Short Communication: The effect of in ovo creatine monohydrate on early pen use activity in chicks.

First-week survival and egg hatchability are lower in chicks from younger broiler breeder hen flocks. Creatine is a naturally occurring compound synthesised from the amino acid arginine or obtained from the diet and is important in the storage and transport of energy. Previous research found an improvement in the hatch rate but no posthatch performance improvements when fertile eggs from young breeder hens were injected with creatine monohydrate (CrM) on embryonic day 14. This pilot study aimed to further investigate the possibility of early posthatch improvements by examining the activity of chicks during the 1st week posthatch. Behaviours were broadly classified as active or inactive, the pen was split into three areas, and the amount of time spent in the heat lamp, feed hopper, or drinker line areas was recorded. Chicks given in ovo CrM spent less time in the heat lamp area over the whole 7 days compared to saline (t = 2.352, P = 0.021) and control groups (t = 3.336, P = 0.003) and more time in the feed hopper area during the first 4 days compared to the control group (t = 2.174, P = 0.033). This finding suggests that creatine may improve energy reserves in young chicks allowing them to spend more time away from the heat lamp.

Modulating non-native aggregation and electrostatic protein-protein interactions with computationally designed single-point mutations.

Non-native protein aggregation is a ubiquitous challenge in the production, storage and administration of protein-based biotherapeutics. This study focuses on altering electrostatic protein-protein interactions as a strategy to modulate aggregation propensity in terms of temperature-dependent aggregation rates, using single-charge variants of human γ-D crystallin. Molecular models were combined to predict amino acid substitutions that would modulate protein-protein interactions with minimal effects on conformational stability. Experimental protein-protein interactions were quantified by the Kirkwood-Buff integrals (G22) from laser scattering, and G22 showed semi-quantitative agreement with model predictions. Experimental initial-rates for aggregation showed that increased (decreased) repulsive interactions led to significantly increased (decreased) aggregation resistance, even based solely on single-point mutations. However, in the case of a particular amino acid (E17), the aggregation mechanism was altered by substitution with R or K, and this greatly mitigated improvements in aggregation resistance. The results illustrate that predictions based on native protein-protein interactions can provide a useful design target for engineering aggregation resistance; however, this approach needs to be balanced with consideration of how mutations can impact aggregation mechanisms.

Effect of CYP3A51/3 polymorphism on blood pressure in renal transplant recipients.

BACKGROUND: The cytochrome P450 3A5 (CYP3A5) enzyme has been implicated to determine blood pressure (BP) in humans. Different results have been reported concerning CYP3A5 gene polymorphisms and posttransplantation hypertension in kidney recipients. Our objective was to investigate whether CYP3A5 1/3 polymorphism was associated with ambulatory BP among a population of renal transplant recipients receiving the calcineurin inhibitor tacrolimus for immunosuppression. METHODS: Sixty primary kidney transplant recipients undergoing treatment with tacrolimus were genotyped for the CYP3A5 1/3 polymorphism. We analysed the association of the CYP3A5 alleles with ambulatory systolic and diastolic BP measured at 6 and 24 months posttransplantation. RESULTS: We observed that 23.3% of the patients were CYP3A5 1 carriers and 76.7% were homozygous for CYP3A5 3. CYP3A5 1 carriers showed higher adjusted systolic BP and diastolic BP at 6 and 24 months posttransplantation, and they were prescribed more antihypertensive drugs compared with non CYP3A5 1 carrier patients, albeit not significant. No significant differences were found comparing the distribution of the hypertension classes. CONCLUSION: We did not observe a significant association of CYP3A5 1/3 polymorphism with posttransplantation hypertension, although there were some differences in BP associated with the presence of the CYP3A5 1 allele.

Lack of association of immune cell function test with rejection in kidney transplantation.

BACKGROUND: The Cylex Immuknow assay provides a rapid assessment of global immune function in immunocompromised patients by measuring the global immune responses of CD4 T cells from a whole-blood sample. It may help to monitor the immune status of immunosuppressed transplant patients. However, earlier studies have shown that there is no consensus on the utility of the Immuknow assay in renal transplant rejection. METHODS: T-cell activation was determined by measuring an increase of intracellular adenosine triphosphate (iATP) from CD4 cells in 227 samples from 116 kidney transplant patients. The results were analyzed regarding patient clinical status, namely, rejection, infection, or stability. In addition, we measured the immunologic response of 108 healthy control subjects. RESULTS: There were 24 infectious and 36 rejection episodes. iATP concentrations differed significantly between stable and infected patients (180.5 ± 55.2 vs 375.3 ± 140.1 ng/mL; P < .001) and between infected patients and control subjects (180.5 ± 55.2 vs 436.5 ± 112 ng/mL; P < .001). No correlation was observed between patients suffering an acute rejection episode with this response. CONCLUSIONS: Our results confirmed that the Immuknow assay identified transplant patients at risk for infection. It may provide information to guide immunosuppressive therapy, but the assay did not seem to have the potential to differentiate subjects experiencing rejection.

Changes in solvent exposure reveal the kinetics and equilibria of adsorbed protein unfolding in hydrophobic interaction chromatography.

Hydrogen exchange has been a useful technique for studying the conformational state of proteins, both in bulk solution and at interfaces, for several decades. Here, we propose a physically based model of simultaneous protein adsorption, unfolding and hydrogen exchange in HIC. An accompanying experimental protocol, utilizing mass spectrometry to quantify deuterium labeling, enables the determination of both the equilibrium partitioning between conformational states and pseudo-first order rate constants for folding and unfolding of adsorbed protein. Unlike chromatographic techniques, which rely on the interpretation of bulk phase behavior, this methodology utilizes the measurement of a molecular property (solvent exposure) and provides insight into the nature of the unfolded conformation in the adsorbed phase. Three model proteins of varying conformational stability, alpha-chymotrypsinogen A, beta-lactoglobulin B, and holo alpha-lactalbumin, are studied on Sepharose HIC resins possessing assorted ligand chemistries and densities. alpha-Chymotrypsinogen, conformationally the most stable protein in the set, exhibits no change in solvent exposure at all the conditions studied, even when isocratic pulse-response chromatography suggests nearly irreversible adsorption. Apparent unfolding energies of adsorbed beta-lactoglobulin B and holo alpha-lactalbumin range from -4 to 3 kJ/mol and are dependent on resin properties and salt concentration. Characteristic pseudo-first order rate constants for surface-induced unfolding are 0.2-0.9 min(-1). While poor protein recovery in HIC is often associated with irreversible unfolding, this study documents that non-eluting behavior can occur when surface unfolding is reversible or does not occur at all. Further, this hydrogen exchange technique can be used to assess the conformation of adsorbed protein under conditions where the protein is non-eluting and chromatographic methods are not applicable.

A new thermodynamic model describes the effects of ligand density and type, salt concentration and protein species in hydrophobic interaction chromatography.

A new thermodynamic model is derived that describes both loading and pulse-response behavior of proteins in hydrophobic interaction chromatography (HIC). The model describes adsorption in terms of protein and solvent activities, and water displacement from hydrophobic interfaces, and distinguishes contributions from ligand density, ligand type and protein species. Experimental isocratic response and loading data for a set of globular proteins on Sepharose resins of various ligand types and densities are described by the model with a limited number of parameters. The model is explicit in ligand density and may provide insight into the sensitivity of protein retention to ligand density in HIC as well as the limited reproducibility of HIC data.

Protein instability during HIC: evidence of unfolding reversibility, and apparent adsorption strength of disulfide bond-reduced alpha-lactalbumin variants.

A two-conformation, four-state model has been proposed to describe protein adsorption and unfolding behavior on hydrophobic interaction chromatography (HIC) resins. In this work, we build upon previous study and application of a four-state model to the effect of salt concentration on the adsorption and unfolding of the model protein alpha-lactalbumin in HIC. Contributions to the apparent adsorption strength of the wild-type protein from native and unfolded conformations, obtained using a deuterium labeling technique, reveal the free energy change and kinetics of unfolding on the resin, and demonstrate that surface unfolding is reversible. Additionally, variants of alpha-lactalbumin in which one of the disulfide bonds is reduced were synthesized to examine the effects of conformational stability on apparent retention. Below the melting temperatures of the wild-type protein and variants, reduction of a single disulfide bond significantly increases the apparent adsorption strength (approximately 6-8 kJ/mol) due to increased instability of the protein. Finally, the four-state model is used to accurately predict the apparent adsorption strength of a disulfide bond-reduced variant.

[Hemoperfusion in the treatment of acute valproic acid intoxication]. / Tratamiento con hemoperfusión de la intoxicación aguda por ácido valproico.

Valproic acid is increasingly used in the treatment of epilepsy, and also prescribed for bipolar affective disorders, schizoaffective disorders, schizophrenia and migraine prophylaxis. Valproic acid intoxication with suicide attempt is a relatively common clinical problem that can result in coma, respiratory depression, pancytopenia, hemodynamic instability and death. The drug's relatively low molecular weight, small volume of distribution and saturable protein-binding render it potentially amenable to exracorporeal removal (hemodialysis, hemoperfusion or hemofiltration ), but published experience is scarce. We describe a case report involving valproic acid intoxication with ingestion of ethanol, who was successfully treated with charcoal hemoperfusion. With this treatment the half-life of valproic acid was reduced with rapid lowering of valproic acid levels and clinical improvement. Based on our experience in this patient and a review of previously reported cases, charcoal hemoperfusion should be considered for serious valproic acid intoxication because free as well as bound drug fractions are eliminated via this technique.

[Calcification in nonfunctioning transplanted kidneys]. / Calcificación del injerto renal no funcionante.

Failed renal allografts often are left in situ in patients who revert to chronic dialysis therapy or who undergo retransplantation. These organs may be the site of massive calcification despite their lack of physiological function. Calcification of an endstage renal allograft is sometimes found incidentally. We report here two patients who developed extensive calcification of the renal graft, one was on chronic hemodialysis and the other had a second renal transplantation with normal renal function. The precise pathogenesis of calcification and the factors which determine its tissue localization are unclear. Factors postulated to promote the development of metastatic calcification include an elevated calcium phosphate product, severe secondary hyperparathyroidism, aluminium toxicity and duration of dialytic therapy. In some cases local factors related with the chronic inflammatory rejection process are probably involved as well. However, the exact relative contribution of these factors remains unresolved. Unless specific clinical indications are present, transplant nephrectomy is not necessary for calcified end-stage renal allografts.

[Treatment of acute lithium intoxication with high-flux haemodialysis membranes]. / Tratamiento de la intoxicación aguda por litio mediante hemodiálisis con dializadores de alta eficiencia.

Lithium carbonate is commonly prescribed for the treatment of bipolar (manic-depressive) disorders. However, because of its narrow therapeutic index an excessive elevation of serum lithium concentration, either during chronic maintenance therapy or after an acute overdose, can result in serious toxicity. In addition to supportive care, the established treatment of severe lithium toxicity is haemodialysis. Conventional haemodialysis can reduce serum lithium rapidly, but post-dialysis rebound elevations with recurrent toxicity have been documented in old publications. High-flux membranes should be capable of removing more lithium per hour of haemodialysis, but published values are not available. We report here three patients with acute lithium intoxication who were treated successfully with bicarbonate and high-flux haemodialysis membranes. Our patients presented with a severe degree of intoxication, based on the amount of drug ingested, the initial serum lithium level, the severity of neurologic symptoms and systemic manifestations. Two patients developed acute renal failure probably as a result of volume depletion since it was rapidly reversible by haemodialysis and infusion therapy. In addition, consecutive haemodialysis sessions and improvement of renal function allowed a rapid decrease in serum lithium levels without haemodynamic instability or rebound elevations in lithium concentration. The effectiveness of the procedure in these cases can be attributed to the use of bicarbonate dialysate and high-efficiency dialysers. This is the first report describing the effect of high-efficiency dialysers on lithium pharmacokinetic. Using this technique the elimination rate of lithium was found to be greater than previously reported with haemodialysis.

Construction of a tethered poly(ethylene glycol) surface gradient for studies of cell adhesion kinetics.

Surface gradients can be used to perform a wide range of functions and represent a novel experimental platform for combinatorial discovery and analysis. In this work, a gradient in the coverage of a surface-immobilized poly(ethylene glycol) (PEG) layer is constructed to interrogate cell adhesion on a solid surface. Variation of surface coverage is achieved by controlled transport of a reactive PEG precursor from a point source through a hydrated gel. Immobilization of PEG is achieved by covalent attachment of the PEG molecule via direct coupling chemistry to a cystamine self-assembled monolayer on gold. This represents a simple method for creating spatial gradients in surface chemistry that does not require special instrumentation or microfabrication procedures. The structure and spatial distribution of the PEG gradient are evaluated via ellipsometry and atomic force microscopy. A cell adhesion assay using bovine arteriole endothelium cells is used to study the influence of PEG thickness and chain density on biocompatibility. The kinetics of cell adhesion are quantified as a function of the thickness of the PEG layer. Results depict a surface in which the variation in layer thickness along the PEG gradient strongly modifies the biological response.

[Malignant laryngeal fibrous histiocytoma (MLFH). Report of two unusual cases]. / Fibrohistiocitoma maligno laríngeo. A propósito de dos casos de localización infrecuente.

Laryngeal presentation of Malignat Fibrous Histiocytoma (MFH) is uncommon. It is more prevalent in elderly males. The most frequent form of laryngeal presentation is as subepithelial nodules, and its clinical behaviour is variable and unpredictable. Microscopically, it is a tumour with two well differentiated components: histiocitic and fibroblastic, with several different structural patterns that can make histologic diagnosis a difficult one. We present two cases of MFH of the larynx, and a review of the literature.

Adaptive-optics ultrahigh-resolution optical coherence tomography.

Merging of ultrahigh-resolution optical coherence tomography (UHR OCT) and adaptive optics (AO), resulting in high axial (3 microm) and improved transverse resolution (5-10 microm) is demonstrated for the first time to our knowledge in in vivo retinal imaging. A compact (300 mm x 300 mm) closed-loop AO system, based on a real-time Hartmann-Shack wave-front sensor operating at 30 Hz and a 37-actuator membrane deformable mirror, is interfaced to an UHR OCT system, based on a commercial OCT instrument, employing a compact Ti:sapphire laser with 130-nm bandwidth. Closed-loop correction of both ocular and system aberrations results in a residual uncorrected wave-front rms of 0.1 microm for a 3.68-mm pupil diameter. When this level of correction is achieved, OCT images are obtained under a static mirror configuration. By use of AO, an improvement of the transverse resolution of two to three times, compared with UHR OCT systems used so far, is obtained. A significant signal-to-noise ratio improvement of up to 9 dB in corrected compared with uncorrected OCT tomograms is also achieved.

Detection of Fusarium oxysporum f. sp. melonis Race 1 in Soil in Colima State, Mexico.

During the winters of 2002 and 2003, a wilt occurred in melons cultivated on 1,500 ha in Colima State, Mexico. Yield losses reached 25% of final production, despite soil disinfestation with 60% methyl bromide and 40% chloropicrin. On the basis of the observation of plants with necrotic xylem, yellowing, and wilting of leaves, this disease was identified provisionally as Fusarium wilt. During February 2003, four soil samples from affected fields were plated onto a Fusarium-selective medium (1), which resulted in the detection of 2,260 ± 357, 179 ± 76, 668 ± 357, and 1,391 ± 256 CFU/g of F. oxysporum (3). Thirty-one randomly chosen isolates were used to inoculate differential cultivars of melon as described by Risser et al. (4). The cultivars were Amarillo Canario (susceptible to all races), Diana (resistant to races 0 and 2), Tango (resistant to races 0 and 1), and Vulcano (resistant to races 0, 1, and 2) (2). Ten plants of each cultivar, grown on sterilized vermiculite, were inoculated at the first true-leaf stage by drenching with 200 ml of a conidial suspension (1 × 105 CFU/ml) of each isolate. Noninoculated plants of each cultivar served as controls. Plants were maintained in a growth chamber with a 16-h photoperiod (18 × 103 lux) and temperatures at 23 to 25°C. Yellowing, wilt, and vascular discoloration symptoms developed on cvs. Amarillo Canario and Diana following inoculation with each of the 31 isolates, while noninoculated plants remained symptomless. F. oxysporum was consistently reisolated on potato dextrose agar from the affected plants. On the basis of the combination of affected cultivars, all isolates were identified as F. oxysporum f. sp. melonis race 1. To our knowledge, this is the first report of F. oxysporum f. sp. melonis race 1 in Colima State, Mexico. References: (1) H. Komada. Rev. Plant Prot. Res. 8:114, 1975. (2) J. Marín Rodríquez. Portagrano 2004. Vadmecum de Variedades Hortícolas. Agrobook, Spain. 2004. (3) P. E. Nelson et al. Fusarium Species: An Illustrated Manual for Identification. Pennsylvania State University Press, University Park, 1983. (4) G. Risser et al. Phytopathology 66:1105, 1976.

Analytical methods for food-contact materials additives in olive oil simulant at sub-mg kg-1 level.

Methods of analysis for four additives (two antioxidants, IRGANOX 245 and 1035; an ultraviolet absorber, CHIMMASORB 81; and an optical brightening agent, UVITEX OB) in olive oil are reported. These additives have the potential to migrate from food-contact materials into the European Union fatty food simulant olive oil, which is the most difficult matrix for analysis. The additives were chosen because they differed in their chemically active groups, had different functions within the polymer, have low proposed specific migration limits and are commonly used in food-contact materials such as polystyrenes and polyolefins. The proposed analytical methods for the additives are simple, rapid, inexpensive and also broadly applicable to the aqueous food simulants. All methods were evaluated by constructing calibration curves, measurement of recovery and precision, and determining the limits of detection. Most of the methods involve direct injection of an olive oil solution for high-performance liquid chromatography analysis with ultraviolet-visible or fluorescence detection. The methods allowed establishment of additive stability and measurement of migration of the selected additives into olive oil at different time-temperature conditions used in migration studies into food simulants.