Impact of Glycosylation on the Local Backbone Flexibility of Well-Defined IgG1-Fc Glycoforms Using Hydrogen Exchange-Mass Spectrometry

We have used hydrogen exchange–mass spectrometry to characterize local backbone flexibility of 4 well-defined IgG1-Fc glycoforms expressed and purified from Pichia pastoris, 2 of which were prepared using subsequent in vitro enzymatic treatments. Progressively decreasing the size of the N-linked N297 oligosaccharide from high mannose (Man8-Man12), to Man5, to GlcNAc, to nonglycosylated N297Q resulted in progressive increases in backbone flexibility. Comparison of these results with recently published physicochemical stability and Fcγ receptor binding data with the same set of glycoproteins provide improved insights into correlations between glycan structure and these pharmaceutical properties. Flexibility significantly increased upon glycan truncation in 2 potential aggregation-prone regions. In addition, a correlation was established between increased local backbone flexibility and increased deamidation at asparagine 315. Interestingly, the opposite trend was observed for oxidation of tryptophan 277 where faster oxidation correlated with decreased local backbone flexibility. Finally, a trend of increasing C'E glycopeptide loop flexibility with decreasing glycan size was observed that correlates with their FcγRIIIa receptor binding properties. These well-defined IgG1-Fc glycoforms serve as a useful model system to identify physicochemical stability and local backbone flexibility data sets potentially discriminating between various IgG glycoforms for potential applicability to future comparability or biosimilarity assessments.     

Demonstration of In Vitro Host-Guest Complex Formation and Safety of para-Sulfonatocalix[8]arene as a Delivery Vehicle for Two Antibiotic Drugs

The macrocycle para-sulfonatocalix[8]arene, sCX[8], was examined with 2 antibiotic drugs, ciprofloxacin (CIP) and isoniazid. The drugs were shown to form complexes with sCX[8] using proton nuclear magnetic resonance, thermogravimetric analysis, fluorescence spectroscopy, and molecular modeling. Both drugs form 1:1 hydrated (H₂O: 13%-14% w/w) host-guest complexes, with sCX[8] binding around the pyridine ring of isoniazid, and around the piperazine and cyclopropane rings of CIP. From proton nuclear magnetic resonance, the binding constant of isoniazid to sCX[8] was 6.8 (±0.3) × 10³ M^?1. Addition of 2 equivalents of sCX[8] to CIP resulted in a 58% decrease in fluorescence, and time-resolved fluorescence anisotropy of CIP doubles with sCX[8]. Each drug binds into the cavity of the macrocycle, with binding stabilized via combinations of hydrogen bonding, electrostatic interactions, π-π stacking, and hydrophobic effects. The safety of sCX[8] was examined in vitro with human embryonic kidney 293 cells. The IC₅₀ of sCX[8] was 559 μM, which is a minimum of 5-fold higher than the concentration that would be used in the clinic. The in vitro effect of sCX[8] on the action of CIP was examined on a panel of bacterial lines. The results showed that sCX[8] has no inherent antibiotic activity and had no negative effect on the action of CIP.     

Dibucaine in Ionic-Gradient Liposomes: Biophysical,Toxicological, and Activity Characterization

Administration of local anesthetics is one of the most effective pain control techniques for postoperative analgesia. However, anesthetic agents easily diffuse into the injection site, limiting the time of anesthesia. One approach to prolong analgesia is to entrap local anesthetic agents in nanostructured carriers (e.g., liposomes). Here, we report that using an ammonium sulphate gradient was the best strategy to improve the encapsulation (62.6%) of dibucaine (DBC) into liposomes. Light scattering and nanotracking analyses were used to characterize vesicle properties, such as, size, polydispersity, zeta potentials, and number. In vitro kinetic experiments revealed the sustained release of DBC (50% in 7 h) from the liposomes. In addition, in vitro (3T3 cells in culture) and in vivo (zebrafish) toxicity assays revealed that ionic-gradient liposomes were able to reduce DBC cyto/cardiotoxicity and morphological changes in zebrafish larvae. Moreover, the anesthesia time attained after infiltrative administration in mice was longer with encapsulated DBC (27 h) than that with free DBC (11 h), at 320 μM (0.012%), confirming it as a promising long-acting liposome formulation for parenteral drug administration of DBC.     

Effect of correcting for gestational age at birth on population prevalence of early childhood undernutrition

Background

Postmenstrual and/or gestational age-corrected age (CA) is required to apply child growth standards to children born preterm (< 37 weeks gestational age). Yet, CA is rarely used in epidemiologic studies in low- and middle-income countries (LMICs), which may bias population estimates of childhood undernutrition. To evaluate the effect of accounting for GA in the application of growth standards, we used GA-specific standards at birth (INTERGROWTH-21st newborn size standards) in conjunction with CA for preterm-born children in the application of World Health Organization Child Growth Standards postnatally (referred to as ‘CA’ strategy) versus postnatal age for all children, to estimate mean length-for-age (LAZ) and weight-for-age (WAZ) z scores at 0, 3, 12, 24, and 48-months of age in the 2004 Pelotas (Brazil) Birth Cohort.

Results

At birth (n = 4066), mean LAZ was higher and the prevalence of stunting (LAZ < ?2) was lower using CA versus postnatal age (mean ± SD): ? 0.36 ± 1.19 versus ? 0.67 ± 1.32; and 8.3 versus 11.6%, respectively. Odds ratio (OR) and population attributable risk (PAR) of stunting due to preterm birth were attenuated and changed inferences using CA versus postnatal age at birth [OR, 95% confidence interval (CI): 1.32 (95% CI 0.95, 1.82) vs 14.7 (95% CI 11.7, 18.4); PAR 3.1 vs 42.9%]; differences in inferences persisted at 3-months. At 12, 24, and 48-months, preterm birth was associated with stunting, but ORs/PARs remained attenuated using CA compared to postnatal age. Findings were similar for weight-for-age z scores.

Conclusions

Population-based epidemiologic studies in LMICs in which GA is unused or unavailable may overestimate the prevalence of early childhood undernutrition and inflate the fraction of undernutrition attributable to preterm birth.

     

A high frequency missense SULT1B1 allelic variant (L145V) selectively expressed in African descendants exhibits altered kinetic properties

1.?Human cytosolic sulfotransferase 1B1 (SULT1B1) sulfates small phenolic compounds and bioactivates polycyclic aromatic hydrocarbons. To date, no SULT1B1 allelic variants have been well-characterized.

2.?While cloning SULT1B1 from human endometrial specimens, an allelic variant resulting in valine instead of leucine at the 145th amino acid position (L145V) was detected. NCBI reported this alteration as the highest frequency SULT1B1 allelic variant.

3.?L145V frequency comprised 9% of 37 mixed-population human patients and was specific to African Americans with an allelic frequency of 25%. Structurally, replacement of leucine with valine potentially destabilizes a conserved helix (α8) that forms the “floor” of both the substrate and PAPS binding domains. This destabilization results in altered kinetic properties including a four-fold decrease in affinity for PAP (3′, 5′-diphosphoadenosine). K_ms for 3′-phosphoadenosine- 5′-phosphosulfate (PAPS) are similar; however, maximal turnover rate of the variant isoform (0.86?pmol/(min*μg)) is slower than wild-type (WT) SULT1B1 (1.26?pmol/(min*μg)). The L145V variant also displays altered kinetics toward small phenolic substrates, including a diminished p-nitrophenol K_m and increased susceptibility to 1-naphthol substrate inhibition.

4.?No significant correlation between genotype and prostate or colorectal cancer was observed in patients; however, the variant isoform could underlie specific pathologies in sub-Saharan African carriers.     

PROBLEMATIC INTERNET USE AMONG ADULT JAPANESE PSYCHIATRIC PATIENTS

Background

A large number of studies have reported the high prevalence of problematic internet use (PIU) among adolescents and students (13-50%)¹, and PIU has been associated with many psychiatric symptoms². In contrast, only a few studies have investigated its prevalence among the adult population. To the best of our knowledge, there have been no studies investigating the prevalence and comorbidity of PIU in a psychiatric population although psychiatric symptoms might either induce PIU in patients with psychiatric illnesses, or PIU might induce or aggravate psychiatric symptoms. The aims of this study are to investigate the prevalence of PIU and psychiatric co-morbidity among adult psychiatric patients.