Weak interactions govern the viscosity of concentrated antibody solutions: high-throughput analysis using the diffusion interaction parameter

Weak protein-protein interactions are thought to modulate the viscoelastic properties of concentrated antibody solutions. Predicting the viscoelastic behavior of concentrated antibodies from their dilute solution behavior is of significant interest and remains a challenge. Here, we show that the diffusion interaction parameter (k(D)), a component of the osmotic second virial coefficient (B(2)) that is amenable to high-throughput measurement in dilute solutions, correlates well with the viscosity of concentrated monoclonal antibody (mAb) solutions. We measured the k(D) of 29 different mAbs (IgG(1) and IgG(4)) in four different solvent conditions (low and high ion normality) and found a linear dependence between k(D) and the exponential coefficient that describes the viscosity concentration profiles (|R| ≥ 0.9). Through experimentally measured effective charge measurements, under low ion normality where the electroviscous effect can dominate, we show that the mAb solution viscosity is poorly correlated with the mAb net charge (|R| ≤ 0.6). With this large data set, our results provide compelling evidence in support of weak intermolecular interactions, in contrast to the notion that the electroviscous effect is important in governing the viscoelastic behavior of concentrated mAb solutions. Our approach is particularly applicable as a screening tool for selecting mAbs with desirable viscosity properties early during lead candidate selection.     

A method for regenerating gold surface for prolonged reuse of gold-coated surface plasmon resonance chip

We developed a method to completely regenerate the gold (Au) surface of 3-aminopropyltriethoxysilane (APTES)-functionalized Au-coated surface plasmon resonance (SPR) chip that had been used for human fetuin A (HFA) immunoassay. It involved treatment of the used SPR chip with freshly prepared piranha solution (concentrated H(2)SO(4)/30% H(2)O(2)=3:1, v/v) for 15 min followed by extensive rinsing with ethanol and deionized water. The developed method enabled prolonged reuse of the regenerated SPR chip that increased its cost-effectiveness without affecting the reproducibility of HFA immunoassays.     

Studies of histidine residues in soybean (Glycine max) urease

Soybean urease has been investigated extensively to reveal the presence of histidine residue (s) in the active site and their potential role in the catalysis. The spectrophotometric studies using diethylpyrocarbonate (DEP) showed the modification of 11.76 ± 0.1 histidine residues per mole of native urease. Therefore, the results are indicative of the presence of twelve histidine residues per urease molecule. It is presumed that the soybean urease, being a hexameric protein possess two histidine residues per subunit. Correlation plot showed that the complete inactivation of soybean urease corresponds to the modification of 1.97 histidine residues per subunit. Further, double logarithmic plot of kapp versus DEP concentration has resulted in a linear correlation and thereby demonstrating that only one of the two histidine residues per subunit is catalytically essential. Significant protection has been observed against inactivation when urea or acetohydroxamate (AHA) is incubated with DEP treated urease. The studies have demonstrated the presence of one histidine residue at the active site of soybean urease and its significance in catalysis.     

Adverse fetal and neonatal outcomes associated with a life-long high fat diet: role of altered development of the placental vasculature

Maternal obesity results in a number of obstetrical and fetal complications with both immediate and long-term consequences. The increased prevalence of obesity has resulted in increasing numbers of women of reproductive age in this high-risk group. Since many of these obese women have been subjected to hypercaloric diets from early childhood we have developed a rodent model of life-long maternal obesity to more clearly understand the mechanisms that contribute to adverse pregnancy outcomes in obese women. Female Sprague Dawley rats were fed a control diet (CON--16% of calories from fat) or high fat diet (HF--45% of calories from fat) from 3 to 19 weeks of age. Prior to pregnancy HF-fed dams exhibited significant increases in body fat, serum leptin and triglycerides. A subset of dams was sacrificed at gestational day 15 to evaluate fetal and placental development. The remaining animals were allowed to deliver normally. HF-fed dams exhibited a more than 3-fold increase in fetal death and decreased neonatal survival. These outcomes were associated with altered vascular development in the placenta, as well as increased hypoxia in the labyrinth. We propose that the altered placental vasculature may result in reduced oxygenation of the fetal tissues contributing to premature demise and poor neonatal survival.     

Phase I evaluation of intravenous ascorbic acid in combination with gemcitabine and erlotinib in patients with metastatic pancreatic cancer

Background

Preclinical data support further investigation of ascorbic acid in pancreatic cancer. There are currently insufficient safety data in human subjects, particularly when ascorbic acid is combined with chemotherapy.

Methods and Findings

14 subjects with metastatic stage IV pancreatic cancer were recruited to receive an eight week cycle of intravenous ascorbic acid (three infusions per week), using a dose escalation design, along with standard treatment of gemcitabine and erlotinib. Of 14 recruited subjects enrolled, nine completed the study (three in each dosage tier). There were fifteen non-serious adverse events and eight serious adverse events, all likely related to progression of disease or treatment with gemcitabine or erlotinib. Applying RECIST 1.0 criteria, seven of the nine subjects had stable disease while the other two had progressive disease.

Conclusions

These initial safety data do not reveal increased toxicity with the addition of ascorbic acid to gemcitabine and erlotinib in pancreatic cancer patients. This, combined with the observed response to treatment, suggests the need for a phase II study of longer duration.

Trial Registration

Clinicaltrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00954525","term_id":"NCT00954525"}}NCT00954525     

Obesity negatively impacts aerobic capacity improvements both acutely and 1-year following cardiac rehabilitation

Cardiac rehabilitation (CR) produces a host of health benefits related to modifiable cardiovascular risk factors. The purpose of the present investigation was to determine the influence of body weight, assessed through BMI, on acute and long-term improvements in aerobic capacity following completion of CR. Three thousand nine hundred and ninety seven subjects with coronary artery disease (CAD) participated in a 12-week multidisciplinary CR program. Subjects underwent an exercise test to determine peak estimated metabolic equivalents (eMETs) and BMI assessment at baseline, immediately following CR completion and at 1-year follow-up. Normal weight subjects at 1-year follow-up demonstrated the greatest improvement in aerobic fitness and best retention of those gains (gain in peak METs: 0.95 ± 1.1, P < 0.001). Although the improvement was significant (P < 0.001), subjects who were initially classified as obese had the lowest aerobic capacity and poorest retention in CR fitness gains at 1-year follow-up (gain in peak eMETs: 0.69 ± 1.2). Subjects initially classified as overweight by BMI had a peak eMET improvement that was also significantly better (P < 0.05) than obese subjects at 1-year follow-up (gain in peak eMETs: 0.82 ± 1.1). Significant fitness gains, one of the primary beneficial outcomes of CR, can be obtained by all subjects irrespective of BMI classification. However, obese patients have poorer baseline fitness and are more likely to "give back" fitness gains in the long term. Obese CAD patients may therefore benefit from additional interventions to enhance the positive adaptations facilitated by CR.     

Effects of deoxycholylglycine, a conjugated secondary bile acid, on myogenic tone and agonist-induced contraction in rat resistance arteries

Background

Bile acids (BAs) regulate cardiovascular function via diverse mechanisms. Although in both health and disease serum glycine-conjugated BAs are more abundant than taurine-conjugated BAs, their effects on myogenic tone (MT), a key determinant of systemic vascular resistance (SVR), have not been examined.

Methodology/Principal Findings

Fourth-order mesenteric arteries (170–250 µm) isolated from Sprague-Dawley rats were pressurized at 70 mmHg and allowed to develop spontaneous constriction, i.e., MT. Deoxycholylglycine (DCG; 0.1–100 µM), a glycine-conjugated major secondary BA, induced reversible, concentration-dependent reduction of MT that was similar in endothelium-intact and -denuded arteries. DCG reduced the myogenic response to stepwise increase in pressure (20 to 100 mmHg). Neither atropine nor the combination of L-NAME (a NOS inhibitor) plus indomethacin altered DCG-mediated reduction of MT. K⁺ channel blockade with glibenclamide (K_ATP), 4-aminopyradine (K_V), BaCl₂ (K_IR) or tetraethylammonium (TEA, K_Ca) were also ineffective. In Fluo-2-loaded arteries, DCG markedly reduced vascular smooth muscle cell (VSM) Ca²⁺ fluorescence (∼50%). In arteries incubated with DCG, physiological salt solution (PSS) with high Ca²⁺ (4 mM) restored myogenic response. DCG reduced vascular tone and VSM cytoplasmic Ca²⁺ responses (∼50%) of phenylephrine (PE)- and Ang II-treated arteries, but did not affect KCl-induced vasoconstriction.

Conclusion

In rat mesenteric resistance arteries DCG reduces pressure- and agonist-induced vasoconstriction and VSM cytoplasmic Ca²⁺ responses, independent of muscarinic receptor, NO or K⁺ channel activation. We conclude that BAs alter vasomotor responses, an effect favoring reduced SVR. These findings are likely pertinent to vascular dysfunction in cirrhosis and other conditions associated with elevated serum BAs.     

Low plasma albumin levels are associated with increased plasma protein glycation and HbA1c in diabetes

Albumin is one of the most abundant plasma proteins and is heavily glycated in diabetes. In this study, we have addressed whether variation in the albumin levels influence glycation of plasma proteins and HbA1c. The study was performed in three systems: (1) streptozotocin (STZ)-induced diabetic mice plasma, (2) diabetic clinical plasma, and (3) in vitro glycated plasma. Diabetic mice and clinical plasma samples were categorized as diabetic high albumin plasma (DHAP) and diabetic low albumin plasma (DLAP) on the basis of their albumin levels. For the in vitro experiment, two albumin levels, high albumin plasma (HAP) and low albumin plasma (LAP), were created by differential depletion of plasma albumin. Protein glycation was studied by using a combination of two-dimensional electrophoresis (2DE), Western blotting, and LC-MS(E). In both mice and clinical experiments, an increased plasma protein glycation was observed in DLAP than in DHAP. Additionally, plasma albumin levels were negatively correlated with HbA1c. The in vitro experiment with differential depletion of albumin mechanistically showed that the low albumin levels are associated with increased plasma protein glycation and that albumin competes for glycation with other plasma proteins.     

Safety evaluation of genetically modified mustard (V4) seeds in terms of allergenicity: Comparison with native crop

Genetically modified (GM) mustard line (V4) with increased carotenoid content was compared with native mustard to find the difference in allergenic potential, if any. Simulated gastric fluid (SGF) digestibility of crude protein extract from GM as well as its native counterpart mustard crop was envisaged to understand the intended or unintended changes in GM crop along with IgE immunoblotting. BALB/c mice were used as model for allergenicity studies for monitoring total and specific IgE, specific IgG1, histamine level, histopathology, and systemic anaphylaxis score. Allergenicity of mustard was checked in humans by clinical history, skin prick test and IgE levels. Similar results were evident by significant increase in total IgE, specific IgE, IgG1, histamine levels, in GM and native mustard in comparison to control group. Prominent anaphylactic symptoms (score 2: 60%; score 3: 20%; score 4: 20% in native mustard and score 2: 40%; score 3: 40%; score 4: 20% in GM mustard) and eruptive histopathological changes were observed in both GM and native mustard when compared with controls. One protein of approximately 16 kDa was found stable up to 1 h in both GM as well as non GM mustard. IgE immunoblotting detected three protein components of approximately 29, 24 and 16 kDa in both GM and non GM varieties. Collectively, our data demonstrate substantially equivalent allergic responses against GM as well as its native counterpart. Therefore, the GM mustard may be as safe as its native counterpart with reference to allergenic responses.