Sedimentation velocity FDS studies of antibodies in pooled human serum.

The biotech industry has great interest in investigating therapeutic proteins in high concentration environments like human serum. The fluorescence detection system (Aviv-FDS) allows the performance of analytical ultracentrifuge (AUC) sedimentation velocity (SV) experiments in tracer or BOLTS protocols. Here, we compare six pooled human serum samples by AUC SV techniques and demonstrate the potential of this technology for characterizing therapeutic antibodies in serum. Control FDS SV experiments on serum alone reveal a bilirubin-HSA complex whose sedimentation is slowed by solution nonideality and exhibits a Johnston-Ogston (JO) effect due to the presence of high concentrations of IgG. Absorbance SV experiments on diluted serum samples verify the HSA-IgG composition as well as a significant IgM pentamer boundary at 19 s. Alexa-488 labeled Simponi (Golimumab) is used as a tracer to investigate the behavior of a therapeutic monoclonal antibody (mAb) in serum, and the sedimentation behavior of total IgG in serum. Serum dilution experiments allow extrapolation to zero concentration to extract so, while global direct boundary fitting with SEDANAL verifies the utility of a matrix of self- and cross-term phenomenological nonideality coefficients (ks and BM1) and the source of the JO effect. The best fits include weak reversible association (~ 4 × 103 M-1) between Simponi and total human IgG. Secondary mAbs to human IgG and IgM verify the formation of a 10.2 s 1:1 complex with human IgG and a 19 s complex with human IgM pentamers. These results demonstrate that FDS AUC allows a range of approaches for investigating therapeutic antibodies in human serum.

Analysis of nonideality: insights from high concentration simulations of sedimentation velocity data.

The Aviv fluorescence detection system (Aviv-FDS) has allowed the performance of sedimentation velocity experiments on therapeutic antibodies in highly concentrated environments like formulation buffers and serum. Methods were implemented in the software package SEDANAL for the analysis of nonideal, weakly associating AUC data acquired on therapeutic antibodies and proteins (Wright et al. Eur Biophys J 47:709-722, 2018, Anal Biochem 550:72-83, 2018). This involved fitting both hydrodynamic, ks, and thermodynamic, BM1, nonideality where concentration dependence is expressed as s = so/(1 + ksc) and D = Do(1 + 2BM1c)/(1 + ksc) and so and Do are values extrapolated to c = 0 (mg/ml). To gain insight into the consequences of these phenomenological parameters, we performed simulations with SEDANAL of a monoclonal antibody as a function of ks (0-100 ml/g) and BM1 (0-100 ml/g). This provides a visual understanding of the separate and joint impact of ks and BM1 on the shape of high-concentration sedimentation velocity boundaries and the challenge of their unique determination by finite element methods. In addition, mAbs undergo weak self- and hetero-association (Yang et al. Prot Sci 27:1334-1348, 2018) and thus we have simulated examples of nonideal weak association over a wide range of concentrations (1-120 mg/ml). Here we demonstrate these data are best analyzed by direct boundary global fitting to models that account for ks, BM1 and weak association. Because a typical clinical dose of mAb is 50-200 mg/ml, these results have relevance for biophysical understanding of concentrated therapeutic proteins.

Determination of the nitrogen vacancy as a shallow compensating center in GaN doped with divalent metals.

We report accurate energetics of defects introduced in GaN on doping with divalent metals, focusing on the technologically important case of Mg doping, using a model that takes into consideration both the effect of hole localization and dipolar polarization of the host material, and includes a well-defined reference level. Defect formation and ionization energies show that divalent dopants are counterbalanced in GaN by nitrogen vacancies and not by holes, which explains both the difficulty in achieving p-type conductivity in GaN and the associated major spectroscopic features, including the ubiquitous 3.46 eV photoluminescence line, a characteristic of all lightly divalent-metal-doped GaN materials that has also been shown to occur in pure GaN samples. Our results give a comprehensive explanation for the observed behavior of GaN doped with low concentrations of divalent metals in good agreement with relevant experiment.

The maturation of photoreceptors in the avian retina is stimulated by thyroid hormone.

During retinal development, the cell-fate of photoreceptors is committed long before maturation, which entails the expression of opsins and functional transduction of light. The mechanisms that delay the maturation of photoreceptors remain unknown. We have recently reported that immature photoreceptors express the LIM domain transcription factors Islet2 and Lim3, as well as the cell-surface glycoprotein axonin1 [Fischer et al., (2008a) J Comp Neurol 506:584-603]. As the photoreceptors mature to form outer segments and express photopigments, the expression of the Islet2, Lim3 and axonin1 is diminished. The purpose of this study was to investigate whether thyroid hormone (TH) influences the maturation of photoreceptors. We studied the maturation of photoreceptors across the gradient of maturity that exists in far peripheral regions of the post-natal chicken retina [Ghai et al., (2008) Brain Res 1192:76-89]. We found that intraocular injections of TH down-regulated Islet2, Lim3 and axonin1 in photoreceptors in far peripheral regions of the retina. By contrast, TH stimulated the up-regulation of red-green opsin, violet opsin, rhodopsin and calbindin in photoreceptors. We found a correlation between the onset of RLIM (RING finger LIM-domain binding protein) and down-regulation of Islet2 and Lim3 in maturing photoreceptors; RLIM is known to interfere with the transcriptional activity of LIM-domain transcription factors. We conclude that TH stimulates the maturation of photoreceptors in the avian retina. We propose that TH inhibits the expression of Islet2 and Lim3, which thereby permits photoreceptor maturation and the onset of photopigment-expression.

Structure, optical properties and defects in nitride (III-V) nanoscale cage clusters.

Density Functional Theory calculations are reported on cage structured BN, AlN, GaN and InN sub- and low nanosize stoichiometric clusters, including two octahedral families of T(d) and T(h) symmetry. The structures and energetics are determined, and we observe that BN clusters in particular show high stability with respect to the bulk phase. The cluster formation energy is demonstrated to include a constant term that we attribute to the curvature energy and the formation of six tetragonal defects. The (BN)(60) onion double-bubble structure was found to be particularly unstable. In contrast, similar or greater stability was found for double and single shell cages for the other nitrides. The optical absorption spectra have been first characterised by the one-electron Kohn-Sham orbital energies for all compounds, after which we concentrated on BN where we employed a recently developed Time Dependent Density Functional Theory approach. The one-electron band gaps do not show a strong and consistent size dependency, in disagreement with the predictions of quantum confinement theory. The density of excited bound states and absorption spectrum have been calculated for four smallest BN clusters within the first ionisation potential cut-off energy. The relative stability of different BN clusters has been further explored by studying principal point defects and their complexes including topological B-N bond rotational defects, vacancies, antisites and interstititials. The latter have the lowest energy of formation.

Primary malignant brain tumor incidence and Medicaid enrollment.

BACKGROUND: The relationship between socioeconomic status and health care disparities in the incidence of brain tumors is unclear. OBJECTIVE: To identify the associations between age, sex, and Medicaid enrollment and the incidence of primary malignant brain tumors in Michigan in 1996 and 1997. METHODS: Records were obtained from the Michigan Cancer Surveillance Program on the 1,006 incident cases during this period and cross-checked with Medicaid enrollment files. RESULTS: Persons enrolled in Medicaid were more likely than non-enrolled persons to develop a malignant brain tumor of any type, a glioblastoma multiforme, and an astrocytoma for certain subgroups. In addition, incidence rates for malignant brain tumors in persons enrolled in Medicaid peaked at a younger age. CONCLUSION: Sociodemographic status may be associated with cerebral malignancy and should be considered when targeting treatment and educational interventions at persons at risk.

Impact of acid secretion, gastritis, and mucus thickness on gastric transfer of antibiotics in rats.

BACKGROUND AND AIMS: The success of Helicobacter pylori eradication regimens depends on gastric pH, inflammation, and mucus thickness. Our aim was to investigate the effects of acid secretion, inflammation, and mucolysis on gastric antibiotic transfer. SUBJECTS AND METHODS: A total of 134 anaesthetised rats were given metronidazole, amoxicillin, or clarithromycin intravenously and gastric contents were aspirated via an indwelling cannula. Acid secretion was controlled by either omeprazole or pentagastrin while gastritis was induced by infection with H pylori or dosing with iodoacetamide. Mucolysis was achieved by instilling pronase into the gastric lumen. RESULTS: Metronidazole transfer increased with acid secretion and fell with omeprazole, independently of gastric pH. Clarithromycin was also transferred with acid but was then rapidly degraded. Omeprazole prevented this degradation, raising gastric luminal concentrations. Omeprazole did not alter amoxicillin transfer. Gastritis induced by H pylori did not alter gastric transfer of metronidazole and amoxicillin but that of clarithromycin was increased by 23%. However, gastritis induced by iodoacetamide reduced clarithromycin transfer without any effect on metronidazole or amoxicillin transfer. Pronase treatment increased amoxicillin transfer fourfold and metronidazole by 66% but reduced clarithromycin transfer by 35%. CONCLUSIONS: Metronidazole and clarithromycin are predominantly transferred with gastric acid rather than by an acid trapping mechanism. Pronase increases the appearance of amoxicillin and metronidazole in gastric secretions.

Analytical utility of valence band X-ray photoelectron spectroscopy of iron and its oxides, with spectral interpretation by cluster and band structure calculations.

The valence band and core-level X-ray photoelectron spectroscopy (XPS) of iron and its oxides are reported, and the valence band spectra interpreted by various calculation models. The paper focuses upon the valence band region, which shows significant differences between the metal and the following oxidized iron species: FeO, Fe(3)O(4), alpha-Fe(2)O(3), gamma-Fe(2)O(3), alpha-FeOOH and gamma-FeOOH. The core region is of little analytical value as a means of distinguishing between these species, but the valence band region shows significant differences. These differences are consistent with spectra predicted by cluster and band structure calculations. Cluster calculations are valuable as a means for interpreting the spectra of iron oxides with multiple iron sites and defect characteristics.

X-ray photoelectron spectroscopic studies of the oxidation of aluminium by liquid water monitored in an anaerobic cell.

The oxidation of clean polycrystalline aluminium foil was performed in a previously described anaerobic cell and studied by core level and valence band X-ray photoelectron spectroscopy. Oxide free sample surfaces were exposed under inert atmosphere at room temperature to increasingly oxidative environments of water vapor and liquid water beginning with an oxide free metal foil each time. Minor oxidative environments were found to produce a film of boehmite with a thinner outer film of gibbsite. Harsher oxidation in liquid water resulted in an oxide film much thicker than that found on the as received sample which contained an inner composition of gamma alumina with an outer film of gibbsite. As expected, the oxide film observed for ultra high purity polycrystalline aluminiurm foil in the as received state was gibbsite. The valence band of the cleaned metal is discussed in terms of residual surface species. The evaluation and determination of these residual surface species is evaluated following an analysis of two different band structure calculations which use different d orbital exponents. It is found that while the density of states is almost unaltered by changes in the d exponent, the predicted spectrum is substantially changed. The work makes extensive use of valence band X-ray photoelectron spectroscopy.

How are abnormal results for liver function tests dealt with in primary care? Audit of yield and impact.

OBJECTIVE: To determine whether abnormal results for liver function tests are investigated in primary care and findings on full investigation. DESIGN: Retrospective audit and prospective clinical investigation. SETTING: University hospital and surrounding general practices serving around 330 000 people. SUBJECTS: Adults with abnormal results for liver function based on tests requested by their doctor between 1 January and 30 June 1995. INTERVENTIONS: All patients with gamma-glutamyltransferase, alanine aminotransferase, or alkaline phosphatase concentrations at least twice the upper limit of the reference range were studied. A median of 15 months later (range 12-21) records of hospital attendances and further investigations were examined. Where investigations were incomplete the records from the general practice were examined, and suitable patients were invited to attend the liver clinic. MAIN OUTCOME MEASURES: Investigations requested by the doctor and final diagnoses reached. RESULTS: 933 patients with abnormal liver function tests were identified; follow up data were obtained in 873 (94%). 531 patients were already under hospital review. Of the remaining 342 patients, 157 were suitable for investigation; the others had died, moved away, were elderly, or had repeat liver function tests with normal results. No further tests were requested for 91 (58%) of these patients. 66 had been partially investigated by their doctor, and in seven patients results suggesting a treatable chronic liver disease had not been followed up. On investigation, 97 (62%) had an identifiable diagnosis requiring hospital intervention or follow up, or both. CONCLUSIONS: Abnormal results for liver function are often not adequately investigated, missing an important chance of identifying treatable chronic liver disease.

A glucose transporter chimera confers a dominant negative glucose starvation phenotype in Saccharomyces cerevisiae.

A family of glucose transporters mediates glucose uptake in Saccharomyces cerevisiae. We show that the dominant mutation GSF4-1, which impairs glucose repression of SUC2, results in a nonfunctional chimera of the transporters Hxt1p and Hxt4p. Hxt1/4p inhibits the function of wild-type glucose transporters. Similar mutations may facilitate analysis of the major facilitator superfamily.

Analysis of the mechanism by which glucose inhibits maltose induction of MAL gene expression in Saccharomyces.

Expression of the MAL genes required for maltose fermentation in Saccharomyces cerevisiae is induced by maltose and repressed by glucose. Maltose-inducible regulation requires maltose permease and the MAL-activator protein, a DNA-binding transcription factor encoded by MAL63 and its homologues at the other MAL loci. Previously, we showed that the Mig1 repressor mediates glucose repression of MAL gene expression. Glucose also blocks MAL-activator-mediated maltose induction through a Mig1p-independent mechanism that we refer to as glucose inhibition. Here we report the characterization of this process. Our results indicate that glucose inhibition is also Mig2p independent. Moreover, we show that neither overexpression of the MAL-activator nor elimination of inducer exclusion is sufficient to relieve glucose inhibition, suggesting that glucose acts to inhibit induction by affecting maltose sensing and/or signaling. The glucose inhibition pathway requires HXK2, REG1, and GSF1 and appears to overlap upstream with the glucose repression pathway. The likely target of glucose inhibition is Snf1 protein kinase. Evidence is presented indicating that, in addition to its role in the inactivation of Mig1p, Snf1p is required post-transcriptionally for the synthesis of maltose permease whose function is essential for maltose induction.

Patterns of use of complementary health services in the south-west of Western Australia.

The objective of this research was to identify patterns of complementary health service usage by rural Western Australians in the south-west of the State. Complementary health providers identified by health users included homeopaths, chiropractors, naturopaths, acupuncturists, faith healers, herbalists, reiki or energy workers, counsellors, physiotherapists, osteopaths, podiatrists and reflexologists. More than half of the health provider usage in the region was with complementary therapists and the remainder with medical doctors. The main reason identified for using complementary therapists was their level of skills and the main reason identified for not using them was a lack of knowledge about what their services could provide.

Experience awareness tools for preventing burnout in nurses.

Philophonetics counselling is a radically new method of coping with stress through the development of individually tailored self-controlling emotional and cognitive strategies. This paper proposes that philophonetics counselling provides an innovative holistic approach of dealing with burnout in the nursing profession.

Combined forward error control and packetized zerotree wavelet encoding for transmission of images over varying channels.

One method of transmitting wavelet based zerotree encoded images over noisy channels is to add channel coding without altering the source coder. A second method is to reorder the embedded zerotree bitstream into packets containing a small set of wavelet coefficient trees. We consider a hybrid mixture of these two approaches and demonstrate situations in which the hybrid image coder can outperform either of the two building block methods, namely on channels that can suffer packet losses as well as statistically varying bit errors.

Efficient export of the glucose transporter Hxt1p from the endoplasmic reticulum requires Gsf2p.

Mutations in the GSF2 gene cause glucose starvation phenotypes in Saccharomyces cerevisiae. We have isolated the HXT1 gene, which encodes a low-affinity, high-capacity glucose transporter, as a multicopy suppressor of a gsf2 mutation. We show that gsf2 mutants accumulate Hxt1p in the endoplasmic reticulum (ER) and that Gsf2p is a 46-kDa integral membrane protein localized to the ER. gsf2 mutants also display a galactose growth defect and abnormal localization of the galactose transporter Gal2p but are not defective in function or localization of the high-affinity glucose transporter Hxt2p. These findings suggest that Gsf2p functions in the ER to promote the secretion of certain hexose transporters.