Digital cell quantification identifies global immune cell dynamics during influenza infection

Hundreds of immune cell types work in coordination to maintain tissue homeostasis. Upon infection, dramatic changes occur with the localization, migration, and proliferation of the immune cells to first alert the body of the danger, confine it to limit spreading, and finally extinguish the threat and bring the tissue back to homeostasis. Since current technologies can follow the dynamics of only a limited number of cell types, we have yet to grasp the full complexity of global in vivo cell dynamics in normal developmental processes and disease. Here, we devise a computational method, digital cell quantification (DCQ), which combines genome‐wide gene expression data with an immune cell compendium to infer in vivo changes in the quantities of 213 immune cell subpopulations. DCQ was applied to study global immune cell dynamics in mice lungs at ten time points during 7 days of flu infection. We find dramatic changes in quantities of 70 immune cell types, including various innate, adaptive, and progenitor immune cells. We focus on the previously unreported dynamics of four immune dendritic cell subtypes and suggest a specific role for CD103⁺ CD11b⁻ DCs in early stages of disease and CD8⁺ pDC in late stages of flu infection.     

Rituximab-induced direct inhibition of T-cell activation

Background

Rituximab, an anti-CD20 monoclonal antibody, is reported to increase the T-cell-dependent infection risk. The current study was designed to investigate whether rituximab interferes with T-cell activation.

Patients and methods

Patients with non-Hodgkin lymphoma receiving 4–6 courses of 375?mg/m² rituximab underwent detailed assessment of T-cell activation pre- and post-rituximab. A similar analysis assessed the in vitro effect of rituximab on T-cell activation in response to allogeneic dendritic cells (allo-DCs) and other stimuli.

Results

Patients receiving rituximab exhibited a significant decline in IL-2 and IFN-γ levels in peripheral blood, most prominent after repeated rituximab courses. Evaluation at 3?months after rituximab therapy showed restoration of inflammatory cytokine production. Similarly, in vitro stimulation of peripheral blood mononuclear cells in the presence of rituximab resulted in a significant decrease in T-cell activation markers, inflammatory cytokine production and proliferative capacity. These effects were also observed using B-cell-depleted T cells (CD3⁺CD25^?CD19^?) and were accompanied with disappearance of CD3⁺CD20^dim T-cell population.

Conclusion

Rituximab administration results in transient, dose-dependent T-cell inactivation. This effect is obtained even in B-cell absence and may increase the infection risk.     

Oocyte-directed depletion of connexin43 using the Cre-LoxP system leads to subfertility in female mice

A T-DNA insertion mutant of FUSCA3 (fus3-T) in Arabidopsis thaliana exhibits several of the expected deleterious effects on seed development, but not the formation of brown seeds, a colouration which results from the accumulation of large amounts of anthocyanin. A detailed phenotypic comparison between fus3-T and a known splice point mutant (fus3-3) revealed that the seeds from both mutants do not enter dormancy and can be rescued at an immature stage. Without rescue, mature fus3-3 seeds are non-viable, whereas those of fus3-T suffer only a slight loss in their germinability. A series of comparisons between the two mutants uncovered differences with respect to conditional lethality, in histological and sub-cellular features, and in the relative amounts of various storage compounds and metabolites present, leading to a further dissection of developmental processes in seeds and a partial reinterpretation of the complex seed phenotype. FUS3 function is now known to be restricted to the acquisition of embryo-dependent seed dormancy, the determination of cotyledonary cell identity, and the synthesis and accumulation of storage compounds. Based on DNA binding studies, a model is presented which can explain the differences between the mutant alleles. The fus3-T lesion is responsible for loss of function only, while the fus3-3 mutation induces various pleiotropic effects conditioned by a truncation gene product causing severe mis-differentiation.     

From endosymbionts to host communities: factors determining the reproductive success of arthropod vectors

Elucidating the factors determining reproductive success has challenged scientists since Darwin, but the exact pathways that shape the evolution of life history traits by connecting extrinsic (e.g., landscape structure) and intrinsic (e.g., female’s age and endosymbionts) factors and reproductive success have rarely been studied. Here we collected female fleas from wild rodents in plots differing in their densities and proportions of the most dominant rodent species. We then combined path analysis and model selection approaches to explore the network of effects, ranging from micro to macroscales, determining the reproductive success of these fleas. Our results suggest that female reproductive success is directly and positively associated with their infection by Mycoplasma bacteria and their own body mass, and with the rodent species size and total density. In addition, we found evidence for indirect effects of rodent sex and rodent community diversity on female reproductive success. These results highlight the importance of exploring interrelated factors across organization scales while studying the reproductive success of wild organisms, and they have implications for the control of vector-borne diseases.     

Inhibition of pectin methyl esterase activity by green tea catechins

Pectin methyl esterases (PMEs) and their endogenous inhibitors are involved in the regulation of many processes in plant physiology, ranging from tissue growth and fruit ripening to parasitic plant haustorial formation and host invasion. Thus, control of PME activity is critical for enhancing our understanding of plant physiological processes and regulation. Here, we report on the identification of epigallocatechin gallate (EGCG), a green tea component, as a natural inhibitor for pectin methyl esterases. In a gel assay for PME activity, EGCG blocked esterase activity of pure PME as well as PME extracts from citrus and from parasitic plants. Fluorometric tests were used to determine the IC50 for a synthetic substrate. Molecular docking analysis of PME and EGCG suggests close interaction of EGCG with the catalytic cleft of PME. Inhibition of PME by the green tea compound, EGCG, provides the means to study the diverse roles of PMEs in cell wall metabolism and plant development. In addition, this study introduces the use of EGCG as natural product to be used in the food industry and agriculture.     

Reconstitution of the signal recognition particle of the halophilic archaeon Haloferax volcanii

The signal recognition particle (SRP) is a ribonucleoprotein complex involved in the recognition and targeting of nascent extracytoplasmic proteins in all three domains of life. In Archaea, SRP contains 7S RNA like its eukaryal counterpart, yet only includes two of the six protein subunits found in the eukaryal complex. To further our understanding of the archaeal SRP, 7S RNA, SRP19 and SRP54 of the halophilic archaeon Haloferax volcanii have been expressed and purified, and used to reconstitute the ternary SRP complex. The availability of SRP components from a haloarchaeon offers insight into the structure, assembly and function of this ribonucleoprotein complex at saturating salt conditions. While the amino acid sequences of H.volcanii SRP19 and SRP54 are modified presumably as an adaptation to their saline surroundings, the interactions between these halophilic SRP components and SRP RNA appear conserved, with the possibility of a few exceptions. Indeed, the H.volcanii SRP can assemble in the absence of high salt. As reported with other archaeal SRPs, the limited binding of H.volcanii SRP54 to SRP RNA is enhanced in the presence of SRP19. Finally, immunolocalization reveals that H.volcanii SRP54 is found in the cytosolic fraction, where it is associated with the ribosomal fraction of the cell.     

The RNA helicase DbpA exhibits a markedly different conformation in the ADP-bound state when compared with the ATP- or RNA-bound states

The motor enzymes that belong to the family of RNA helicases catalyze the strand separation of duplex RNA via ATP hydrolysis. Among these enzymes, Escherichia coli DbpA is a unique RNA helicase because it possesses ATPase-specific activity toward the peptidyl transferase center in 23 S ribosomal RNA. For this reason, it has been the subject of numerous biochemical and structure-function studies. The ATP-stimulated unwinding activity of DbpA toward specific and nonspecific RNA duplexes has been demonstrated. However, the underlying molecular and structural basis, which facilitates its helicase activities, is presently not known. We combined time-dependent limited proteolysis digestion, fluorescence spectroscopy, and three-dimensional structural homology modeling techniques to study the structural conformations of DbpA with respect to its binding to stoichiometric ratios of RNA and cofactors. We show that the conformational state of DbpA is markedly different in the ADP-bound state than in any other state (ATP- or RNA-bound). These results, together with structural homology studies, suggest that a hinge region located in the core domain of DbpA mediates such conformational changes.