Influence of FCGRT gene polymorphisms on pharmacokinetics of therapeutic antibodies

The neonatal Fc receptor (FcRn) encoded by FCGRT is known to be involved in the pharmacokinetics (PK) of therapeutic monoclonal antibodies (mAbs). Variability in the expression of FCGRT gene and consequently in the FcRn protein level could explain differences in PK observed between patients treated with mAbs. We studied whether the previously described variable number tandem repeat (VNTR) or copy number variation (CNV) of FCGRT are associated with individual variations of PK parameters of cetuximab. VNTR and CNV were assessed on genomic DNA of 198 healthy individuals and of 94 patients treated with the therapeutic mAb. VNTR and CNV were analyzed by allele-specific PCR and duplex real-time PCR with Taqman^® technology, respectively. The relationship between FCGRT polymorphisms (VNTR and CNV) and PK parameters of patients treated with cetuximab was studied. VNTR3 homozygote patients had a lower cetuximab distribution clearance than VNTR2/VNTR3 and VNTR3/VNTR4 patients (p = 0.021). We observed no affects of VNTR genotype on elimination clearance. One healthy person (0.5%) and 1 patient (1.1%) had 3 copies of FCGRT. The PK parameters of this patient did not differ from those of patients with 2 copies. The FCGRT promoter VNTR may influence mAbs’ distribution in the body. CNV of FCGRT cannot be used as a relevant pharmacogenetic marker because of its low frequency.     

DNA BARCODING OF CHLORARACHNIOPHYTES USING NUCLEOMORPH ITS SEQUENCES1

Chlorarachniophytes are a small group of marine photosynthetic protists. They are best known as examples of an intermediate stage of secondary endosymbiosis: their plastids are derived from green algae and retain a highly reduced nucleus, called a nucleomorph, between the inner and outer pairs of membranes. Chlorarachniophytes can be challenging to identify to the species level, due to their small size, complex life cycles, and the fact that even genus‐level diagnostic morphological characters are observable only by EM. Few species have been formally described, and many available culture collection strains remain unnamed. To alleviate this difficulty, we have developed a barcoding system for rapid and accurate identification of chlorarachniophyte species in culture, based on the internal transcribed spacer (ITS) region of the nucleomorph rRNA cistron. Although this is a multicopy locus, encoded in both subtelomeric regions of each chromosome, interlocus variability is low due to gene conversion by homologous recombination in this region. Here, we present barcode sequences for 39 cultured strains of chlorarachniophytes (>80% of currently available strains). Based on barcode data, other published molecular data, and information from culture records, we were able to recommend names for 21 out of the 24 unidentified, partially identified, or misidentified chlorarachniophyte strains in culture. Most strains could be assigned to previously described species, but at least two to as many as five new species may be present among cultured strains.     

The Role of Host Phylogeny Varies in Shaping Microbial Diversity in the Hindguts of Lower Termites

The hindguts of lower termites and Cryptocercus cockroaches are home to a distinct community of archaea, bacteria, and protists (primarily parabasalids and some oxymonads). Within a host species, the composition of these hindgut communities appears relatively stable, but the evolutionary and ecological factors structuring community composition and stability are poorly understood, as are differential impacts of these factors on protists, bacteria, and archaea. We analyzed the microbial composition of parabasalids and bacteria in the hindguts of Cryptocercus punctulatus and 23 species spanning 4 families of lower termites by pyrosequencing variable regions of the small-subunit rRNA gene. Especially for the parabasalids, these data revealed undiscovered taxa and provided a phylogenetic basis for a more accurate understanding of diversity, diversification, and community composition. The composition of the parabasalid communities was found to be strongly structured by the phylogeny of their hosts, indicating the importance of historical effects, although exceptions were also identified. Particularly, spirotrichonymphids and trichonymphids likely were transferred between host lineages. In contrast, host phylogeny was not sufficient to explain the majority of bacterial community composition, but the compositions of the Bacteroidetes, Elusimicrobia, Tenericutes, Spirochaetes, and Synergistes were structured by host phylogeny perhaps due to their symbiotic associations with protists. All together, historical effects probably resulting from vertical inheritance have had a prominent role in structuring the hindgut communities, especially of the parabasalids, but dispersal and environmental acquisition have played a larger role in community composition than previously expected.     

Evidence for essential thiol groups and disulfide bonds in agonist and antagonist binding to the dopamine receptor

Dithiothreitol (DTT), a disulfide reducing agent, diminished the specific binding of [³H] dopamine to partially purified calf striatal membranes (P₂) but did not have an effect on [³H] spiroperidol binding. The thiol reagents, p-chloromercuribenzoate (PCMB), N-ethylmaleimide (NEM) and iodoacetamide (IA), were also tested for inhibitory effects on agonist and antagonist binding to the dopamine receptor. PCMB inhibited both [³H] dopamine and [³H] spiroperidol binding by changing the affinity (K_d) and the number of binding sites (B_max) for both of these ligands. This effect of PCMB was reversed by the addition of DTT. NEM inhibited binding to the dopamine agonist site but not to the antagonist site, while IA was ineffective on either site. These results indicate that a DTT-reducible disulfide bond may be an essential component for agonist binding to the dopamine receptor. Furthermore, the experiments with PCMB, NEM and IA suggest that the exposure of thiol groups in the dopamine receptor may play an important role in agonist and antagonist binding.     

Comparison of protein quantification and extraction methods suitable for E. coli cultures

Many different extraction and analysis methods exist to determine the protein fraction of microbial cells. For metabolic engineering purposes it is important to have precise and accurate measurements. Therefore six different protein extraction protocols and seven protein quantification methods were tested and compared. Comparison was based on the reliability of the methods and boxplots of the normalized residuals. Some extraction techniques (SDS/chloroform and toluene) should never be used: the measurements are neither precise nor accurate. Bugbuster extraction combined with UV280 quantification gives the best results, followed by the combinations Sonication-UV280 and EasyLyse-UV280. However, if one does not want to use the quantification method UV280, one can opt to use Bugbuster, EasyLyse or sonication extraction combined with any quantification method with exception of the EasyLyse-BCA_P and Sonication-BCA_P combinations.