Structure-selected RBM immunogens prime polyclonal memory responses that neutralize SARS-CoV-2 variants of concern

Successful control of the COVID-19 pandemic depends on vaccines that prevent transmission. The full-length Spike protein is highly immunogenic but the majority of antibodies do not target the virus: ACE2 interface. In an effort to affect the quality of the antibody response focusing it to the receptor-binding motif (RBM) we generated a series of conformationally-constrained immunogens by inserting solvent-exposed RBM amino acid residues into hypervariable loops of an immunoglobulin molecule. Priming C57BL/6 mice with plasmid (p)DNA encoding these constructs yielded a rapid memory response to booster immunization with recombinant Spike protein. Immune sera antibodies bound strongly to the purified receptor-binding domain (RBD) and Spike proteins. pDNA primed for a consistent response with antibodies efficient at neutralizing authentic WA1 virus and three variants of concern (VOC), B.1.351, B.1.617.2, and BA.1. We demonstrate that immunogens built on structure selection can be used to influence the quality of the antibody response by focusing it to a conserved site of vulnerability shared between wildtype virus and VOCs, resulting in neutralizing antibodies across variants.     

Accounting for Errors in Data Improves Divergence Time Estimates in Single-cell Cancer Evolution

Single-cell sequencing provides a new way to explore the evolutionary history of cells. Compared to traditional bulk sequencing, where a population of heterogeneous cells is pooled to form a single observation, single-cell sequencing isolates and amplifies genetic material from individual cells, thereby preserving the information about the origin of the sequences. However, single-cell data are more error-prone than bulk sequencing data due to the limited genomic material available per cell. Here, we present error and mutation models for evolutionary inference of single-cell data within a mature and extensible Bayesian framework, BEAST2. Our framework enables integration with biologically informative models such as relaxed molecular clocks and population dynamic models. Our simulations show that modeling errors increase the accuracy of relative divergence times and substitution parameters. We reconstruct the phylogenetic history of a colorectal cancer patient and a healthy patient from single-cell DNA sequencing data. We find that the estimated times of terminal splitting events are shifted forward in time compared to models which ignore errors. We observed that not accounting for errors can overestimate the phylogenetic diversity in single-cell DNA sequencing data. We estimate that 30–50% of the apparent diversity can be attributed to error. Our work enables a full Bayesian approach capable of accounting for errors in the data within the integrative Bayesian software framework BEAST2.     

CellDynaMo–stochastic reaction-diffusion-dynamics model: Application to search-and-capture process of mitotic spindle assembly

We introduce a Stochastic Reaction-Diffusion-Dynamics Model (SRDDM) for simulations of cellular mechanochemical processes with high spatial and temporal resolution. The SRDDM is mapped into the CellDynaMo package, which couples the spatially inhomogeneous reaction-diffusion master equation to account for biochemical reactions and molecular transport within the Langevin Dynamics (LD) framework to describe dynamic mechanical processes. This computational infrastructure allows the simulation of hours of molecular machine dynamics in reasonable wall-clock time. We apply SRDDM to test performance of the Search-and-Capture of mitotic spindle assembly by simulating, in three spatial dimensions, dynamic instability of elastic microtubules anchored in two centrosomes, movement and deformations of geometrically realistic centromeres with flexible kinetochores and chromosome arms. Furthermore, the SRDDM describes the mechanics and kinetics of Ndc80 linkers mediating transient attachments of microtubules to the chromosomal kinetochores. The rates of these attachments and detachments depend upon phosphorylation states of the Ndc80 linkers, which are regulated in the model by explicitly accounting for the reactions of Aurora A and B kinase enzymes undergoing restricted diffusion. We find that there is an optimal rate of microtubule-kinetochore detachments which maximizes the accuracy of the chromosome connections, that adding chromosome arms to kinetochores improve the accuracy by slowing down chromosome movements, that Aurora A and kinetochore deformations have a small positive effect on the attachment accuracy, and that thermal fluctuations of the microtubules increase the rates of kinetochore capture and also improve the accuracy of spindle assembly.     

Base excision repair is limited by different proteins in male germ cell nuclear extracts prepared from young and old mice

The combined observations of elevated DNA repair gene expression, high uracil-DNA glycosylase-initiated base excision repair, and a low spontaneous mutant frequency for a lacI transgene in spermatogenic cells from young mice suggest that base excision repair activity is high in spermatogenic cell types. Notably, the spontaneous mutant frequency of the lacI transgene is greater in spermatogenic cells obtained from old mice, suggesting that germ line DNA repair activity may decline with age. A paternal age effect in spermatogenic cells is recognized for the human population as well. To determine if male germ cell base excision repair activity changes with age, uracil-DNA glycosylase-initiated base excision repair activity was measured in mixed germ cell (i.e., all spermatogenic cell types in adult testis) nuclear extracts prepared from young, middle-aged, and old mice. Base excision repair activity was also assessed in nuclear extracts from premeiotic, meiotic, and postmeiotic spermatogenic cell types obtained from young mice. Mixed germ cell nuclear extracts exhibited an age-related decrease in base excision repair activity that was restored by addition of apurinic/apyrimidinic (AP) endonuclease. Uracil-DNA glycosylase and DNA ligase were determined to be limiting in mixed germ cell nuclear extracts prepared from young animals. Base excision repair activity was only modestly elevated in pachytene spermatocytes and round spermatids relative to other spermatogenic cells. Thus, germ line short-patch base excision repair activity appears to be relatively constant throughout spermatogenesis in young animals, limited by uracil-DNA glycosylase and DNA ligase in young animals, and limited by AP endonuclease in old animals.     

Genetic and environmental influences on migraine: a twin study across six countries.

Migraine is a common neurovascular brain disorder that is manifested in recurrent episodes of disabling headache. The aim of the present study was to compare the prevalence and heritability of migraine across six of the countries that participate in GenomEUtwin project including a total number of 29,717 twin pairs. Migraine was assessed by questionnaires that differed between most countries. It was most prevalent in Danish and Dutch females (32% and 34%, respectively), whereas the lowest prevalence was found in the younger and older Finnish cohorts (13% and 10%, respectively). The estimated genetic variance (heritability) was significant and the same between sexes in all countries. Heritability ranged from 34% to 57%, with lowest estimates in Australia, and highest estimates in the older cohort of Finland, the Netherlands, and Denmark. There was some indication that part of the genetic variance was non-additive, but this was significant in Sweden only. In addition to genetic factors, environmental effects that are non-shared between members of a twin pair contributed to the liability of migraine. After migraine definitions are homogenized among the participating countries, the GenomEUtwin project will provide a powerful resource to identify the genes involved in migraine.     

Molecular characterization and intracellular distribution of the alpha 5 subunit of Trypanosoma cruzi 20S proteasome

Three different monoclonal antibodies were produced against Trypanosona cruzi proteasomes. These antibodies were shown to react with a single 27-kDa band on immunoblots of purified proteasomes. Using a 7E5 monoclonal antibody (IgG1) that recognized the α5 subunit of protozoan protease we have studied the intracellular distribution of the T. cruzi 20S proteasome. Contrary to all cell types described to date, T. cruzi 20S proteasome was found not only in the cytoplasm and nucleus but also in the kinetoplast. As revealed by confocal microscopy, the reactivity of monoclonal antibody 7E5 was highly specific for protozoan proteasome because the antibody recognized only the proteasomes from parasites and not those from the mammalian host in T. cruzi infected cells. These findings were confirmed by immunoblots or immunoprecipitations, followed by chymotrypsin-like activity detection in kinetoplasts isolated by differential centrifugation and sucrose density gradients. Proteasome 20S was present in all T. cruzi stages and only slight differences in terms of relative abundance were found. The potential role of the proteasome in kinetoplast remodeling remains to be determined.     

Penicillic Acid Production by Blue-Eye Fungi on Various Agricultural Commodities

Of 10 Penicillium species reported to cause blue-eye disease of corn, four (P. martensii, P. palitans, P. cyclopium, P. puberulum) were found capable of producing the mycotoxin penicillic acid on various agricultural commodities. Commodities with high protein contents did not support toxin synthesis. The extent of toxin production varied with the strain of mold, the commodity, and the temperature; low temperatures (1 to 10 C) favored toxin accumulation.     

ABA-regulated promoter activity in stomatal guard cells

CDeT6-19 is an ABA-regulated gene which has been isolated from Craterostigma plantagineum . The CDeT6-19 gene promoter has been fused to the β- glucuronidase reporter gene ( GUS ) and used to stably transform Arabidopsis thaliana and Nicotiana tabacum . This construct has been shown to be expressed in stomatal guard cells and often in the adjacent epidermal cells of both species in response to both exogenous ABA and drought stress. These results indicate that the stomatal guard cell is competent to relay an ABA signal to the nucleus. In contrast GUS expression directed by the promoter from a predominantly seed-specific, ABA-regulated gene, Em , or the promoter from the ABA-regulated CDeT27-45 gene is not detectable in the epidermal or guard cells of tobacco or Arabidopsis in response to ABA. The fact that not all ABA-regulated gene promoters are active in stomatal guard cells suggests that effective transduction of the signal is dependent upon particular regions within the gene promoter or that guard cells lack all or part of the specific transduction apparatus required to couple the ABA signal to these promoters. This suggests that there are multiple ABA stimulus response coupling pathways. The identification of a regulatory sequence from an ABA-induced gene which is expressed in stomatal guard cells creates the possibility of examining the role of Ca²⁺ and other second messengers in ABA-induced gene expression.