Interrogating cellular perception and decision making with optogenetic tools.

Optogenetics promises to deepen our understanding of how cells perceive and respond to complex and dynamic signals and how this perception regulates normal and abnormal function. In this study, we present our vision for how these nascent tools may transform our view of fundamental cell biological processes

The thermal history and hydrocarbon source rock potential of the mid Carboniferous Quebradas Formation in SW Portugal and its correlatives in western Atlantic offshore basins

The mid Carboniferous Quebradas Formation of the ‘South Portuguese Zone’ (SPZ) comprises 80m of post-mature black mudrocks with a mean TOC of 2.5%. Lithostratigraphic units of similar facies and age such as the Holywell Shale, the Edale Shale and the Bowland Shale are important HC source rocks in the UK, having sourced a considerable proportion of the hydrocarbons in the East Irish Sea, East Midlands and Formby oilfields respectively. The kerogen content of the Quebradas Formation is mixed but slightly more oil-prone in its lower part. At outcrop, it is strongly post-mature with vitrinite reflectance (Rr) ca. 4%. Illite crystallinity results from the Quebradas Formation and associated units suggest lower maturity than vitrinite reflectance. Analysis of the optic fabric of very thin coal lenses within the Brejeira Formation which overlies the Quebradas Formation suggests that peak temperatures were attained before the Variscan (late Carboniferous – early Permian) deformation. Triassic rocks unconformably overlying the Carboniferous sequence are much less mature, with Rr ca. 1.2%. Although the the Quebradas Fm has no HC source potential onshore due to its high maturity, Carboniferous rocks offshore may not have experienced the same extreme thermal history as the SPZ

Interrogating cellular perception and decision making with optogenetic tools.

Optogenetics promises to deepen our understanding of how cells perceive and respond to complex and dynamic signals and how this perception regulates normal and abnormal function. In this study, we present our vision for how these nascent tools may transform our view of fundamental cell biological processes

T cells selectively filter oscillatory signals on the minutes timescale.

T cells experience complex temporal patterns of stimulus via receptor-ligand-binding interactions with surrounding cells. From these temporal patterns, T cells are able to pick out antigenic signals while establishing self-tolerance. Although features such as duration of antigen binding have been examined, our understanding of how T cells interpret signals with different frequencies or temporal stimulation patterns is relatively unexplored. We engineered T cells to respond to light as a stimulus by building an optogenetically controlled chimeric antigen receptor (optoCAR). We discovered that T cells respond to minute-scale oscillations of activation signal by stimulating optoCAR T cells with tunable pulse trains of light. Systematically scanning signal oscillation period from 1 to 150 min revealed that expression of CD69, a T cell activation marker, reached a local minimum at a period of ∼25 min (corresponding to 5 to 15 min pulse widths). A combination of inhibitors and genetic knockouts suggest that this frequency filtering mechanism lies downstream of the Erk signaling branch of the T cell response network and may involve a negative feedback loop that diminishes Erk activity. The timescale of CD69 filtering corresponds with the duration of T cell encounters with self-peptide-presenting APCs observed via intravital imaging in mice, indicating a potential functional role for temporal filtering in vivo. This study illustrates that the T cell signaling machinery is tuned to temporally filter and interpret time-variant input signals in discriminatory ways

T cells selectively filter oscillatory signals on the minutes timescale.

T cells experience complex temporal patterns of stimulus via receptor-ligand-binding interactions with surrounding cells. From these temporal patterns, T cells are able to pick out antigenic signals while establishing self-tolerance. Although features such as duration of antigen binding have been examined, our understanding of how T cells interpret signals with different frequencies or temporal stimulation patterns is relatively unexplored. We engineered T cells to respond to light as a stimulus by building an optogenetically controlled chimeric antigen receptor (optoCAR). We discovered that T cells respond to minute-scale oscillations of activation signal by stimulating optoCAR T cells with tunable pulse trains of light. Systematically scanning signal oscillation period from 1 to 150 min revealed that expression of CD69, a T cell activation marker, reached a local minimum at a period of ∼25 min (corresponding to 5 to 15 min pulse widths). A combination of inhibitors and genetic knockouts suggest that this frequency filtering mechanism lies downstream of the Erk signaling branch of the T cell response network and may involve a negative feedback loop that diminishes Erk activity. The timescale of CD69 filtering corresponds with the duration of T cell encounters with self-peptide-presenting APCs observed via intravital imaging in mice, indicating a potential functional role for temporal filtering in vivo. This study illustrates that the T cell signaling machinery is tuned to temporally filter and interpret time-variant input signals in discriminatory ways

Early T cell receptor signals globally modulate ligand:receptor affinities during antigen discrimination.

Antigen discrimination by T cells occurs at the junction between a T cell and an antigen-presenting cell. Juxtacrine binding between numerous adhesion, signaling, and costimulatory molecules defines both the topographical and lateral geometry of this cell-cell interface, within which T cell receptor (TCR) and peptide major histocompatibility complex (pMHC) interact. These physical constraints on receptor and ligand movement have significant potential to modulate their molecular binding properties. Here, we monitor individual ligand:receptor binding and unbinding events in space and time by single-molecule imaging in live primary T cells for a range of different pMHC ligands and surface densities. Direct observations of pMHC:TCR and CD80:CD28 binding events reveal that the in situ affinity of both pMHC and CD80 ligands for their respective receptors is modulated by the steady-state number of agonist pMHC:TCR interactions experienced by the cell. By resolving every single pMHC:TCR interaction it is evident that this cooperativity is accomplished by increasing the kinetic on-rate without altering the off-rate and has a component that is not spatially localized. Furthermore, positive cooperativity is observed under conditions where the T cell activation probability is low. This TCR-mediated feedback is a global effect on the intercellular junction. It is triggered by the first few individual pMHC:TCR binding events and effectively increases the efficiency of TCR scanning for antigen before the T cell is committed to activation

Early detection of high-grade squamous intraepithelial lesions in the cervix with quantitative spectroscopic imaging

Quantitative spectroscopy has recently been extended from a contact-probe to wide-area spectroscopic imaging to enable mapping of optical properties across a wide area of tissue. We train quantitative spectroscopic imaging (QSI) to identify cervical high-grade squamous intraepithelial lesions (HSILs) in 34 subjects undergoing the loop electrosurgical excision procedure (LEEP subjects). QSI’s performance is then prospectively evaluated on the clinically suspicious biopsy sites from 47 subjects undergoing colposcopic-directed biopsy. The results show the per-subject normalized reduced scattering coefficient at 700 nm (A[subscript n]) and the total hemoglobin concentration are significantly different (p<0.05) between HSIL and non-HSIL sites in LEEP subjects. A[subscript n] alone retrospectively distinguishes HSIL from non-HSIL with 89% sensitivity and 83% specificity. It alone applied prospectively on the biopsy sites distinguishes HSIL from non-HSIL with 81% sensitivity and 78% specificity. The findings of this study agree with those of an earlier contact-probe study, validating the robustness of QSI, and specifically A[subscript n], for identifying HSIL. The performance of A[subscript n] suggests an easy to use and an inexpensive to manufacture monochromatic instrument is capable of early cervical cancer detection, which could be used as a screening and diagnostic tool for detecting cervical cancer in low resource countries.National Institutes of Health (U.S.) (Massachusetts Institute of Technology. Laser Biomedical Research Center Grant R01 CA097966)National Institutes of Health (U.S.) (Massachusetts Institute of Technology. Laser Biomedical Research Center Grant P41 RR02594