Increased cortical neuronal responses to NMDA and improved attentional set-shifting performance in rats following prebiotic (B-GOS®) ingestion

We have previously shown that prebiotics (dietary fibres that augment the growth of indigenous beneficial gut bacteria) such as Bimuno^? galacto-oligosaccharides (B-GOS^®), increased N-methyl-D-aspartate (NMDA) receptor levels in the rat brain. The current investigation examined the functional correlates of these changes in B-GOS^®-fed rats by measuring cortical neuronal responses to NMDA using in vivo NMDA micro-iontophoresis electrophysiology, and performance in the attentional set-shifting task. Adult male rats were supplemented with B-GOS^® in the drinking water 3 weeks prior to in vivo iontophoresis or behavioural testing. Cortical neuronal responses to NMDA iontophoresis, were greater (+30%) in B-GOS^® administered rats compared to non-supplemented controls. The intake of B-GOS^® also partially hindered the reduction of NMDA responses by the glycine site antagonist, HA-966. In the attentional set-shifting task, B-GOS^® -fed rats shifted from an intra-dimensional to an extra-dimensional set in fewer trials than controls, thereby indicating greater cognitive flexibility. An initial exploration into the mechanisms revealed that rats ingesting B-GOS^® had increased levels of plasma acetate, and cortical GluN2B subunits and Acetyl Co-A Carboxylase mRNA. These changes were also observed in rats fed daily for 3 weeks with glyceryl triacetate, though unlike B-GOS^®, cortical histone deacetylase (HDAC1, HDAC2) mRNAs were also increased which suggested an additional epigenetic action of direct acetate supplementation. Our data demonstrate that a pro-cognitive effect of B-GOS^® intake in rats is associated with an increase in cortical NMDA receptor function, but the role of circulating acetate derived from gut bacterial fermentation of this prebiotic requires further investigation.     

The Properties of Cysteine-Conjugated Antibody-Drug Conjugates Are Impacted by the IgG Subclass

Among the numerous antibody-drug conjugate (ADC) clinical candidates, one of the most prevalent types utilizes the interchain cysteines in antibodies to conjugate auristatin via a maleimide-containing linker. In this class of ADCs, there are a paucity of systematic studies characterizing how IgG subclass influences the biophysical properties and in vivo pharmacokinetics of the ADC molecules. In the current investigation, we studied cysteine-conjugated ADCs using a model system consisting of human IgG1, IgG2, and IgG4 antibodies with the same variable region. Our findings identified some unforeseen differences among the three ADCs. Drug conjugation profiling by LC-MS revealed that 50% of inter heavy-light chain disulfide bonds are disrupted to conjugate drugs in IgG1 antibody while only 10% in IgG2 antibody and 20% in IgG4 antibody. The solution behavior of the ADCs was interrogated in concentrating experiments and diffusion interaction parameter measurements. We found that drug conjugation affected the solution property of the three antibodies differently, with the IgG2-based ADC having the most increased propensity to aggregate. Rat PK studies using a sensitive LC-MS-based bioanalytical method showed that the IgG1-based ADC has poor peripheral linker-payload stability while the IgG2- and IgG4-based ADCs are stable. The conjugate stability of the IgG2-based ADC was further confirmed in a cynomolgus monkey PK study. Overall, the IgG2-based ADC exhibited the best PK/conjugate stability but also the most deterioration in stability among the three ADCs. Our findings provide important information and present multifactorial considerations for the selection of IgG subclass during ADC drug discovery when employing stochastic cysteine conjugation.     

MRI compatible MS2 nanoparticles designed to cross the blood–brain-barrier: providing a path towards tinnitus treatment

Fundamental challenges of targeting specific brain regions for treatment using pharmacotherapeutic nanoparticle (NP) carriers include circumventing the blood–brain-barrier (BBB) and tracking delivery. Angiopep-2 (AP2) has been shown to facilitate the transport of large macromolecules and synthetic nanoparticles across the BBB. Thus, conjugation of AP2 to an MS2 bacteriophage based NP should also permit transport across the BBB. We have fabricated and tested a novel MS2 capsid-based NP conjugated to the ligand AP2. The reaction efficiency was determined to be over 70%, with up to two angiopep-2 conjugated per MS2 capsid protein. When linked with a porphyrin ring, manganese (Mn²⁺) remained stable within MS2 and was MRI detectable. Nanoparticles were introduced intracerebroventricularly or systemically. Systemic delivery yielded dose dependent, non-toxic accumulation of NPs in the midbrain. Design of a multifunctional MRI compatible NP platform provides a significant step forward for the diagnosis and treatment of intractable brain conditions, such as tinnitus.     

Time-varying coefficient of determination to quantify the explanatory power of biomarkers on longitudinal GFR among children with chronic kidney disease

Purpose

Coefficients of determination (R²) for continuous longitudinal data are typically reported as time constant, if they are reported at all. The widely used mixed model with random intercepts and slopes yields the total outcome variance as a time-varying function. We propose a generalized and intuitive approach based on this variance function to estimate the time-varying predictive power (R²) of a variable on outcome levels and changes.

A Prospective Cohort Study of the Impact of Return-to-Work Coordinators in Getting Injured Workers Back on the Job

Purpose To assess the impact of workplace-based return-to-work (RTW) Coordinators’ interpersonal and functional activities on RTW outcomes. Methods Multivariable logistic regression analyses of cross-sectional and longitudinal survey responses of 632 injured workers with at least 10 days of work absence in Victoria, Australia, adjusting for demographic and other workplace factors. Outcome was being back at work for at least 1 month, measured at both baseline and 6 month follow-up survey. Participant responses to stressfulness of Coordinator interactions were dichotomised into good and poor and evaluated as a proxy for Coordinators’ interpersonal activities, while having a RTW plan was evaluated as a proxy for functional activities. Results At baseline, RTW plans doubled the odds of RTW (OR 2.02; 95% CI 1.40–2.90) and attenuated the impact of good Coordinator interactions (1.14; 0.77–1.70). At 6-month follow-up, the opposite was observed: good interactions nearly doubled odds of RTW (1.90; 1.22–2.95) while RTW plans were non-significant (1.02; 0.68–1.54). Conclusions Differences between when the two Coordinator activities were effective may be due to the nature of claimants who RTW in each survey period. Length of shorter-duration claims are influenced by injury related factors, while psychosocial factors tend to be more important for longer-duration claims. Such factors may determine whether a claimant is more likely to respond to Coordinators’ functional or interpersonal activities. The findings have important implications for increasing Coordinator effectiveness.     

Strategies for phasing and imputation in a population isolate

In the search for genetic associations with complex traits, population isolates offer the advantage of reduced genetic and environmental heterogeneity. In addition, cost‐efficient next‐generation association approaches have been proposed in these populations where only a subsample of representative individuals is sequenced and then genotypes are imputed into the rest of the population. Gene mapping in such populations thus requires high‐quality genetic imputation and preliminary phasing. To identify an effective study design, we compare by simulation a range of phasing and imputation software and strategies. We simulated 1,115,604 variants on chromosome 10 for 477 members of the large complex pedigree of Campora, a village within the established isolate of Cilento in southern Italy. We assessed the phasing performance of identical by descent based software ALPHAPHASE and SLRP, LD‐based software SHAPEIT2, SHAPEIT3, and BEAGLE, and new software EAGLE that combines both methodologies. For imputation we compared IMPUTE2, IMPUTE4, MINIMAC3, BEAGLE, and new software PBWT. Genotyping errors and missing genotypes were simulated to observe their effects on the performance of each software. Highly accurate phased data were achieved by all software with SHAPEIT2, SHAPEIT3, and EAGLE2 providing the most accurate results. MINIMAC3, IMPUTE4, and IMPUTE2 all performed strongly as imputation software and our study highlights the considerable gain in imputation accuracy provided by a genome sequenced reference panel specific to the population isolate.     

Frequency of development and associated physiological cost of azithromycin resistance in Chlamydia psittaci 6BC and C. trachomatis L2

Azithromycin is a major drug used in the treatment and prophylaxis of chlamydial infections. Spontaneous azithromycin-resistant mutants of Chlamydia psittaci 6BC were isolated in vitro in the plaque assay at a frequency of about 10(-8). Isogenic clonal variants with A(2058)C, A(2059)G, or A(2059)C mutations in the unique 23S rRNA gene (Escherichia coli numbering system) displayed MICs for multiple macrolides (i.e., azithromycin, erythromycin, josamycin, and spiramycin) at least 100 times higher than those of the parent strain and were also more resistant to the lincosamide clindamycin. Chlamydia trachomatis L2 variants with a Gln-to-Lys substitution in ribosomal protein L4 at position 66 (E. coli numbering system), conferring an eightfold decrease in azithromycin and erythromycin sensitivities and a fourfold decrease in josamycin and spiramycin sensitivities, were isolated following serial passage in subinhibitory concentrations of azithromycin. Each mutation was stably maintained in the absence of selection but severely affected chlamydial infectivity, as determined by monitoring the development of each isolate over 46 h in the absence of selection, in pure culture or in 1:1 competition with the isogenic parent. Data in this study support the hypothesis that the mechanisms which confer high-level macrolide resistance in chlamydiae carry a prohibitive physiological cost and may thus limit the emergence of highly resistant clones of these important pathogens in vivo.     

High-level telithromycin resistance in a clinical isolate of Streptococcus pneumoniae

A rare clinical isolate of Streptococcus pneumoniae, highly resistant to telithromycin, contained erm(B) with a truncated leader peptide and a mutant ribosomal protein L4. By transformation of susceptible strains, this study shows that high-level telithromycin resistance is conferred by erm(B), wild type or mutant, in combination with a (69)GTG(71)-to-TPS mutation in ribosomal protein L4.