissolves slowly and the texture helps to break down the acidity when rubbed into the lemons. You can also add herbs and spices such as peppercorns, bay leaves, cinnamon and cloves to the preserved lemons.

Use a sterilized glass jar to prevent bacteria from growing, and remember that the lemons must be completely covered with lemon juice or a white mold (harmless but unattractive) might form on the surface of the preserving liquid. When you're ready to use the preserved lemons, rinse them in cool water. Most of the time only the rinds are used, but the pulp can be used, too -- it's simply a matter of the taste and appearance in the dish you're making. Oh, and don't throw away the preserving liquid as it can be used over and over again, as long as you retrieve the lemons using a wooden spoon and don't contaminate the liquid by leaving the container open too long.

Most any kind of lemons can be used, just be sure they are fresh and unblemished. Rinse the lemons in cool water and pat them dry before beginning.

Makes 2 quarts

Two 1-quart sterilized jars with airtight lids

8 medium- to large-size lemons

1 cup coarse rock salt (or about 1/2 cup table salt)

Cut the lemons into quarters and put them in a shallow glass bowl. Sprinkle the salt over the lemons. With your fingertips, rub the salt into the lemons to release the juice. Add the lemon pieces, a few at a time, to the prepared jars. Mash the lemons down into the jar with the handle of clean wooden spoon to release more juice. Add any juice and the salt from the bowl into the jars. If there is not enough juice to cover the lemons, add more freshly squeezed lemon juice. Seal the jars and store in a cool, dark place for 30 days, shaking the jars from time to time to distribute the salt.

I keep them in the refrigerator so I can use slivers of the lemon rinds in salads or to shove in between the skin and breast of a chicken before roasting. Oh, and I sometimes put a chunk of the lemon rind in a Bloody Mary to give an extra perk!


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How To Build A Simple Tabbed Menu

In this tutorial we are going to take a PSD and turn them into HTML and CSS. I previously done this with other tutorials such as building a CSS pricing table.

The style we are going to create is going to look this.
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dc.contributor.authorLouw, Henk
dc.descriptionThesis (M.A. (English))--North-West University, Potchefstroom Campus, 2006
dc.description.abstractThe primary aim of this study is to determine whether it is possible to standardize written feedback on L2 student writing for use in a computerised marking environment. It forms part of a bigger project aimed at enhancing the feedback process as a whole The study attempts to establish "best practice" with regards to feedback on writing, by establishing from the literature what works and what should be avoided. Also, an empirical study was launched to establish what lecturers focus on and what marking techniques they use. A set of randomly selected essays from the Tswana Learner English Corpus and the Afrikaans Learner English Corpus were sent to the English departments of different tertiary institutions across the country. The essays were marked by the English lecturers at the relevant institutions. The conclusion was that lecturers typically focus on surface structures, and use ineffective marking techniques. The best practice (and data from the empirical study) was then used to create a set of standardised feedback comments (tag set) that can be used in a specially programmed software package in which students submit their texts electronically. Lecturers can then mark the student essays on the computer, hopefully speeding up the process, while at the same time giving much more detailed feedback. In later stages of the bigger project, students will get individualized exercises based on the feedback, and there are experiments currently being run to try and automate certain pans of the marking process in order to take some strain off the lecturers when marking. The immense archiving abilities of the computer will also be utilized in order to create opportunities for longitudinal studies. The effectiveness of the feedback tag set was tested in comparison to the marking techniques used by the lecturers in the empirical study and a self-correcting exercise. The conclusion was that the feedback tag set is more effective than the other two techniques. but students seem to perform weak overall when it gets to the revision of cohesive devices and supporting arguments. I argue that students are not used to revising these features, since lecturers seldom (if ever) comment on the structural elements of texts. However, the experiment proves that standardization of written feedback is possible to an extent. The implications of the findings are discussed, and recommendations for further research are made.
dc.publisherNorth-West University
dc.subjectSecond language student writingen
dc.subjectComputer-assisted language learning (CALL)en
dc.subjectLanguage teachingen
dc.subjectError classificationen
dc.titleStandardising written feedback on L2 student writingen

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