Identification of a frit-related sample carryover in newborn screening by tandem mass spectrometry.

The need for high-throughput analysis of multiple analytes for inborn errors of metabolism in newborn screening (NBS) has led to the introduction of tandem mass spectrometry (MS/MS) into the NBS laboratory. In a flow-injection analysis (FIA), the predominant MS/MS method utilized for NBS, samples are introduced directly into the mass spectrometer without chromatographic separation. When a high-throughput FIA-based MS/MS method is implemented on newer generations of mass spectrometers with increased sensitivity, the risk of carryover and contamination increases. In the present study, we report the carryover of ornithine identified during the implementation of the NeoBase™ 2 (PerkinElmer) non-derivatized kits on the Xevo-TQD platform (Waters Corporation) and describe the source of the carryover, which was traced to the stainless-steel frit-type inline filter. Furthermore, a possible compound-dependent interaction with the stainless-steel frit is suggested based on the structure of ornithine and its effect on separation techniques. Investigation and mitigation of carryover can be a time and resource consuming process, and to this end, our report on identification of a stainless-steel frit as the source of delayed elution and carryover of ornithine should be recognized as a rare, albeit possible source of carryover in FIA-MS/MS methods adopted for NST.

PERISCOPE-Opt: Machine learning-based prediction of optimal fermentation conditions and yields of recombinant periplasmic protein expressed in Escherichia coli.

Optimization of the fermentation process for recombinant protein production (RPP) is often resource-intensive. Machine learning (ML) approaches are helpful in minimizing the experimentations and find vast applications in RPP. However, these ML-based tools primarily focus on features with respect to amino-acid-sequence, ruling out the influence of fermentation process conditions. The present study combines the features derived from fermentation process conditions with that from amino acid-sequence to construct an ML-based model that predicts the maximal protein yields and the corresponding fermentation conditions for the expression of target recombinant protein in the Escherichia coli periplasm. Two sets of XGBoost classifiers were employed in the first stage to classify the expression levels of the target protein as high (>50 mg/L), medium (between 0.5 and 50 mg/L), or low (<0.5 mg/L). The second-stage framework consisted of three regression models involving support vector machines and random forest to predict the expression yields corresponding to each expression-level-class. Independent tests showed that the predictor achieved an overall average accuracy of 75% and a Pearson coefficient correlation of 0.91 for the correctly classified instances. Therefore, our model offers a reliable substitution of numerous trial-and-error experiments to identify the optimal fermentation conditions and yield for RPP. It is also implemented as an open-access webserver, PERISCOPE-Opt (http://periscope-opt.erc.monash.edu).

Comprehensive genomics and expression analysis of eceriferum (CER) genes in sunflower (Helianthus annuus).

Sunflower occupies the fourth position among oilseed crops the around the world. Eceriferum (CER) is an important gene family that plays critical role in very-long-chain fatty acids elongation and biosynthesis of epicuticular waxes under both biotic and abiotic stress conditions. The aim of present study was to investigate the effect of sunflower CER genes during drought stress condition. Thus, comparative analysis was undertaken for sunflower CER genes with Arabidopsis genome to determine phylogenetic relationship, chromosomal mapping, gene structures, gene ontology and conserved motifs. Furthermore, we subjected the sunflower cultivars under drought stress and used qRT-PCR analysis to explore the expression pattern of CER genes during drought conditions. We identified thirty-seven unevenly distributed CER genes in the sunflower genome. The phylogenetic analysis revealed that CER genes were grouped into seven clades in Arabidopsis, Helianthus annuus, and Gossypium hirsutum. Expression analysis showed that genes CER10 and CER60 were upregulated in sunflower during drought conditions, indicating that these genes are activated during drought stress. The results obtained will serve to characterize the CER gene family in sunflower and exploit the role of these genes in wax biosynthesis under limited water conditions. KEY MESSAGE: Cuticular waxes protect the plants from drought stress, so we observed the expression of wax bio synthesis genes in recently sequences genome of Helianthus annuus. We observed that expression of wax biosynthesis genes CER10 and CER60 was upregulated when the plants were subjected to drought stress.

Oral delivery of proteins and peptides: Challenges, status quo and future perspectives.

Proteins and peptides (PPs) have gradually become more attractive therapeutic molecules than small molecular drugs due to their high selectivity and efficacy, but fewer side effects. Owing to the poor stability and limited permeability through gastrointestinal (GI) tract and epithelia, the therapeutic PPs are usually administered by parenteral route. Given the big demand for oral administration in clinical use, a variety of researches focused on developing new technologies to overcome GI barriers of PPs, such as enteric coating, enzyme inhibitors, permeation enhancers, nanoparticles, as well as intestinal microdevices. Some new technologies have been developed under clinical trials and even on the market. This review summarizes the history, the physiological barriers and the overcoming approaches, current clinical and preclinical technologies, and future prospects of oral delivery of PPs.

Comparative expression analysis of heavy metal ATPase subfamily genes between Cd-tolerant and Cd-sensitive turnip landraces.

The heavy metal ATPase (HMA) subfamily is mainly involved in heavy metal (HM) tolerance and transport in plants, but an understanding of the definite roles and mechanisms of most HMA members are still limited. In the present study, we identified 14 candidate HMA genes named BrrHMA1-BrrHMA8 from the turnip genome and analyzed the phylogeny, gene structure, chromosome distribution, and conserved domains and motifs of HMAs in turnip (Brassica rapa var. rapa). According to our phylogenetic tree, the BrrHMAs are divided into a Zn/Cd/Co/Pb subclass and Cu/Ag subclass. The BrrHMA members show similar structural characteristics within subclasses. To explore the roles of BrrHMAs in turnip, we compared the gene sequences and expression patterns of the BrrHMA genes between a Cd-tolerant landrace and a Cd-sensitive landrace. Most BrrHMA genes showed similar spatial expression patterns in both Cd-tolerant and Cd-sensitive turnip landraces; some BrrHMA genes, however, were differentially expressed in specific tissue in Cd-tolerant and Cd-sensitive turnip. Specifically, BrrHMA genes in the Zn/Cd/Co/Pb subclass shared the same coding sequence but were differentially expressed in Cd-tolerant and Cd-sensitive turnip landraces under Cd stress. Our findings suggest that the stable expression and up-regulated expression of BrrHMA Zn/Cd/Co/Pb subclass genes under Cd stress may contribute to the higher Cd tolerance of turnip landraces.

Balancing charge in the complementarity-determining regions of humanized mAbs without affecting pI reduces non-specific binding and improves the pharmacokinetics.

Lowering the isoelectric point (pI) through engineering the variable region or framework of an IgG can improve its exposure and half-life via a reduction in clearance mediated through non-specific interactions. As such, net charge is a potentially important property to consider in developing therapeutic IgG molecules having favorable pharmaceutical characteristics. Frequently, it may not be possible to shift the pI of monoclonal antibodies (mAbs) dramatically without the introduction of other liabilities such as increased off-target interactions or reduced on-target binding properties. In this report, we explored the influence of more subtle modifications of molecular charge on the in vivo properties of an IgG1 and IgG4 monoclonal antibody. Molecular surface modeling was used to direct residue substitutions in the complementarity-determining regions (CDRs) to disrupt positive charge patch regions, resulting in a reduction in net positive charge without affecting the overall pI of the mAbs. The effect of balancing the net positive charge on non-specific binding was more significant for the IgG4 versus the IgG1 molecule that we examined. This differential effect was connected to the degree of influence on cellular degradation in vitro and in vivo clearance, distribution and metabolism in mice. In the more extreme case of the IgG4, balancing the charge yielded an ∼7-fold improvement in peripheral exposure, as well as significantly reduced tissue catabolism and subsequent excretion of proteolyzed products in urine. Balancing charge on the IgG1 molecule had a more subtle influence on non-specific binding and yielded only a modest alteration in clearance, distribution and elimination. These results suggest that balancing CDR charge without affecting the pI can lead to improved mAb pharmacokinetics, the magnitude of which is likely dependent on the relative influence of charge imbalance and other factors affecting the molecule's disposition.

The neonatal Fc receptor (FcRn) binds independently to both sites of the IgG homodimer with identical affinity.

The neonatal Fc receptor (FcRn) is expressed by cells of epithelial, endothelial and myeloid lineages and performs multiple roles in adaptive immunity. Characterizing the FcRn/IgG interaction is fundamental to designing therapeutic antibodies because IgGs with moderately increased binding affinities for FcRn exhibit superior serum half-lives and efficacy. It has been hypothesized that 2 FcRn molecules bind an IgG homodimer with disparate affinities, yet their affinity constants are inconsistent across the literature. Using surface plasmon resonance biosensor assays that eliminated confounding experimental artifacts, we present data supporting an alternate hypothesis: 2 FcRn molecules saturate an IgG homodimer with identical affinities at independent sites, consistent with the symmetrical arrangement of the FcRn/Fc complex observed in the crystal structure published by Burmeister et al. in 1994. We find that human FcRn binds human IgG1 with an equilibrium dissociation constant (KD) of 760 ± 60 nM (N = 14) at 25°C and pH 5.8, and shows less than 25% variation across the other human subtypes. Human IgG1 binds cynomolgus monkey FcRn with a 2-fold higher affinity than human FcRn, and binds both mouse and rat FcRn with a 10-fold higher affinity than human FcRn. FcRn/IgG interactions from multiple species show less than a 2-fold weaker affinity at 37°C than at 25°C and appear independent of an IgG's variable region. Our in vivo data in mouse and rat models demonstrate that both affinity and avidity influence an IgG's serum half-life, which should be considered when choosing animals, especially transgenic systems, as surrogates.

Proteomics of old world camelid (Camelus dromedarius): Better understanding the interplay between homeostasis and desert environment.

Life is the interplay between structural-functional integrity of biological systems and the influence of the external environment. To understand this interplay, it is useful to examine an animal model that competes with harsh environment. The dromedary camel is the best model that thrives under severe environment with considerable durability. The current proteomic study on dromedary organs explains a number of cellular mysteries providing functional correlates to arid living. Proteome profiling of camel organs suggests a marked increased expression of various cytoskeleton proteins that promote intracellular trafficking and communication. The comparative overexpression of α-actinin of dromedary heart when compared with rat heart suggests an adaptive peculiarity to sustain hemoconcentration-hemodilution episodes associated with alternative drought-rehydration periods. Moreover, increased expression of the small heat shock protein, α B-crystallin facilitates protein folding and cellular regenerative capacity in dromedary heart. The observed unbalanced expression of different energy related dependent mitochondrial enzymes suggests the possibility of mitochondrial uncoupling in the heart in this species. The evidence of increased expression of H+-ATPase subunit in camel brain guarantees a rapidly usable energy supply. Interestingly, the guanidinoacetate methyltransferase in camel liver has a renovation effect on high energy phosphate with possible concomitant intercession of ion homeostasis. Surprisingly, both hump fat tissue and kidney proteomes share the altered physical distribution of proteins that favor cellular acidosis. Furthermore, the study suggests a vibrant nature for adipose tissue of camel hump by the up-regulation of vimentin in adipocytes, augmenting lipoprotein translocation, blood glucose trapping, and challenging external physical extra-stress. The results obtained provide new evidence of homeostasis in the arid habitat suitable for this mammal.

A water-soluble selenoxide reagent as a useful probe for the reactivity and folding of polythiol peptides.

A water-soluble selenoxide (DHS(ox)) having a five-membered ring structure enables rapid and selective conversion of cysteinyl SH groups in a polypeptide chain into SS bonds in a wide pH and temperature range. It was previously demonstrated that the second-order rate constants for the SS formation with DHS(ox) would be proportional to the number of the free SH groups present in the substrate if there is no steric congestion around the SH groups. In the present study, kinetics of the SS formation with DHS(ox) was extensively studied at pH 4-10 and 25 °C by using reduced ribonuclease A, recombinant hirudin variant (CX-397), insulin A- and B-chains, and relaxin A-chain, which have two to eight cysteine residues, as polythiol substrates. The obtained rate constants showed stochastic SS formation behaviors under most conditions. However, the rate constants for CX-397 at pH 8.0 and 10.0 were not proportional to the number of the free SH groups, suggesting that the SS intermediate ensembles possess densely packed structures under weakly basic conditions. The high two-electron redox potential of DHS(ox) (375 mV at 25 °C) compared to l-cystine supported the high ability of DHS(ox) for SS formation in a polypeptide chain. Interestingly, the rate constants of the SS formation jumped up at a pH around the pK a value of the cysteinyl SH groups. The SS formation velocity was slightly decreased by addition of a denaturant due probably to the interaction between the denaturant and the peptide. The stochastic behaviors as well as the absolute values of the second-order rate constants in comparison to dithiothreitol (DTT(red)) are useful to probe the chemical reactivity and conformation, hence the folding, of polypeptide chains.