Computational design of non-porous pH-responsive antibody nanoparticles.

Programming protein nanomaterials to respond to changes in environmental conditions is a current challenge for protein design and is important for targeted delivery of biologics. Here we describe the design of octahedral non-porous nanoparticles with a targeting antibody on the two-fold symmetry axis, a designed trimer programmed to disassemble below a tunable pH transition point on the three-fold axis, and a designed tetramer on the four-fold symmetry axis. Designed non-covalent interfaces guide cooperative nanoparticle assembly from independently purified components, and a cryo-EM density map closely matches the computational design model. The designed nanoparticles can package protein and nucleic acid payloads, are endocytosed following antibody-mediated targeting of cell surface receptors, and undergo tunable pH-dependent disassembly at pH values ranging between 5.9 and 6.7. The ability to incorporate almost any antibody into a non-porous pH-dependent nanoparticle opens up new routes to antibody-directed targeted delivery.

Nuclear spin effects in biological processes.

Traditionally, nuclear spin is not considered to affect biological processes. Recently, this has changed as isotopic fractionation that deviates from classical mass dependence was reported both in vitro and in vivo. In these cases, the isotopic effect correlates with the nuclear magnetic spin. Here, we show nuclear spin effects using stable oxygen isotopes (16O, 17O, and 18O) in two separate setups: an artificial dioxygen production system and biological aquaporin channels in cells. We observe that oxygen dynamics in chiral environments (in particular its transport) depend on nuclear spin, suggesting future applications for controlled isotope separation to be used, for instance, in NMR. To demonstrate the mechanism behind our findings, we formulate theoretical models based on a nuclear-spin-enhanced switch between electronic spin states. Accounting for the role of nuclear spin in biology can provide insights into the role of quantum effects in living systems and help inspire the development of future biotechnology solutions.

Computational design of non-porous, pH-responsive antibody nanoparticles.

Programming protein nanomaterials to respond to changes in environmental conditions is a current challenge for protein design and important for targeted delivery of biologics. We describe the design of octahedral non-porous nanoparticles with the three symmetry axes (four-fold, three-fold, and two-fold) occupied by three distinct protein homooligomers: a de novo designed tetramer, an antibody of interest, and a designed trimer programmed to disassemble below a tunable pH transition point. The nanoparticles assemble cooperatively from independently purified components, and a cryo-EM density map reveals that the structure is very close to the computational design model. The designed nanoparticles can package a variety of molecular payloads, are endocytosed following antibody-mediated targeting of cell surface receptors, and undergo tunable pH-dependent disassembly at pH values ranging between to 5.9-6.7. To our knowledge, these are the first designed nanoparticles with more than two structural components and with finely tunable environmental sensitivity, and they provide new routes to antibody-directed targeted delivery.

Scaffolding protein functional sites using deep learning.

The binding and catalytic functions of proteins are generally mediated by a small number of functional residues held in place by the overall protein structure. Here, we describe deep learning approaches for scaffolding such functional sites without needing to prespecify the fold or secondary structure of the scaffold. The first approach, "constrained hallucination," optimizes sequences such that their predicted structures contain the desired functional site. The second approach, "inpainting," starts from the functional site and fills in additional sequence and structure to create a viable protein scaffold in a single forward pass through a specifically trained RoseTTAFold network. We use these two methods to design candidate immunogens, receptor traps, metalloproteins, enzymes, and protein-binding proteins and validate the designs using a combination of in silico and experimental tests.

Systemic hypoxia inhibits T cell response by limiting mitobiogenesis via matrix substrate-level phosphorylation arrest.

Systemic oxygen restriction (SOR) is prevalent in numerous clinical conditions, including chronic obstructive pulmonary disease (COPD), and is associated with increased susceptibility to viral infections. However, the influence of SOR on T cell immunity remains uncharacterized. Here we show the detrimental effect of hypoxia on mitochondrial-biogenesis in activated mouse CD8+ T cells. We find that low oxygen level diminishes CD8+ T cell anti-viral response in vivo. We reveal that respiratory restriction inhibits ATP-dependent matrix processes that are critical for mitochondrial-biogenesis. This respiratory restriction-mediated effect could be rescued by TCA cycle re-stimulation, which yielded increased mitochondrial matrix-localized ATP via substrate-level phosphorylation. Finally, we demonstrate that the hypoxia-arrested CD8+ T cell anti-viral response could be rescued in vivo through brief exposure to atmospheric oxygen pressure. Overall, these findings elucidate the detrimental effect of hypoxia on mitochondrial-biogenesis in activated CD8+ T cells, and suggest a new approach for reducing viral infections in COPD.

Eukaryotic Adaptation to Years-Long Starvation Resembles that of Bacteria.

The Growth Advantage in Stationary Phase (GASP) phenomenon, described in bacteria, reflects the genetic adaptation of bacteria to stress, including starvation, for a long time. Unlike in stationary phase where no cell division occurs, GASP harbors active cell division, concurrent with genetic adaptation. Here we show that GASP occurs also in eukaryotes. Two strains of Saccharomyces cerevisiae (Sc404 and Sc424) have been isolated from 2-year-old sealed bottles of beer. These strains presented advantage in survival and growth over the parent during stress. The differences between the strains are irreversible and therefore genetic in origin rather than epigenetic. Direct competition assays show that Sc404 and Sc424 outcompete the parent in direct competition. DNA sequencing shows changes of the genome: the TOR complexes are mutated, and DNA repair gene mutations confer a mutator phenotype. The differences between the strains are reflected in a difference in taste between beers brewed from them.

Isolation and Characterization of Live Yeast Cells from Ancient Vessels as a Tool in Bio-Archaeology.

Ancient fermented food has been studied based on recipes, residue analysis, and ancient-DNA techniques and reconstructed using modern domesticated yeast. Here, we present a novel approach based on our hypothesis that enriched yeast populations in fermented beverages could have become the dominant species in storage vessels and their descendants could be isolated and studied today. We developed a pipeline of yeast isolation from clay vessels and screened for yeast cells in beverage-related and non-beverage-related ancient vessels and sediments from several archaeological sites. We found that yeast cells could be successfully isolated specifically from clay containers of fermented beverages. The findings that genotypically the isolated yeasts are similar to those found in traditional African beverages and phenotypically they grow similar to modern beer-producing yeast strongly suggest that they are descendants of the original fermenting yeast. These results demonstrate that modern microorganisms can serve as a new tool in bio-archaeology research.IMPORTANCE So far, most of the study of ancient organisms has been based mainly on the analysis of ancient DNA. Here we show that it is possible to isolate and study microorganisms-yeast in this case-from ancient pottery vessels used for fermentation. We demonstrate that it is highly likely that these cells are descendants of the original yeast strains that participated in the fermentation process and were absorbed into the clay matrix of the pottery vessels. Moreover, we characterized the isolated yeast strains, their genomes, and the beer they produced. These results open new and exciting avenues in the study of domesticated microorganisms and contribute significantly to the fields of bio- and experimental archaeology that aim to reconstruct ancient artifacts and products.

Long Noncoding RNA MALAT1 Regulates Cancer Glucose Metabolism by Enhancing mTOR-Mediated Translation of TCF7L2.

Reprogrammed glucose metabolism of enhanced aerobic glycolysis (or the Warburg effect) is known as a hallmark of cancer. The roles of long noncoding RNAs (lncRNA) in regulating cancer metabolism at the level of both glycolysis and gluconeogenesis are mostly unknown. We previously showed that lncRNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) acts as a proto-oncogene in hepatocellular carcinoma (HCC). Here, we investigated the role of MALAT1 in regulating cancer glucose metabolism. MALAT1 upregulated the expression of glycolytic genes and downregulated gluconeogenic enzymes by enhancing the translation of the metabolic transcription factor TCF7L2. MALAT1-enhanced TCF7L2 translation was mediated by upregulation of SRSF1 and activation of the mTORC1-4EBP1 axis. Pharmacological or genetic inhibition of mTOR and Raptor or expression of a hypophosphorylated mutant version of eIF4E-binding protein (4EBP1) resulted in decreased expression of TCF7L2. MALAT1 expression regulated TCF7L2 mRNA association with heavy polysomes, probably through the TCF7L2 5'-untranslated region (UTR), as determined by polysome fractionation and 5'UTR-reporter assays. Knockdown of TCF7L2 in MALAT1-overexpressing cells and HCC cell lines affected their metabolism and abolished their tumorigenic potential, suggesting that the effects of MALAT1 on glucose metabolism are essential for its oncogenic activity. Taken together, our findings suggest that MALAT1 contributes to HCC development and tumor progression by reprogramming tumor glucose metabolism. SIGNIFICANCE: These findings show that lncRNA MALAT1 contributes to HCC development by regulating cancer glucose metabolism, enhancing glycolysis, and inhibiting gluconeogenesis via elevated translation of the transcription factor TCF7L2.

Inhibition of nervous necrosis virus by ribavirin in a zebrafish larvae model.

The guanosine analog ribavirin is a broad-spectrum antiviral drug, mostly used in human clinical practice. It has in vitro and in vivo activity against a broad range of RNA and DNA viruses. Here, we report that treatment of zebrafish larvae with ribavirin prior to infection with nervous necrosis virus (NNV) significantly reduces the mortality caused by the virus during the first 10 days post-infection. The RNA genome of NNV harvested from ribavirin-treated infected larvae contains three synonymous and one single non-synonymous mutation, resulting in the replacement of a serine codon with a glycine codon in the RNA-dependent RNA polymerase gene. Adding increasing amounts of guanosine to ribavirin prior to larvae infection did not impede the antiviral activity. Ribavirin treatment of uninfected larvae reduces the basal level of IFNγ, but increases the level of IL-1ß mRNA expression. Furthermore, infecting larvae with NNV following ribavirin treatment reduces the expression levels of IFNγ, IFN-I, Mx, and TNF-α genes, while the expression of IL-1ß is increased. These results suggest that cytokine modulation plays an important role in the activity of ribavirin against NNV. Mortality of more than 40 species of teleost fish, mostly larvae and juveniles, from NNV is a major obstacle in hatcheries, and impedes the supply of young fish to farms. Hence, cost-effective ribavirin treatment should be considered as an efficient means to reduce the peril of NNV.

Clinical Significance of Circulating CD33+CD11b+HLA-DR- Myeloid Cells in Patients with Stage IV Melanoma Treated with Ipilimumab.

PURPOSE: High levels of circulating myeloid-derived suppressor cells (MDSCs) in various cancer types, including melanoma, were shown to correlate with poor survival. We investigated whether frequencies of circulating CD33+CD11b+HLA-DR- MDSCs could be used as immune system monitoring biomarkers to predict response and survival of patients with stage IV melanoma treated with anti-CTLA4 (ipilimumab) therapy. EXPERIMENTAL DESIGN: Peripheral blood samples from 56 patients and 50 healthy donors (HDs) were analyzed for CD33+CD11b+HLA-DR- MDSC percentage, NO-, and hROS levels by flow cytometry. We determined whether MDSC levels and suppressive features detected before anti-CTLA4 therapy correlate with the patients' response and overall survival (OS). RESULTS: Patients with melanoma had significantly higher levels of circulating CD33+CD11b+HLA-DR- MDSCs with suppressive phenotype when compared with HDs. Low levels of MDSCs before CTLA-4 therapy correlated with an objective clinical response, long-term survival, increased CD247 expression in T cells, and an improved clinical status. No predictive impact was observed for lactate dehydrogenase (LDH). Kaplan-Meier and log-rank tests performed on the 56 patients showed that the presence of more than 55.5% of circulating CD33+CD11b+ out of the HLA-DR- cells, were associated with significant short OS (P < 0.003), a median of 6.5 months, in comparison with the group showing lower MDSC frequencies, with a median survival of 15.6 months. CONCLUSIONS: Our study suggests the use of CD33+CD11b+HLA-DR- cells as a predictive and prognostic biomarker in patients with stage IV melanoma treated with anti-CTLA4 therapy. This monitoring system may aid in the development of combinatorial modalities, targeting the suppressive environment in conjunction with iplimumab, toward facilitating better disease outcomes. Clin Cancer Res; 22(23); 5661-72. ©2016 AACR.