Mass Balance Model with Donnan Equilibrium Accurately Describes Unusual pH and Excipient Profiles during Diafiltration of Monoclonal Antibodies

There is extensive experimental data showing that the final pH and buffer composition after protein diafiltration (DF), particularly with monoclonal antibodies, can be considerably different than that in the DF buffer due to electrostatic interactions between the charged protein and the charged ions. Previous models for this behavior have focused on the final (equilibrium) partitioning and are unable to explain the complex pH and concentration profiles during the DF process. The objective of this study is to develop a new model for antibody DF based on solution of the transient mass balance equations, with the permeate concentrations of the charged species evaluated assuming Donnan equilibrium across the semipermeable membrane in combination with electroneutrality constraints. Model predictions are in excellent agreement with experimental data obtained during DF of both acidic and basic monoclonal antibodies, with the protein charge determined from independent electrophoretic mobility measurements. The model is able to predict the entire pH/histidine concentration profiles during DF, providing a framework for the development of DF processes that yield the desired antibody formulation.     

Mathematical model for pressure losses in the hemodialysis graft vascular circuit

Stenosis-induced thrombosis and abandonment of the hemodialysis synthetic graft is an important cause of morbidity and mortality. The graft vascular circuit is a unique low-resistance shunt that has not yet been systematically evaluated. In this study, we developed a mathematical model of this circuit. Pressure losses (deltaPs) were measured in an in vitro experimental apparatus and compared with losses predicted by equations from the engineering literature. We considered the inflow artery, arterial and venous anastomoses, graft, stenosis, and outflow vein. We found significant differences between equations and experimental results, and attributed these differences to the transitional nature of the flow. Adjustment of the equations led to good agreement with experimental data. The resulting mathematical model predicts relations between stenosis, blood flow, intragraft pressure, and important clinical variables such as mean arterial blood pressure and hematocrit. Application of the model should improve understanding of the hemodynamics of the stenotic graft vascular circuit.     

Intracellular heterogeneity in adhesiveness of endothelium affects early steps in leukocyte adhesion

Endothelial cell junctions are thought to be preferential sites for transmigration. However, the factors that determine the site of transmigration are not well defined. Our data show that the preferential role of endothelial cell junctions is not limited to transmigration but extends to earlier steps of leukocyte recruitment, such as rolling and arrest. We used primary mouse neutrophils and mouse aortic endothelium in a flow chamber system to compare adhesive interactions near endothelial cell junctions to interactions over endothelial cell centers. We found differences in both rolling velocity and arrest frequency for neutrophils at endothelial cell junctions vs. more central areas of endothelial cells. Differences were governed by adhesion molecule interactions, not local topography. Interestingly, the role of particular adhesion molecules depended on their location on the endothelial cell surface. Although ICAM-1 stabilized and slowed rolling over central areas of the cell, it did not influence rolling velocity over endothelial cell junctions. P-selectin and VCAM-1 were more important for rolling near endothelial cell junctions than E-selectin. This demonstrates that adhesive properties of endothelial cell junctions influence early events in the adhesion cascade, which may help explain how leukocytes are localized to sites of eventual transmigration. endothelial cells; rolling; selectins; integrins     

The ability of land plants to synthesize glucuronoxylans predates the evolution of tracheophytes

Glucuronoxylans with a backbone of 1,4-linked β-D-xylosyl residues are ubiquitous in the secondary walls of gymnosperms and angiosperms. Xylans have been reported to be present in hornwort cell walls, but their structures have not been determined. In contrast, the presence of xylans in the cell walls of mosses and liverworts remains a subject of debate. Here we present data that unequivocally establishes that the cell walls of leafy tissue and axillary hair cells of the moss Physcomitrella patens contain a glucuronoxylan that is structurally similar to glucuronoxylans in the secondary cell walls of vascular plants. Some of the 1,4-linked β-D-xylopyranosyl residues in the backbone of this glucuronoxylan bear an α-D-glucosyluronic acid (GlcpA) sidechain at O-2. In contrast, the lycopodiophyte Selaginella kraussiana synthesizes a glucuronoxylan substituted with 4-O-Me-α-D-GlcpA sidechains, as do many hardwood species. The monilophyte Equisetum hyemale produces a glucuronoxylan with both 4-O-Me-α-D-GlcpA and α-D-GlcpA sidechains, as does Arabidopsis. The seedless plant glucuronoxylans contain no discernible amounts of the reducing-end sequence that is characteristic of gymnosperm and eudicot xylans. Phylogenetic studies showed that the P. patens genome contains genes with high sequence similarity to Arabidopsis CAZy family GT8, GT43 and GT47 glycosyltransferases that are likely involved in xylan synthesis. We conclude that mosses synthesize glucuronoxylan that is structurally similar to the glucuronoxylans present in the secondary cell walls of lycopodiophytes, monilophytes, and many seed-bearing plants, and that several of the glycosyltransferases required for glucuronoxylan synthesis evolved before the evolution of tracheophytes.     

The RicAFT (YmcA‐YlbF‐YaaT) complex carries two [4Fe‐4S]2+ clusters and may respond to redox changes

During times of environmental insult, Bacillus subtilis undergoes developmental changes leading to biofilm formation, sporulation and competence. Each of these states is regulated in part by the phosphorylated form of the master response regulator Spo0A (Spo0A～P). The phosphorylation state of Spo0A is controlled by a multi‐component phosphorelay. RicA, RicF and RicT (previously YmcA, YlbF and YaaT) have been shown to be important regulatory proteins for multiple developmental fates. These proteins directly interact and form a stable complex, which has been proposed to accelerate the phosphorelay. Indeed, this complex is sufficient to stimulate the rate of phosphotransfer amongst the phosphorelay proteins in vitro. In this study, we demonstrate that two [4Fe‐4S]²⁺ clusters can be assembled on the complex. As with other iron‐sulfur cluster‐binding proteins, the complex was also found to bind FAD, hinting that these cofactors may be involved in sensing the cellular redox state. This work provides the first comprehensive characterization of an iron‐sulfur protein complex that regulates Spo0A～P levels. Phylogenetic and genetic evidence suggests that the complex plays a broader role beyond stimulation of the phosphorelay.     

P. falciparum RH5‐Basigin interaction induces changes in the cytoskeleton of the host RBC

The successful invasion of Plasmodium is an essential step in their life cycle. The parasite reticulocyte‐binding protein homologues (RHs) and erythrocyte‐binding like proteins are two families involved in the invasion leading to merozoite‐red blood cell (RBC) junction formation. Ca²⁺ signaling has been shown to play a critical role in the invasion. RHs have been linked to Ca²⁺ signaling, which triggers the erythrocyte‐binding like proteins release ahead of junction formation, consistent with RHs performing an initial sensing function in identifying suitable RBCs. RH5, the only essential RHs, is a highly promising vaccine candidate. RH5‐basigin interaction is essential for merozoite invasion and also important in determining host tropism. Here, we show that RH5 has a distinct function from the other RHs. We show that RH5‐Basigin interaction on its own triggers a Ca²⁺ signal in the RBC resulting in changes in RBC cytoskeletal proteins phosphorylation and overall alterations in RBC cytoskeleton architecture. Antibodies targeting RH5 that block the signal prevent invasion before junction formation consistent with the Ca²⁺ signal in the RBC leading to rearrangement of the cytoskeleton required for invasion. This work provides the first time a functional context for the essential role of RH5 and will now open up new avenues to target merozoite invasion.     

Structural diversity of xylans in the cell walls of monocots

Main conclusion

Xylans in the cell walls of monocots are structurally diverse. Arabinofuranose-containing glucuronoxylans are characteristic of commelinids. However, other structural features are not correlated with the major transitions in monocot evolution. Most studies of xylan structure in monocot cell walls have emphasized members of the Poaceae (grasses). Thus, there is a paucity of information regarding xylan structure in other commelinid and in non-commelinid monocot walls. Here, we describe the major structural features of the xylans produced by plants selected from ten of the twelve monocot orders. Glucuronoxylans comparable to eudicot secondary wall glucuronoxylans are abundant in non-commelinid walls. However, the α-d-glucuronic acid/4-O-methyl-α-d-glucuronic acid is often substituted at O-2 by an α-l-arabinopyranose residue in Alismatales and Asparagales glucuronoxylans. Glucuronoarabinoxylans were the only xylans detected in the cell walls of five different members of the Poaceae family (grasses). By contrast, both glucuronoxylan and glucuronoarabinoxylan are formed by the Zingiberales and Commelinales (commelinids). At least one species of each monocot order, including the Poales, forms xylan with the reducing end sequence -4)-β-d-Xylp-(1,3)-α-l-Rhap-(1,2)-α-d-GalpA-(1,4)-d-Xyl first identified in eudicot and gymnosperm glucuronoxylans. This sequence was not discernible in the arabinopyranose-containing glucuronoxylans of the Alismatales and Asparagales or the glucuronoarabinoxylans of the Poaceae. Rather, our data provide additional evidence that in Poaceae glucuronoarabinoxylan, the reducing end xylose residue is often substituted at O-2 with 4-O-methyl glucuronic acid or at O-3 with arabinofuranose. The variations in xylan structure and their implications for the evolution and biosynthesis of monocot cell walls are discussed.

     

Phenotype guided characterization and molecular analysis of Indian patients with long QT syndromes

BackgroundLong QT syndromes (LQTS) are characterized by prolonged QTc interval on electrocardiogram (ECG) and manifest with syncope, seizures or sudden cardiac death. Long QT 1–3 constitute about 75% of all inherited LQTS. We classified a cohort of Indian patients for the common LQTS based on T wave morphology and triggering factors to prioritize the gene to be tested. We sought to identify the causative mutations and mutation spectrum, perform genotype-phenotype correlation and screen family members.MethodsThirty patients who fulfilled the criteria were enrolled. The most probable candidate gene among KCNQ1, KCNH2 and SCN5A were sequenced.ResultsOf the 30 patients, 22 were classified at LQT1, two as LQT2 and six as LQT3. Mutations in KCNQ1 were identified in 17 (77%) of 22 LQT1 patients, KCNH2 mutation in one of two LQT2 and SCN5A mutations in two of six LQT3 patients. We correlated the presence of the specific ECG morphology in all mutation positive cases. Eight mutations in KCNQ1 and one in SCN5A were novel and predicted to be pathogenic by in-silico analysis. Of all parents with heterozygous mutations, 24 (92%) of 26 were asymptomatic. Ten available siblings of nine probands were screened and three were homozygous and symptomatic, five heterozygous and asymptomatic.ConclusionsThis study in a cohort of Asian Indian patients highlights the mutation spectrum of common Long QT syndromes. The clinical utility for prevention of unexplained sudden cardiac deaths is an important sequel to identification of the mutation in at-risk family members.