No abstract

Survival of naive T cells is dependent upon IL-7, which is present in vivo in limiting amounts with the result that naive T cells must compete for IL-7-mediated survival signals. It would seem imperative during T cell homeostasis that limiting IL-7 be shared by the greatest possible number of T cells. We now describe a novel regulatory mechanism that specifically suppresses IL7Ralpha transcription in response to IL-7 and other prosurvival cytokines (IL-2, IL-4, IL-6, and IL-15). Consequently, IL7R expression is reduced on T cells that have received cytokine-mediated survival signals so they do not compete with unsignaled T cells for remaining IL-7. Interestingly, cytokine-mediated suppression of IL7Ralpha transcription involves different molecular mechanisms in CD4+ and CD8+ T cells, as CD8+ T cells utilize the transcriptional repressor GFI1 while CD4+ T cells do not. We suggest that this homeostatic regulatory mechanism promotes survival of the maximum possible number of T cells for the amount of IL-7 available.

There is considerable interest in novel cell imaging tools that avoid the use of fluorescent proteins. One widely used class of such reagents are “pro-fluorescent” biarsenical dyes such as FlAsH, ReAsH, CrAsH, and Cy3As. Despite their utility, biarsenicals are plagued by high background labeling and cytotoxicity, and are challenging to apply in oxidizing cellular locale. Here we demonstrate that [(3-oxospiro[isobenzofuran-1(3H),9′-[9H]xanthene]-3′,6′-diyl)bis(iminomethylene-2,1-phenylene)]bis-(9CI), a rhodamine-derived bisboronic acid (RhoBo) described initially as a monosaccharide sensor, functions as a cell-permeable, turn-on fluorescent sensor for a tetraserine-motifs in recombinant proteins. RhoBo binds peptides or proteins containing Ser-Ser-Pro-Gly-Ser-Ser with affinities in the nanomolar concentration range, and prefers this sequence to simple monosaccharides by >10,000-fold. RhoBo fails to form fluorescent complexes with constituents of the mammalian cell surface, as judged by epifluorescent, confocal, and TIRF microscopy, but fluoresces brightly within the Ser-Ser-Pro-Gly-Ser-Ser-rich cell interior. These results suggest that current efforts to identify optimal serine-rich sequences for RhoBo will allow it to function effectively as a selective small-molecule label for appropriately tagged proteins either upon or within living cells.

Aberrant activation of the epidermal growth factor receptor (EGFR) is critical to the biology of many common cancers. The molecular events that define how EGFR transmits an extracellular ligand binding event through the membrane are not understood. Here we use a chemical tool, bipartite tetracysteine display, to report on ligand-specific conformational changes that link ligand binding and kinase activation for full-length EGFR on the mammalian cell surface. We discover that EGF binding is communicated to the cytosol through formation of an antiparallel coiled coil within the intracellular juxtamembrane (JM) domain. This interaction is functionally coupled to receptor activation by EGF. In contrast, TGFα binding is communicated to the cytosol through formation of a discrete, alternative helical interface. These findings suggest that the JM region can differentially decode extracellular signals and transmit them to the cell interior, and provide new insight into how EGFR communicates ligand-specific information across the membrane.

β-peptides possess several features that are desirable in peptidomimetics; they are easily synthesized, fold into stable secondary structures in physiologic buffers, and resist proteolysis. They can also bind to a diverse array of proteins to inhibit their interactions with α–helical ligands. β–peptides are not usually cell permeable, however, and this feature limits their utility as research tools and potential therapeutics. Appending an Arg8 sequence to a β–peptide improves uptake but adds considerable mass. We reported that embedding a small cationic patch within a PPII, α– or β–peptide helix improves uptake without the addition of significant mass. In another mass-neutral strategy, Verdine, Walensky, and others have reported that insertion of a hydrocarbon bridge between the i and i+4 positions of an α–helix also increases cell uptake. Here we describe a series of β–peptides containing diether and hydrocarbon bridges and compare them on the basis of cell uptake and localization, affinities for hDM2, and 14-helix structure. Our results highlight the relative merits of cationic patch and hydrophobic bridge strategies for improving β–peptide uptake and identify a surprising correlation between uptake efficiency and hDM2 affinity.

Abstract. We present a refined chosen-prefix collision construction for MD5 that allowed creation of a rogue Certification Authority (CA) certificate, based on a collision with a regular end-user website certificate provided by a commercial CA. Compared to the previous construction from Eurocrypt 2007, this paper describes a more flexible family of differential paths and a new variable birthdaying search space. Combined with a time-memory trade-off, these improvements lead to just three pairs of near-collision blocks to generate the collision, enabling construction of RSA moduli that are sufficiently short to be accepted by current CAs. The entire construction is fast enough to allow for adequate prediction of certificate serial number and validity period: it can be made to require about 2 49 MD5 compression function calls. Finally, we improve the complexity of identical-prefix collisions for MD5 to about 2 16 MD5 compression function calls and use it to derive a practical single-block chosen-prefix collision construction of which an example is given.

Proteins and other macromolecules that cross biological membranes have great potential as tools for research and next-generation therapeutics. Here we describe two assays that effectively quantify the cytosolic localization of a number of previously reported peptides and protein domains. One assay, which we call GIGI (Glucocorticoid-Induced eGFP Induction), is an amplified assay that informs on relative cytosolic access without need for sophisticated imaging equipment or adherent cells. The second, GIGT (Glucocorticoid-Induced eGFP Translocation), is a non-amplified assay that informs on relative cytosolic access and exploits sophisticated imaging equipment to facilitate high-content screens in live cells. Each assay was employed to quantify the cytosolic delivery of several canonical “cell permeable peptides”, as well as more recently reported minimally cationic miniature proteins and zinc finger nuclease domains. Our results show definitively that both overall charge as well as charge distribution influence cytosolic access, and that small protein domains containing a discrete, helical, penta-arg motif can dramatically improve the cytosolic delivery of small folded proteins such as zinc finger domains. We anticipate that the assays described herein will prove useful to explore and discover the fundamental physicochemical and genetic properties that influence both the uptake and endosomal release of peptidic molecules and their mimetics.

Contrary to widespread assumption, dynamic RAM (DRAM), the main memory in most modern computers, retains its contents for several seconds after power is lost, even at room temperature and even if removed from a motherboard. Although DRAM becomes less reliable when it is not refreshed, it is not immediately erased, and its contents persist sufficiently for malicious (or forensic) acquisition of usable full-system memory images. We show that this phenomenon limits the ability of an operating system to protect cryptographic key material from an attacker with physical access to a machine. It poses a particular threat to laptop users who rely on disk encryption: we demonstrate that it could be used to compromise several popular disk encryption products without the need for any special devices or materials. We experimentally characterize the extent and predictability of memory retention and report that remanence times can be increased dramatically with simple cooling techniques. We offer new algorithms for finding cryptographic keys in memory images and for correcting errors caused by bit decay. Though we discuss several strategies for mitigating these risks, we know of no simple remedy that would eliminate them.