Signaling properties of CD38 in the mouse immune system: enzyme-dependent and -independent roles in immunity

The 5th international CD38 meeting, held in Torino, Italy, spanned a range of topics from the role of CD38 as a signaling receptor in lymphocytic tumors to the importance of CD38-derived metabolites in NAD(+) metabolism, calcium signaling, and immune function. This meeting was particularly exciting as data were presented demonstrating that collaborative experiments between enzymologists, biochemists, cell biologists, immunologists, and clinicians have started to unravel the secrets of CD38 biology. It is now clear that all of the products of the CD38 enzyme reaction regulate calcium signal transduction in cell types as diverse as sea urchin oocytes and mammalian lymphocytes. It is also apparent that CD38 plays important immunomodulatory role(s), however there is still much debate on how CD38 mediates its immunoregulatory functions and whether the enzymatic products generated by CD38 are important for immunity. The data presented at this meeting have begun to resolve some of these controversies. First, CD38 regulates the function of leukocytes by enzyme-dependent and enzyme-independent mechanisms. Second, CD38 regulates inflammatory responses by modulating the activity of the responding leukocytes and by altering the activity of non-hematopoietic cells in the inflamed tissue. Finally, crosstalk between CD38 and other NAD(+) utilizing enzymes such as ART2, SIRT1, and PARP-1 impacts NAD(+) homeostasis, inflammation, and immunity. Thus, immunity is regulated by CD38 in multiple and unexpected ways and the new research challenge will be to determine whether we can exploit the complex biology of CD38 to therapeutically regulate the immune system.     

IPSE/alpha-1, a major secretory glycoprotein antigen from schistosome eggs, expresses the Lewis X motif on core-difucosylated N-glycans

Schistosomes are parasitic flatworms that infect millions of people in (sub)tropical areas around the world. Glycoconjugates of schistosomes play a critical role in the interaction of the different developmental stages of the parasite with the host. In particular, glycosylated components of the eggs produced by the adult worm pairs living in the bloodstream are strongly immunogenic. We have investigated the glycosylation of interleukin-4-inducing factor from schistosome eggs (IPSE/alpha-1), a major secretory egg antigen from Schistosoma mansoni that triggers interleukin-4 production in human basophils, by MS analysis of tryptic glycopeptides. Nanoscale LC-MS(/MS) and MALDI-TOF(/TOF)-MS studies combined with enzymatic degradations showed that monomeric IPSE/alpha-1 contains two N-glycosylation sites, which are each occupied for a large proportion with core-difucosylated diantennary glycans that carry one or more Lewis X motifs. Lewis X has been reported as a major immunogenic glycan element of schistosomes. This is the first report both on the expression of Lewis X on a specific schistosome egg protein and on a protein-specific glycosylation analysis of schistosome eggs.     

Sex and virulence in Escherichia coli: an evolutionary perspective

Pathogenic Escherichia coli cause over 160 million cases of dysentery and one million deaths per year, whereas non-pathogenic E. coli constitute part of the normal intestinal flora of healthy mammals and birds. The evolutionary pathways underlying this dichotomy in bacterial lifestyle were investigated by multilocus sequence typing of a global collection of isolates. Specific pathogen types [enterohaemorrhagic E. coli, enteropathogenic E. coli, enteroinvasive E. coli, K1 and Shigella] have arisen independently and repeatedly in several lineages, whereas other lineages contain only few pathogens. Rates of evolution have accelerated in pathogenic lineages, culminating in highly virulent organisms whose genomic contents are altered frequently by increased rates of homologous recombination; thus, the evolution of virulence is linked to bacterial sex. This long-term pattern of evolution was observed in genes distributed throughout the genome, and thereby is the likely result of episodic selection for strains that can escape the host immune response.     

Detection of bacterial toxins with monosaccharide arrays

A large number of bacterial toxins, viruses and bacteria target carbohydrate derivatives on the cell surface to attach and gain entry into the cell. We report here the use of a monosaccharide-based array to detect protein toxins. The array-based technique provides the capability to perform simultaneous multianalyte analyses. Arrays of N-acetyl galactosamine (GalNAc) and N-acetylneuraminic acid (Neu5Ac) derivatives were immobilized on the surface of a planar waveguide and were used as receptors for protein toxins. These arrays were probed with fluorescently labeled bacterial cells and protein toxins. While Salmonella typhimurium, Listeria monocytogenes, Escherichia coli and staphylococcal enterotoxin B (SEB) did not bind to either of the monosaccharides, both cholera toxin and tetanus toxin bound to GalNAc and Neu5Ac. The results show that the binding of the toxins to the carbohydrates is density dependent and semi-selective. Both toxins were detectable at 100 ng/ml.     

Sequence analysis of the lactococcal plasmid pNP40: a mobile replicon for coping with environmental hazards

The conjugative lactococcal plasmid pNP40, identified in Lactococcus lactis subsp. diacetylactis DRC3, possesses a potent complement of bacteriophage resistance systems, which has stimulated its application as a fitness-improving, food-grade genetic element for industrial starter cultures. The complete sequence of this plasmid allowed the mapping of previously known functions including replication, conjugation, bacteriocin resistance, heavy metal tolerance, and bacteriophage resistance. In addition, functions for cold shock adaptation and DNA damage repair were identified, further confirming pNP40's contribution to environmental stress protection. A plasmid cointegration event appears to have been part of the evolution of pNP40, resulting in a "stockpiling" of bacteriophage resistance systems.     

High Nucleotide Divergence in Developmental Regulatory Genes Contrasts With the Structural Elements of Olfactory Pathways in Caenorhabditis

The “middle-class neighborhood” is a breeding design intended to allow new mutations to accumulate by lessening the effects of purifying selection through the elimination of among-line fitness variation. We show that this design effectively applies soft selection to the experimental population, potentially causing biased estimates of mutational effects if social effects contribute to fitness.BECAUSE mutations shape adaptation, there is much interest in describing the distribution of effects of de novo mutations on phenotypes. Mutation-accumulation experiments are performed to estimate these mutational parameters. Geneticists employ at least three different approaches to accumulate naturally occurring mutations by mitigating the effects of purifying selection: inbred lines (Vassilieva and Lynch 1999; Lynch et al. 2008), balancer chromosomes in Drosophila (Mukai et al. 1972; Houle et al. 1994), and the “middle-class neighborhood” (MCN) breeding design (Shabalina et al. 1997). The last approach applies extreme and invariant bottlenecks (N = 2) to each replicate full-sib family at every generation. It is given its name to suggest the mythical lack of reproductive variation among human families of intermediate socioeconomic rank.The MCN approach was motivated by a desire to accumulate mutations on genetic backgrounds uncompromised by “weak, genetically altered chromosomes” used by alternative approaches (Shabalina et al. 1997). Selection on individual phenotypes was expected to be reduced because at every generation, every full-sib family was sampled for new individuals exactly twice (one male and one female)—thereby eliminating among-family variation for fitness. Sampled individuals were crossed among families to avoid inbreeding. This method of mutation accumulation has become popular over the past decade (e.g., Bryant and Reed 1999; Mack et al. 2000; Yampolsky et al. 2000; Radwan et al. 2004; Roles and Conner 2008).In the MCN, the fitness of an individual is its probability of being selected for breeding (one per sex per family). Obviously this probability depends upon the survival of the focal individual, but it also is affected by the frequency with which its siblings survive, meaning that its fitness is frequency-dependent. Fitness is also density-dependent. For example, an individual from a highly productive group is less likely to be bred than if it came from a less productive group. Thus, the MCN design is a form of soft selection, or density- and frequency-dependent selection such that there is no variation in fitness among groups (Wallace 1968, 1975; Wade 1985). Significantly, Goodnight et al. (1992) interpreted soft selection in the context of multilevel selection. This perspective views the group-mean fitness as the sum of two components: (1) the individuals'' contributions toward their own fitness averaged over all group members and (2) any contextual, or group-level, contributions to the group-mean fitness. The latter includes selection on effects arising from social interactions. Goodnight et al. (1992) showed that soft selection is a particularly interesting case of multilevel selection because the condition that all groups have the same fitness requires that individual- and group-level selection have equal but opposite effects on total selection. Below, we show how this perspective reveals problems with how the results of MCN experiments may be interpreted if group-level effects caused by social interactions contribute to fitness in control populations.We assume that the MCN leaves the social structure of the population intact, ensuring that selection on group-level effects is not affected by the experiment design (group-level selection is the same in the control and MCN populations). For example, if full-siblings were raised interacting with their mothers and their siblings before the experiment, then the same maternal and sib-social effects are also acting during the MCN experiment. Applying Goodnight et al.''s (1992) findings to the MCN shows us that the strength of individual-level selection will change. Shabalina et al. (1997), who ignore group-level selection, expect that the strength of individual-level selection will be reduced and more deleterious mutations will accumulate. In fact, this change can be very different depending upon the strength and direction of group-level selection relative to individual-level selection prior to the application of the MCN. When group-level selection is initially stronger, for example, the intensity of individual-level selection will increase because the parity conditions of soft selection require it to do so. When the two levels of selection act in concert, the MCN will cause the direction of individual-level selection to reverse. Both types of change can happen simultaneously: if group-level selection is relatively stronger but in the same direction as individual-level selection, then the MCN will cause individual-level selection to change direction and become more intense.

Dynamic in vivo 3-dimensional moment arms of the individual quadriceps components

The purpose of this study was to provide the first in vivo 3-dimensional (3D) measures of knee extensor moment arms, measured during dynamic volitional activity. The hypothesis was that the vastus lateralis (VL) and vastus medialis (VM) have significant off-axis moment arms compared to the central quadriceps components. After obtaining informed consent, three 3D dynamic cine phase contrast (PC) MRI sets (x,y,z velocity and anatomic images) were acquired from 22 subjects during active knee flexion and extension. Using a sagittal-oblique and two coronal-oblique imaging planes, the origins and insertions of each quadriceps muscle were identified and tracked through each time frame by integrating the cine-PC velocity data. The moment arm (MA) and relative moment (RM, defined as the cross product of the tendon line-of-action and a line connecting the line-of-action with the patellar center of mass) were calculated for each quadriceps component. The tendencies of the VM and VL to produce patellar tilt were evenly balanced. Interestingly, the magnitude of RM-P_Spin for the VM and VL is approximately four times greater than the magnitude of RM-P_Tilt for the same muscles suggesting that patellar spin may play a more important role in patellofemoral kinematics than previously thought. Thus, a force imbalance that leads to excessive lateral tilt, such as VM weakness in patellofemoral pain syndrome, would produce excessive negative spin (positive spin: superior patellar pole rotates laterally) and to a much greater degree. This would explain the increased negative spin found in recent studies of patellar maltracking. Assessing the contribution of each quadriceps component in three dimensions provides a more complete understanding of muscle functionality.     

Using two palpable measurements improves the subject-specific femoral modeling

Subject-specific musculoskeletal models are essential to biomedical research and clinical applications, such as customized joint replacement, computer-aided surgical planning, gait analysis and automated segmentation. Generating these models from CT or magnetic resonance imaging (MRI) is time and resource intensive, requiring special skills. Therefore, in many studies individual bone models are approximated by scaling a generic template. Thus, the primary goal of this study was to determine a set of clinically available parameters (palpable measures and demographic data) that could improve the prediction of femoral dimensions, as compared to predicting these variables using uniform scaling based on palpable length. Similar to previous non-homogenous anthropometric scaling methods, the non-homogenous scaling method proposed in this study improved the prediction over uniform scaling of five key femoral measures. Homogenous scaling forces all dimensions of an object to be scaled equally, whereas non-homogenous scaling allows the dimensions to be scaled independently. The largest improvement was in femoral depth, where the coefficient of determination (r²) improved from 0.22 (homogenous) to 0.60 (non-homogeneous). In general, the major advantage of this non-homogenous scaling method is its ability to support the accurate and rapid generation of subject-specific femoral models since all parameters can be collected clinically, without imaging or invasive methods.