Targeted tandem affinity purification of PSD‐95 recovers core postsynaptic complexes and schizophrenia susceptibility proteins

The molecular complexity of mammalian proteomes demands new methods for mapping the organization of multiprotein complexes. Here, we combine mouse genetics and proteomics to characterize synapse protein complexes and interaction networks. New tandem affinity purification (TAP) tags were fused to the carboxyl terminus of PSD‐95 using gene targeting in mice. Homozygous mice showed no detectable abnormalities in PSD‐95 expression, subcellular localization or synaptic electrophysiological function. Analysis of multiprotein complexes purified under native conditions by mass spectrometry defined known and new interactors: 118 proteins comprising crucial functional components of synapses, including glutamate receptors, K⁺ channels, scaffolding and signaling proteins, were recovered. Network clustering of protein interactions generated five connected clusters, with two clusters containing all the major ionotropic glutamate receptors and one cluster with voltage‐dependent K⁺ channels. Annotation of clusters with human disease associations revealed that multiple disorders map to the network, with a significant correlation of schizophrenia within the glutamate receptor clusters. This targeted TAP tagging strategy is generally applicable to mammalian proteomics and systems biology approaches to disease.     

Pro-Leu-Ser/Thr-Pro is a consensus primary sequence for substrate protein phosphorylation. Characterization of the phosphorylation of c-myc and c-jun proteins by an epidermal growth factor receptor threonine 669 protein kinase.

A growth factor-stimulated (MAP2-related) protein kinase, ERT, that phosphorylates the epidermal growth factor receptor at Thr669 has been purified from KB human tumor cells by Northwood and co-workers (Northwood, I. C., Gonzalez, F. A., Wartmann, M., Raden, D. L., and Davis, R. J. (1991) J. Biol. Chem. 266, 15266-15276). The ERT protein kinase has a restricted substrate specificity, and the structural determinants employed for substrate recognition by this enzyme have not been defined. As an approach toward understanding the specificity of substrate phosphorylation, we have used an in vitro assay to identify additional substrates for the ERT protein kinase. In this report we describe two novel substrates: (a) the human c-myc protein at Ser62 and (b) the rat c-jun protein at Ser246. Alignment of the primary sequences surrounding the phosphorylation sites located within the epidermal growth factor receptor (Thr669), Myc (Ser62), and Jun (Ser246) demonstrated a marked similarity. The observed consensus sequence was Pro-Leu-Ser/Thr-Pro. We propose that this sequence forms part of a substrate structure that is recognized by the ERT protein kinase.     

Visualization and ablation of phenylethanolamineN-methyltransferase producing cells in transgenic mice

We cloned and sequenced the mouse phenylethanolamineN-methyltransferase (PNMT) gene which encodes the enzyme that catalyses the conversion of norepinephrine to epinephrine. The ability of various length sequences flanking the mouse or human PNMT genes to direct expression of reporter genes in transgenic mice was examined. We show that 9 kb of 5 flanking sequences from the cloned mouse PNMT gene can direct expression of theEscherichia coli -galactosidase (lacZ) gene to predicted regions of the adrenal, eye can direct in the adult transgenic mouse. The transgene was also expressed during development, in the myelencephalon, adrenal medulla and dorsal root ganglia. PNMT-producing cells were ablated by expression of the diphtheria toxin (DT-A) gene driven by the human PNMT promoter, resulting in abnormalities in the adrenal medulla, eye and testis. The hPNMT_{8 kb}-DT-A line presents a model with which to examine the developmental ramifications of deletion of PNMT-producing cell populations from the adrenal medulla and retina.     

The murine pan T cell marker CD96 is an adhesion receptor for CD155 and nectin-1

The CD155 ligand CD96 is an immunoglobulin-like protein tentatively allocated to the repertoire of human NK receptors. We report here that the CD96/CD155-interaction is preserved between man and mouse although both receptors are only moderately conserved in amino acid sequence. Moreover, murine CD96 (mCD96) binds to nectin-1, a receptor related to CD155. Applying newly generated monoclonal antibodies specifically recognizing mCD96, an expression profile is revealed resembling closely that of human CD96 (hCD96) on cells of hematopoietic origin. A panel of anti-mCD96 but also recently established anti-mCD155 antibodies effectively prevents formation of CD96/CD155-complexes. This was exploited to demonstrate that the only available receptor for mCD96 present on thymocytes is mCD155. Moreover, T cell adhesion to insect cells expressing mCD155 is blocked by these antibodies depending on the T cell subtype. These results suggest a function of the CD96/CD155-adhesion system in T cell biology.     

Major histocompatibility complex based resistance to a common bacterial pathogen of amphibians

Given their well-developed systems of innate and adaptive immunity, global population declines of amphibians are particularly perplexing. To investigate the role of the major histocompatibility complex (MHC) in conferring pathogen resistance, we challenged Xenopus laevis tadpoles bearing different combinations of four MHC haplotypes (f, g, j, and r) with the bacterial pathogen Aeromonas hydrophila in two experiments. In the first, we exposed ff, fg, gg, gj, and jj tadpoles, obtained from breeding MHC homozygous parents, to one of three doses of A. hydrophila or heat-killed bacteria as a control. In the second, we exposed ff, fg, fr, gg, rg, and rr tadpoles, obtained from breeding MHC heterozygous parents and subsequently genotyped by PCR, to A. hydrophila, heat-killed bacteria or media alone as controls. We thereby determined whether the same patterns of MHC resistance emerged within as among families, independent of non-MHC heritable differences. Tadpoles with r or g MHC haplotypes were more likely to die than were those with f or j haplotypes. Growth rates varied among MHC types, independent of exposure dose. Heterozygous individuals with both susceptible and resistant haplotypes were intermediate to either homozygous genotype in both size and survival. The effect of the MHC on growth and survival was consistent between experiments and across families. MHC alleles differentially confer resistance to, or tolerance of, the bacterial pathogen, which affects tadpoles' growth and survival.     

Characterization of culturable bacterial endophytes of switchgrass (Panicum virgatum L.) and their capacity to influence plant growth

Switchgrass (Panicum virgatum L.) is a perennial warm season grass that is native to the plains of North America and is widely grown as a forage, bioenergy or groundcover crop. Despite its importance, a bottleneck in switchgrass production is poor seedling vigor, which as a perennial crop represents an important time for management. Herein, data identify a suite of culturable bacterial microflora extracted from switchgrass, and show their capability to influence host plant growth and development. A total of 307 bacterial isolates were cultured and isolated from surface sterilized switchgrass biomass and sequence identified into 76 strains (subspecies classification), 36 species and 5 phyla. Approximately 58% of bacterial strains, when reintroduced into surface‐sterilized switchgrass seeds, were documented to increase lamina length (cm from base to tip after 60 days growth) relative to uninoculated controls. Ecologically, Phylum Firmicutes was the most abundant bacterial classification and encompassed 75% of all isolates. Although the culturable bacterial community studies herein represent an unknown and assumedly minor proportion of the total microbiome, by focusing on culturable bacteria, we delineate functional feedback between the presence of isolated bacteria and switchgrass seedling growth.     

An evaluation of cellulose saccharification and fermentation with an engineered Saccharomyces cerevisiae capable of cellobiose and xylose utilization

Commercial-scale cellulosic ethanol production has been hindered by high costs associated with cellulose-to-glucose conversion and hexose and pentose co-fermentation. Simultaneous saccharification and fermentation (SSF) with a yeast strain capable of xylose and cellobiose co-utilization has been proposed as a possible avenue to reduce these costs. The recently developed DA24-16 strain of Saccharomyces cerevisiae incorporates a xylose assimilation pathway and a cellodextrin transporter (CDT) that permit rapid growth on xylose and cellobiose. In the current work, a mechanistic kinetic model of cellulase-catalyzed hydrolysis of cellulose was combined with a multi-substrate model of microbial growth to investigate the ability of DA24-16 and improved cellobiose-consuming strains to obviate the need for exogenously added β-glucosidase and to assess the impact of cellobiose utilization on SSF and separate hydrolysis and fermentation (SHF). Results indicate that improved CDT-containing strains capable of growing on cellobiose as rapidly as on glucose produced ethanol nearly as rapidly as non-CDT-containing yeast supplemented with β-glucosidase. In producing 75 g/L ethanol, SSF with any strain did not result in shorter residence times than SHF with a 12 h saccharification step. Strains with improved cellobiose utilization are therefore unlikely to allow higher titers to be reached more quickly in SSF than in SHF.     

Modeling scenarios for mitigating outbreaks in congregate settings

The explosive outbreaks of COVID-19 seen in congregate settings such as prisons and nursing homes, has highlighted a critical need for effective outbreak prevention and mitigation strategies for these settings. Here we consider how different types of control interventions impact the expected number of symptomatic infections due to outbreaks. Introduction of disease into the resident population from the community is modeled as a stochastic point process coupled to a branching process, while spread between residents is modeled via a deterministic compartmental model that accounts for depletion of susceptible individuals. Control is modeled as a proportional decrease in the number of susceptible residents, the reproduction number, and/or the proportion of symptomatic infections. This permits a range of assumptions about the density dependence of transmission and modes of protection by vaccination, depopulation and other types of control. We find that vaccination or depopulation can have a greater than linear effect on the expected number of cases. For example, assuming a reproduction number of 3.0 with density-dependent transmission, we find that preemptively reducing the size of the susceptible population by 20% reduced overall disease burden by 47%. In some circumstances, it may be possible to reduce the risk and burden of disease outbreaks by optimizing the way a group of residents are apportioned into distinct residential units. The optimal apportionment may be different depending on whether the goal is to reduce the probability of an outbreak occurring, or the expected number of cases from outbreak dynamics. In other circumstances there may be an opportunity to implement reactive disease control measures in which the number of susceptible individuals is rapidly reduced once an outbreak has been detected to occur. Reactive control is most effective when the reproduction number is not too high, and there is minimal delay in implementing control. We highlight the California state prison system as an example for how these findings provide a quantitative framework for understanding disease transmission in congregate settings. Our approach and accompanying interactive website (https://phoebelu.shinyapps.io/DepopulationModels/) provides a quantitative framework to evaluate the potential impact of policy decisions governing infection control in outbreak settings.