Explicit Kinetic Heterogeneity: Mathematical Models for Interpretation of Deuterium Labeling of Heterogeneous Cell Populations

Estimation of division and death rates of lymphocytes in different conditions is vital for quantitative understanding of the immune system. Deuterium, in the form of deuterated glucose or heavy water, can be used to measure rates of proliferation and death of lymphocytes in vivo. Inferring these rates from labeling and delabeling curves has been subject to considerable debate with different groups suggesting different mathematical models for that purpose. We show that the three most common models, which are based on quite different biological assumptions, actually predict mathematically identical labeling curves with one parameter for the exponential up and down slope, and one parameter defining the maximum labeling level. By extending these previous models, we here propose a novel approach for the analysis of data from deuterium labeling experiments. We construct a model of “kinetic heterogeneity” in which the total cell population consists of many sub-populations with different rates of cell turnover. In this model, for a given distribution of the rates of turnover, the predicted fraction of labeled DNA accumulated and lost can be calculated. Our model reproduces several previously made experimental observations, such as a negative correlation between the length of the labeling period and the rate at which labeled DNA is lost after label cessation. We demonstrate the reliability of the new explicit kinetic heterogeneity model by applying it to artificially generated datasets, and illustrate its usefulness by fitting experimental data. In contrast to previous models, the explicit kinetic heterogeneity model 1) provides a novel way of interpreting labeling data; 2) allows for a non-exponential loss of labeled cells during delabeling, and 3) can be used to describe data with variable labeling length.     

Oocyte Scoring Enhances Embryo-Scoring in Predicting Pregnancy Chances with IVF Where It Counts Most

Context

Our center’s quality improvement optimization process on many occasions anecdotally suggested that oocyte assessments might enhance embryo assessment in predicting pregnancy chances with in vitro fertilization (IVF).

Objective

To prospectively compare a morphologic oocyte grading system to standard day-3 morphologic embryo assessment.

Design, Setting, Patients

We prospectively investigated in a private academically-affiliated infertility center 94 consecutive IVF cycles based on 6 criteria for oocyte quality: morphology, cytoplasm, perivitelline space (PVS), zona pellucida (ZP), polar body (PB) and oocyte size, each assigned a value of -1 (worst), 0 (average) or +1 (best), so establishing an average total oocyte score (TOS). Embryo assessment utilized grade and cell numbers of each embryo on day-3 after oocyte retrieval. Clinical pregnancy was defined by presence of at least one intrauterine gestational sac.

Interventions

Standard IVF cycles in infertile women.

Main Outcome Measures

Predictability of pregnancy based on oocyte and embryo-grading systems.

Results

Average age for all patients was 36.5 ± 7.3 years; mean oocyte yield was 7.97± 5.76; Patient specific total oocyte score (PTOS) was -1.05 ± 2.24. PTOS, adjusted for patient age, was directly related to odds of increased embryo cell numbers (OR 1.12, P = 0.025), embryo grade (OR 1.19, P < 0.001) and clinical pregnancy [OR 1.58 (95%CI 1.23 to 2.02), P < 0.001]. Restricting the analysis to day three embryos of high quality (8-cell/ good grades), TOS was still predictive of clinical pregnancy (OR 2.08 (95%CI 1.26 to 3.44, P = 0.004). Among the 69 patients with embryos of Grade 4 or better available for transfer 23 achieved Clinical Pregnancy. When the analysis was restricted to the 69 transfers with good quality embryos (≥ Grade 4) the Oocyte Scoring System (TOS) (AUC±SE 0.863±0.044, oocyte score) provided significantly greater predictive value for clinical pregnancy compared to the embryo grade alone (AUC 0.646 ± 0.072, embryo grade) p = 0.015.

Conclusions

Oocyte-scoring, thus, provides useful clinical information especially in good prognosis patients with large numbers of high quality embryos. This finding appears of particular importance at a time when many IVF centers are committing sizable investments to closed incubation systems with time-lapse photography, which are exclusively meant to define embryo morphology.     

Placebo-Induced Somatic Sensations: A Multi-Modal Study of Three Different Placebo Interventions

Somatic sensations induced by placebos are a frequent phenomenon whose etiology and clinical relevance remains unknown. In this study, we have evaluated the quantitative, qualitative, spatial, and temporal characteristics of placebo-induced somatic sensations in response to three different placebo interventions: (1) placebo irritant solution, (2) placebo laser stimulation, and (3) imagined laser stimulation. The quality and intensity of evoked sensations were assessed using the McGill pain questionnaire and visual analogue scales (VAS), while subjects’ sensation drawings processed by a geographic information system (GIS) were used to measure their spatial characteristics. We found that all three interventions are capable of producing robust sensations most frequently described as “tingling” and “warm” that can reach consider-able spatial extent (≤ 205mm^²) and intensity (≤ 80/100 VAS). Sensations from placebo stimulation were often referred to areas remote from the stimulation site and exhibit considerable similarity with referred pain. Interestingly, there was considerable similarity of qualitative features as well as spatial patterns across subjects and placebos. However, placebo laser stimulation elicited significantly stronger and more widespread sensations than placebo irritant solution. Finally, novelty seeking, a character trait assessed by the Temperament and Character Inventory and associated with basal dopaminergic activity, was less pronounced in subjects susceptible to report placebo-induced sensations. Our study has shown that placebo-induced sensations are frequent and can reach considerable intensity and extent. As multiple somatosensory subsystems are involved despite the lack of peripheral stimulus, we propose a central etiology for this phenomenon.     

Competition for Antigen between Th1 and Th2 Responses Determines the Timing of the Immune Response Switch during Mycobaterium avium Subspecies paratuberulosis Infection in Ruminants

Johne''s disease (JD), a persistent and slow progressing infection of ruminants such as cows and sheep, is caused by slow replicating bacilli Mycobacterium avium subspecies paratuberculosis (MAP) infecting macrophages in the gut. Infected animals initially mount a cell-mediated CD4 T cell response against MAP which is characterized by the production of interferon (Th1 response). Over time, Th1 response diminishes in most animals and antibody response to MAP antigens becomes dominant (Th2 response). The switch from Th1 to Th2 response occurs concomitantly with disease progression and shedding of the bacteria in feces. Mechanisms controlling this Th1/Th2 switch remain poorly understood. Because Th1 and Th2 responses are known to cross-inhibit each other, it is unclear why initially strong Th1 response is lost over time. Using a novel mathematical model of the immune response to MAP infection we show that the ability of extracellular bacteria to persist outside of macrophages naturally leads to switch of the cellular response to antibody production. Several additional mechanisms may also contribute to the timing of the Th1/Th2 switch including the rate of proliferation of Th1/Th2 responses at the site of infection, efficiency at which immune responses cross-inhibit each other, and the rate at which Th1 response becomes exhausted over time. Our basic model reasonably well explains four different kinetic patterns of the Th1/Th2 responses in MAP-infected sheep by variability in the initial bacterial dose and the efficiency of the MAP-specific T cell responses. Taken together, our novel mathematical model identifies factors of bacterial and host origin that drive kinetics of the immune response to MAP and provides the basis for testing the impact of vaccination or early treatment on the duration of infection.     

Tools for Gene-Regulatory Analyses in the Marine Annelid Platynereis dumerilii

The advent of high-throughput sequencing technology facilitates the exploration of a variety of reference species outside the few established molecular genetic model systems. Bioinformatic and gene expression analyses provide new ways for comparative analyses between species, for instance, in the field of evolution and development. Despite these advances, a critical bottleneck for the exploration of new model species remains the establishment of functional tools, such as the ability to experimentally express genes in specific cells of an organism. We recently established a first transgenic strain of the annelid Platynereis, using a Tc1/mariner-type Mos1 transposon vector. Here, we compare Mos1 with Tol2, a member of the hAT family of transposons. In Platynereis, Tol2-based constructs showed a higher frequency of nuclear genome insertion and sustained gene expression in the G0 generation. However, in contrast to Mos1-mediated transgenes, Tol2-mediated insertions failed to retain fluorescence in the G1 generation, suggesting a germ line-based silencing mechanism. Furthermore, we present three novel expression constructs that were generated by a simple fusion-PCR approach and allow either ubiquitous or cell-specific expression of a reporter gene. Our study indicates the versatility of Tol2 for transient transgenesis, and provides a template for transgenesis work in other emerging reference species.     

Genetic and chemical characterization of an EMS induced mutation in Cucumis melo CRTISO gene

In order to broaden the available genetic variation of melon, we developed an ethyl methanesulfonate mutation library in an orange-flesh ‘Charentais’ type melon line that accumulates β-carotene. One mutagenized M₂ family segregated for a novel recessive trait, a yellow–orange fruit flesh (‘yofI’). HPLC analysis revealed that ‘yofI’ accumulates pro-lycopene (tetra-cis-lycopene) as its major fruit pigment. The altered carotenoid composition of ‘yofI’ is associated with a significant change of the fruit aroma since cleavage of β-carotene yields different apocarotenoids than the cleavage of pro-lycopene. Normally, pro-lycopene is further isomerized by CRTISO (carotenoid isomerase) to yield all-trans-lycopene, which is further cyclized to β-carotene in melon fruit. Cloning and sequencing of ‘yofI’ CRTISO identified two mRNA sequences which lead to truncated forms of CRTISO. Sequencing of the genomic CRTISO identified an A–T transversion in ‘yofI’ which leads to a premature STOP codon. The early carotenoid pathway genes were up regulated in yofI fruit causing accumulation of other intermediates such as phytoene and ζ-carotene. Total carotenoid levels are only slightly increased in the mutant. Mutants accumulating pro-lycopene have been reported in both tomato and watermelon fruits, however, this is the first report of a non-lycopene accumulating fruit showing this phenomenon.     

The Novel Approach to the Protein Design: Active Truncated Forms of Human 1-CYS Peroxiredoxin

Abstract

The object of the present study is the verification of a new approach to the design of the active truncated forms of enzymes. The method is based on a new way of investigating the protein sequences—the ANalysis of Informational Structure (ANIS). The analysis of informational structure allows to determine the hierarchically organized structures (IDIC-trees) formed by the sites with the Increased Degree of Informational Coordination between residues. The proposed approach involves the consequent removal of the fragments corresponding to the individual IDIC-trees from the wild-type enzyme sequences. The described procedure was applied to the design of the active truncated form of human 1-CYS peroxiredoxin (PrxVI). Two variants of the PrxVI truncated sequences were proposed according to ANIS method. These truncated forms of the enzyme were expressed in E. coli and purified. The respective antioxidant activities were measured. It was shown that one of the truncated recombinant proteins retains more than 90% of the wild-type PrxVI enzymatic activity. According to the results of our study we can assume that ANIS method can be an effective tool for the design of the active truncated forms of the enzymes or the chimeric proteins which combine the enzymatic activities of their wild-type prototypes.     

Characterisation of conditioning layers formed by exopolymeric substances of Pseudomonas NCIMB 2021 on surfaces of AISI 316 stainless steel

This investigation aimed to characterise conditioning layers formed on AISI 316 stainless steel by different types of extracellular polymeric substances (EPS), i.e. biofilm, planktonic and capsular exopolymers, isolated from continuous cultures of marine Pseudomonas received from the National Collection of Industrial and Marine Bacteria (strain NCIMB 2021). Colorimetric assays and gas chromatography‐mass spectrometry analysis confirmed previously obtained results based on a FTIR and SDS‐PAGE study of Pseudomonas NCIMB 2021 EPS demonstrating the presence of protein, neutral and amino sugars and uronic acids. The content and the ratio of these macromolecules differed depending on the type of EPS. X‐ray photoelectron spectroscopy revealed that conditioning layers formed upon exposure of steel to EPS solutions were chemically dissimilar. It is proposed that the observed difference in the chemistry of conditioning layers is the likely reason for reported differences in attachment of Pseudomonas cells to EPS‐conditioned steel surfaces.     

On the combined analysis of ²H and ¹⁵N/¹H solid-state NMR data for determination of transmembrane peptide orientation and dynamics

The dynamics of membrane-spanning peptides have a strong affect on the solid-state NMR observables. We present a combined analysis of ²H-alanine quadrupolar splittings together with ¹⁵N/¹H dipolar couplings and ¹⁵N chemical shifts, using two models to treat the dynamics, for the systematic evaluation of transmembrane peptides based on the GWALP23 sequence (acetyl-GGALW(LA)₆LWLAGA-amide). The results indicate that derivatives of GWALP23 incorporating diverse guest residues adopt a range of apparent tilt angles that span 5°–35° in lipid bilayer membranes. By comparing individual and combined analyses of specifically ²H- or ¹⁵N-labeled peptides incorporated in magnetically or mechanically aligned lipid bilayers, we examine the influence of data-set size/identity, and of explicitly modeled dynamics, on the deduced average orientations of the peptides. We conclude that peptides with small apparent tilt values (<∼10°) can be fitted by extensive families of solutions, which can be narrowed by incorporating additional ¹⁵N as well as ²H restraints. Conversely, peptides exhibiting larger tilt angles display more narrow distributions of tilt and rotation that can be fitted using smaller sets of experimental constraints or even with ²H or ¹⁵N data alone. Importantly, for peptides that tilt significantly more than 10° from the bilayer-normal, the contribution from rigid body dynamics can be approximated by a principal order parameter.     

Naphthalene-degrading bacteria of the genus <Emphasis Type="Italic">Rhodococcus</Emphasis> from the Verkhnekamsk salt mining region of Russia

Eight moderately halotolerant naphthalene-degrading strains of the genus Rhodococcus isolated from soil samples and slime pit bottom sediment of the Verkhnekamsk salt mining region of Russia were characterized by PCR amplification of repetitive bacterial DNA elements (rep-PCR) and identified by 16S ribosomal RNA gene sequence analysis. The diversity of their dioxygenase (nar-like) genes was investigated as these genes are known to be involved in naphthalene-degradation. The analysis of the nar-like genes identified revealed their heterogeneity in the strains under study and identity to the known sequences of nar-like genes of previously characterized from members of the genus Rhodococcus.