Cheetah:a computational toolkit for cybergenetic control

Abstract Advances in microscopy, microfluidics, and optogenetics enable single-cell monitoring and environmental regulation and offer the means to control cellular phenotypes. The development of such systems is challenging and often results in bespoke setups that hinder reproducibility. To address this, we introduce Cheetah, a flexible computational toolkit that simplifies the integration of real-time microscopy analysis with algorithms for cellular control. Central to the platform is an image segmentation system based on the versatile U-Net convolutional neural network. This is supplemented with functionality to robustly count, characterize, and control cells over time. We demonstrate Cheetah’s core capabilities by analyzing long-term bacterial and mammalian cell growth and by dynamically controlling protein expression in mammalian cells. In all cases, Cheetah’s segmentation accuracy exceeds that of a commonly used thresholding-based method, allowing for more accurate control signals to be generated. Availability of this easy-to-use platform will make control engineering techniques more accessible and offer new ways to probe and manipulate living cells

Stage-Specific Inhibition of MHC Class I Presentation by the Epstein-Barr Virus BNLF2a Protein during Virus Lytic Cycle

gamma-herpesvirus Epstein-Barr virus (EBV) persists for life in infected individuals despite the presence of a strong immune response. During the lytic cycle of EBV many viral proteins are expressed, potentially allowing virally infected cells to be recognized and eliminated by CD8+ T cells. We have recently identified an immune evasion protein encoded by EBV, BNLF2a, which is expressed in early phase lytic replication and inhibits peptide- and ATP-binding functions of the transporter associated with antigen processing. Ectopic expression of BNLF2a causes decreased surface MHC class I expression and inhibits the presentation of indicator antigens to CD8+ T cells. Here we sought to examine the influence of BNLF2a when expressed naturally during EBV lytic replication. We generated a BNLF2a-deleted recombinant EBV (ΔBNLF2a) and compared the ability of ΔBNLF2a and wild-type EBV-transformed B cell lines to be recognized by CD8+ T cell clones specific for EBV-encoded immediate early, early and late lytic antigens. Epitopes derived from immediate early and early expressed proteins were better recognized when presented by ΔBNLF2a transformed cells compared to wild-type virus transformants. However, recognition of late antigens by CD8+ T cells remained equally poor when presented by both wild-type and ΔBNLF2a cell targets. Analysis of BNLF2a and target protein expression kinetics showed that although BNLF2a is expressed during early phase replication, it is expressed at a time when there is an upregulation of immediate early proteins and initiation of early protein synthesis. Interestingly, BNLF2a protein expression was found to be lost by late lytic cycle yet ΔBNLF2a-transformed cells in late stage replication downregulated surface MHC class I to a similar extent as wild-type EBV-transformed cells. These data show that BNLF2a-mediated expression is stage-specific, affecting presentation of immediate early and early proteins, and that other evasion mechanisms operate later in the lytic cycle

Cluster M Mycobacteriophages Bongo, PegLeg, and Rey with Unusually Large Repertoires of tRNA Isotopes

Genomic analysis of a large set of phages infecting the common hostMycobacterium smegmatis mc2155 shows that they span considerable genetic diversity. There are more than 20 distinct types that lack nucleotide similarity with each other, and there is considerable diversity within most of the groups. Three newly isolated temperate mycobacteriophages, Bongo, PegLeg, and Rey, constitute a new group (cluster M), with the closely related phages Bongo and PegLeg forming subcluster M1 and the more distantly related Rey forming subcluster M2. The cluster M mycobacteriophages have siphoviral morphologies with unusually long tails, are homoimmune, and have larger than average genomes (80.2 to 83.7 kbp). They exhibit a variety of features not previously described in other mycobacteriophages, including noncanonical genome architectures and several unusual sets of conserved repeated sequences suggesting novel regulatory systems for both transcription and translation. In addition to containing transfer-messenger RNA and RtcB-like RNA ligase genes, their genomes encode 21 to 24 tRNA genes encompassing complete or nearly complete sets of isotypes. We predict that these tRNAs are used in late lytic growth, likely compensating for the degradation or inadequacy of host tRNAs. They may represent a complete set of tRNAs necessary for late lytic growth, especially when taken together with the apparent lack of codons in the same late genes that correspond to tRNAs that the genomes of the phages do not obviously encode

First-in-Human Phase I Study of GSK2126458, an Oral Pan-Class I Phosphatidylinositol-3-Kinase Inhibitor, in Patients with Advanced Solid Tumor Malignancies

GSK2126458 (GSK458) is a potent inhibitor of PI3K (α, β, γ, and δ), with preclinical studies demonstrating broad antitumor activity. We performed a first-in-human phase I study in patients with advanced solid tumors

The Epstein-Barr Virus G-Protein-Coupled Receptor Contributes to Immune Evasion by Targeting MHC Class I Molecules for Degradation

Epstein-Barr virus (EBV) is a human herpesvirus that persists as a largely subclinical infection in the vast majority of adults worldwide. Recent evidence indicates that an important component of the persistence strategy involves active interference with the MHC class I antigen processing pathway during the lytic replication cycle. We have now identified a novel role for the lytic cycle gene, BILF1, which encodes a glycoprotein with the properties of a constitutive signaling G-protein-coupled receptor (GPCR). BILF1 reduced the levels of MHC class I at the cell surface and inhibited CD8+ T cell recognition of endogenous target antigens. The underlying mechanism involves physical association of BILF1 with MHC class I molecules, an increased turnover from the cell surface, and enhanced degradation via lysosomal proteases. The BILF1 protein of the closely related CeHV15 c1-herpesvirus of the Rhesus Old World primate (80% amino acid sequence identity) downregulated surface MHC class I similarly to EBV BILF1. Amongst the human herpesviruses, the GPCR encoded by the ORF74 of the KSHV c2-herpesvirus is most closely related to EBV BILF1 (15% amino acid sequence identity) but did not affect levels of surface MHC class I. An engineered mutant of BILF1 that was unable to activate G protein signaling pathways retained the ability to downregulate MHC class I, indicating that the immune-modulating and GPCR-signaling properties are two distinct functions of BILF1. These findings extend our understanding of the normal biology of an important human pathogen. The discovery of a third EBV lytic cycle gene that cooperates to interfere with MHC class I antigen processing underscores the importance of the need for EBV to be able to evade CD8+ T cell responses during the lytic replication cycle, at a time when such a large number of potential viral targets are expressed

Predicting Responses of Geo-ecological Carbonate Reef Systems to Climate Change: A Conceptual Model and Review

[Chapter Abstract] 230Coral reefs provide critical ecological and geomorphic (e.g. sediment production for reef-fronted shoreline maintenance) services, which interact in complex and dynamic ways. These services are under threat from climate change, requiring dynamic modelling approaches that predict how reef systems will respond to different future climate scenarios. Carbonate budgets, which estimate net reef calcium carbonate production, provide a comprehensive ‘snap-shot’ assessment of reef accretionary potential and reef stability. These budgets, however, were not intended to account for the full suite of processes that maintain coral reef services or to provide predictive capacity on longer timescales (decadal to centennial). To respond to the dual challenges of enhancing carbonate budget assessments and advancing their predictive capacity, we applied a novel model elicitation and review method to create a qualitative geo-ecological carbonate reef system model that links geomorphic, ecological and physical processes. Our approach conceptualizes relationships between net carbonate production, sediment transport and landform stability, and rates knowledge confidence to reveal major knowledge gaps and critical future research pathways. The model provides a blueprint for future coral reef research that aims to quantify net carbonate production and sediment dynamics, improving our capacity to predict responses of reefs and reef-fronted shorelines to future climate change.https://nsuworks.nova.edu/occ_facbooks/1116/thumbnail.jp

Gastrokine-1, an anti-amyloidogenic protein secreted by the stomach, regulates diet-induced obesity

Obesity and its sequelae have a major impact on human health. The stomach contributes to obesity in ways that extend beyond its role in digestion, including through effects on the microbiome. Gastrokine-1 (GKN1) is an anti-amyloidogenic protein abundantly and specifically secreted into the stomach lumen. We examined whether GKN1 plays a role in the development of obesity and regulation of the gut microbiome. Gkn1−/− mice were resistant to diet-induced obesity and hepatic steatosis (high fat diet (HFD) fat mass (g) = 10.4 ± 3.0 (WT) versus 2.9 ± 2.3 (Gkn1−/−) p < 0.005; HFD liver mass (g) = 1.3 ± 0.11 (WT) versus 1.1 ± 0.07 (Gkn1−/−) p < 0.05). Gkn1−/− mice also exhibited increased expression of the lipid-regulating hormone ANGPTL4 in the small bowel. The microbiome of Gkn1−/− mice exhibited reduced populations of microbes implicated in obesity, namely Firmicutes of the class Erysipelotrichia. Altered metabolism consistent with use of fat as an energy source was evident in Gkn1−/− mice during the sleep period. GKN1 may contribute to the effects of the stomach on the microbiome and obesity. Inhibition of GKN1 may be a means to prevent obesity

Effects of antiplatelet therapy on stroke risk by brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases: subgroup analyses of the RESTART randomised, open-label trial

Background Findings from the RESTART trial suggest that starting antiplatelet therapy might reduce the risk of recurrent symptomatic intracerebral haemorrhage compared with avoiding antiplatelet therapy. Brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases (such as cerebral microbleeds) are associated with greater risks of recurrent intracerebral haemorrhage. We did subgroup analyses of the RESTART trial to explore whether these brain imaging features modify the effects of antiplatelet therapy