Transposon DNA sequences facilitate the tissue-specific gene transfer of circulating tumor DNA between human cells.

The exchange of genes between cells is known to play an important physiological and pathological role in many organisms. We show that circulating tumor DNA (ctDNA) facilitates cell-specific gene transfer between human cancer cells and explain part of the mechanisms behind this phenomenon. As ctDNA migrates into the nucleus, genetic information is transferred. Cell targeting and ctDNA integration require ERVL, SINE or LINE DNA sequences. Chemically manufactured AluSp and MER11C sequences replicated multiple myeloma (MM) ctDNA cell targeting and integration. Additionally, we found that ctDNA may alter the treatment response of MM and pancreatic cancer models. This study shows that retrotransposon DNA sequences promote cancer gene transfer. However, because cell-free DNA has been detected in physiological and other pathological conditions, our findings have a broader impact than just cancer. Furthermore, the discovery that transposon DNA sequences mediate tissue-specific targeting will open up a new avenue for the delivery of genes and therapies.

Monocytes promote acute neuroinflammation and become pathological microglia in neonatal hypoxic-ischemic brain injury.

Rationale: Monocytes belong to the mononuclear phagocyte system and are immune responders to tissue injury and infection. There were also reports of monocytes transforming to microglia-like cells. Here we explore the roles of monocytes in microglia ontogeny and the pathogenesis of neonatal cerebral hypoxic-ischemic (HI) brain injury in mice. Methods: We used three genetic methods to track the development of monocytes, including CX3CR1GFP/+; CCR2RFP/+ reporter mice, adoptive transfer of GFP+ monocytes, and fate-mapping with CCR2-CreER mice, in neonatal mouse brains with or without lipopolysaccharide (LPS, 0.3 mg/kg)-sensitized Vannucci HI. We also used genetic (CCR2RFP/ RFP, CCR2 knockout) and pharmacological methods (RS102895, a CCR2 antagonist) to test the roles of monocytic influx in LPS/HI brain injury. Results: CCR2+ monocytes entered the late-embryonic brains via choroid plexus, but rapidly became CX3CR1+ amoeboid microglial cells (AMCs). The influx of CCR2+ monocytes declined after birth, but recurred after HI or LPS-sensitized HI (LPS/HI) brain injury, particularly in the hippocampus. The CCR2-CreER-based fate-mapping showed that CCR2+ monocytes became CD68+ TNFα+ macrophages within 4 d after LPS/HI, and maintained as TNFα+ MHCII+ macrophages or persisted as Tmem119+ Sall1+ P2RY12+ ramified microglia for at least five months after injury. Genetic deletion of the chemokine receptor CCR2 markedly diminished monocytic influx, the expression of pro- and anti-inflammatory cytokines, and brain damage. Post-LPS/HI application of RS102895 also reduced inflammatory responses and brain damage, leading to better cognitive functions. Conclusion: These results suggest that monocytes promote acute inflammatory responses and may become pathological microglia long after the neonatal LPS/HI insult. Further, blocking the influx of monocytes may be a potential therapy for neonatal brain injury.

The level of oncogenic Ras determines the malignant transformation of Lkb1 mutant tissue in vivo.

The genetic and metabolic heterogeneity of RAS-driven cancers has confounded therapeutic strategies in the clinic. To address this, rapid and genetically tractable animal models are needed that recapitulate the heterogeneity of RAS-driven cancers in vivo. Here, we generate a Drosophila melanogaster model of Ras/Lkb1 mutant carcinoma. We show that low-level expression of oncogenic Ras (RasLow) promotes the survival of Lkb1 mutant tissue, but results in autonomous cell cycle arrest and non-autonomous overgrowth of wild-type tissue. In contrast, high-level expression of oncogenic Ras (RasHigh) transforms Lkb1 mutant tissue resulting in lethal malignant tumors. Using simultaneous multiview light-sheet microcopy, we have characterized invasion phenotypes of Ras/Lkb1 tumors in living larvae. Our molecular analysis reveals sustained activation of the AMPK pathway in malignant Ras/Lkb1 tumors, and demonstrate the genetic and pharmacologic dependence of these tumors on CaMK-activated Ampk. We further show that LKB1 mutant human lung adenocarcinoma patients with high levels of oncogenic KRAS exhibit worse overall survival and increased AMPK activation. Our results suggest that high levels of oncogenic KRAS is a driving event in the malignant transformation of LKB1 mutant tissue, and uncovers a vulnerability that may be used to target this aggressive genetic subset of RAS-driven tumors.

Catalytic diversity in self-propagating peptide assemblies.

The protein-only infectious agents known as prions exist within cellular matrices as populations of assembled polypeptide phases ranging from particles to amyloid fibres. These phases appear to undergo Darwinian-like selection and propagation, yet remarkably little is known about their accessible chemical and biological functions. Here we construct simple peptides that assemble into well-defined amyloid phases and define paracrystalline surfaces able to catalyse specific enantioselective chemical reactions. Structural adjustments of individual amino acid residues predictably control both the assembled crystalline order and their accessible catalytic repertoire. Notably, the density and proximity of the extended arrays of enantioselective catalytic sites achieve template-directed polymerization of new polymers. These diverse amyloid templates can now be extended as dynamic self-propagating templates for the construction of even more complex functional materials.

Design of Asymmetric Peptide Bilayer Membranes.

Energetic insights emerging from the structural characterization of peptide cross-ß assemblies have enabled the design and construction of robust asymmetric bilayer peptide membranes. Two peptides differing only in their N-terminal residue, phosphotyrosine vs lysine, coassemble as stacks of antiparallel ß-sheets with precisely patterned charged lattices stabilizing the bilayer leaflet interface. Either homogeneous or mixed leaflet composition is possible, and both create nanotubes with dense negative external and positive internal solvent exposed surfaces. Cross-seeding peptide solutions with a preassembled peptide nanotube seed leads to domains of different leaflet architecture within single nanotubes. Architectural control over these cross-ß assemblies, both across the bilayer membrane and along the nanotube length, provides access to highly ordered asymmetric membranes for the further construction of functional mesoscale assemblies.

Gammaherpesvirus Co-infection with Malaria Suppresses Anti-parasitic Humoral Immunity.

Immunity to non-cerebral severe malaria is estimated to occur within 1-2 infections in areas of endemic transmission for Plasmodium falciparum. Yet, nearly 20% of infected children die annually as a result of severe malaria. Multiple risk factors are postulated to exacerbate malarial disease, one being co-infections with other pathogens. Children living in Sub-Saharan Africa are seropositive for Epstein Barr Virus (EBV) by the age of 6 months. This timing overlaps with the waning of protective maternal antibodies and susceptibility to primary Plasmodium infection. However, the impact of acute EBV infection on the generation of anti-malarial immunity is unknown. Using well established mouse models of infection, we show here that acute, but not latent murine gammaherpesvirus 68 (MHV68) infection suppresses the anti-malarial humoral response to a secondary malaria infection. Importantly, this resulted in the transformation of a non-lethal P. yoelii XNL infection into a lethal one; an outcome that is correlated with a defect in the maintenance of germinal center B cells and T follicular helper (Tfh) cells in the spleen. Furthermore, we have identified the MHV68 M2 protein as an important virus encoded protein that can: (i) suppress anti-MHV68 humoral responses during acute MHV68 infection; and (ii) plays a critical role in the observed suppression of anti-malarial humoral responses in the setting of co-infection. Notably, co-infection with an M2-null mutant MHV68 eliminates lethality of P. yoelii XNL. Collectively, our data demonstrates that an acute gammaherpesvirus infection can negatively impact the development of an anti-malarial immune response. This suggests that acute infection with EBV should be investigated as a risk factor for non-cerebral severe malaria in young children living in areas endemic for Plasmodium transmission.

Mapping amyloid-ß(16-22) nucleation pathways using fluorescence lifetime imaging microscopy.

The cross-ß peptide architecture is associated with numerous functional biomaterials and deleterious disease related aggregates. While these diverse and ubiquitous paracrystalline assemblies have been widely studied, a fundamental understanding of the nucleation and aggregation pathways to these structures remains elusive. Here we highlight a novel application of fluorescence lifetime imaging microscopy in characterising the critical stages of peptide aggregation. Using the central nucleating core of the amyloid-ß (Aß), Aß(16-22), as a model cross-ß system, and utilising a small fraction of rhodamine labelled peptide (Rh110-Aß(17-22)), we map out a folding pathway from monomer to paracrystalline nanotube. Using this intrinsic fluorescence reporter, we demonstrate the effects of interfaces and evaporation on the nucleation of sub-critical concentration solutions, providing access to previously uncharacterised intermediate morphologies. Using fluorescence lifetime we follow the local peptide environment through the stages of nucleation and hydrophobic collapse, ending in a stable final structure. This work provides a metric for future implementations of measuring fluorescence lifetimes of intrinsic fluorescence reporters during the very dynamic processes relating to peptide nucleation and maturation.

τFCS: multi-method global analysis enhances resolution and sensitivity in fluorescence fluctuation measurements.

Fluorescence fluctuation methods have become invaluable research tools for characterizing the molecular-level physical and chemical properties of complex systems, such as molecular concentrations, dynamics, and the stoichiometry of molecular interactions. However, information recovery via curve fitting analysis of fluctuation data is complicated by limited resolution and challenges associated with identifying accurate fit models. We introduce a new approach to fluorescence fluctuation spectroscopy that couples multi-modal fluorescence measurements with multi-modal global curve fitting analysis. This approach yields dramatically enhanced resolution and fitting model discrimination capabilities in fluctuation measurements. The resolution enhancement allows the concentration of a secondary species to be accurately measured even when it constitutes only a few percent of the molecules within a sample mixture, an important new capability that will allow accurate measurements of molecular concentrations and interaction stoichiometry of minor sample species that can be functionally important but difficult to measure experimentally. We demonstrate this capability using τFCS, a new fluctuation method which uses simultaneous global analysis of fluorescence correlation spectroscopy and fluorescence lifetime data, and show that τFCS can accurately recover the concentrations, diffusion coefficients, lifetimes, and molecular brightness values for a two component mixture over a wide range of relative concentrations.

Global analysis in fluorescence correlation spectroscopy and fluorescence lifetime microscopy.

Fluorescence correlation spectroscopy (FCS) and related fluctuation spectroscopy and microscopy methods have become important research tools that enable detailed investigations of the chemical and physical properties of molecules and molecular systems in a variety of complex environments. Information recovery via curve fitting of fluctuation data can present complicating challenges due to limited resolution and/or problems with fitting model verification. We discuss a new approach to data analysis called τFCS that couples multiple modes of signal acquisition, here specifically FCS and fluorescence lifetimes, with global analysis. We demonstrate enhanced resolution using τFCS, including the capability to recover the concentration of both molecular species in a two-component mixture even when the species have identical diffusion coefficients and molecular brightness values, provided their fluorescent lifetimes are distinct. We also demonstrate how τFCS provides useful tools for model discrimination in FCS curve fitting.

Phase networks of cross-ß peptide assemblies.

Recent evidence suggests that simple peptides can access diverse amphiphilic phases, and that these structures underlie the robust and widely distributed assemblies implicated in nearly 40 protein misfolding diseases. Here we exploit a minimal nucleating core of the Aß peptide of Alzheimer's disease to map its morphologically accessible phases that include stable intermolecular molten particles, fibers, twisted and helical ribbons, and nanotubes. Analyses with both fluorescence lifetime imaging microscopy (FLIM) and transmission electron microscopy provide evidence for liquid-liquid phase separations, similar to the coexisting dilute and dense protein-rich liquid phases so critical for the liquid-solid transition in protein crystallization. We show that the observed particles are critical for transitions to the more ordered cross-ß peptide phases, which are prevalent in all amyloid assemblies, and identify specific conditions that arrest assembly at the phase boundaries. We have identified a size dependence of the particles in order to transition to the para-crystalline phase and a width of the cross-ß assemblies that defines the transition between twisted fibers and helically coiled ribbons. These experimental results reveal an interconnected network of increasing molecularly ordered cross-ß transitions, greatly extending the initial computational models for cross-ß assemblies.