Improving the photostability of bright monomeric orange and red fluorescent proteins

All organic fluorophores undergo irreversible photobleaching during prolonged illumination. Although fluorescent proteins typically bleach at a substantially slower rate than many small-molecule dyes, in many cases the lack of sufficient photostability remains an important limiting factor for experiments requiring large numbers of images of single cells. Screening methods focusing solely on brightness or wavelength are highly effective in optimizing both properties, but the absence of selective pressure for photostability in such screens leads to unpredictable photobleaching behavior in the resulting fluorescent proteins. Here we describe an assay for screening libraries of fluorescent proteins for enhanced photostability. With this assay, we developed highly photostable variants of mOrange (a wavelength-shifted monomeric derivative of DsRed from Discosoma sp.) and TagRFP (a monomeric derivative of eqFP578 from Entacmaea quadricolor) that maintain most of the beneficial qualities of the original proteins and perform as reliably as Aequorea victoria GFP derivatives in fusion constructs.     

An essential role for DYF-11/MIP-T3 in assembling functional intraflagellar transport complexes

MIP-T3 is a human protein found previously to associate with microtubules and the kinesin-interacting neuronal protein DISC1 (Disrupted-in-Schizophrenia 1), but whose cellular function(s) remains unknown. Here we demonstrate that the C. elegans MIP-T3 ortholog DYF-11 is an intraflagellar transport (IFT) protein that plays a critical role in assembling functional kinesin motor-IFT particle complexes. We have cloned a loss of function dyf-11 mutant in which several key components of the IFT machinery, including Kinesin-II, as well as IFT subcomplex A and B proteins, fail to enter ciliary axonemes and/or mislocalize, resulting in compromised ciliary structures and sensory functions, and abnormal lipid accumulation. Analyses in different mutant backgrounds further suggest that DYF-11 functions as a novel component of IFT subcomplex B. Consistent with an evolutionarily conserved cilia-associated role, mammalian MIP-T3 localizes to basal bodies and cilia, and zebrafish mipt3 functions synergistically with the Bardet-Biedl syndrome protein Bbs4 to ensure proper gastrulation, a key cilium- and basal body-dependent developmental process. Our findings therefore implicate MIP-T3 in a previously unknown but critical role in cilium biogenesis and further highlight the emerging role of this organelle in vertebrate development.     

N-terminal labeling of proteins by the Pictet-Spengler reaction

The Pictet-Spengler reaction was applied to the N-terminal labeling of horse heart myoglobin. This was performed in the following two steps: (1) conversion of the N-terminal glycine residue to an alpha-keto aldehyde by a transamination reaction and (2) condensation of the resulting activated myoglobin with tryptamine analogues by the Pictet-Spengler reaction. Ultraviolet (UV)/visible (vis) absorption and circular dichroism (CD) spectral data revealed that the tertiary structure of myoglobin was not altered by the Pictet-Spengler reaction.     

Synthesis and SAR of analogs of the M1 allosteric agonist TBPB. Part II: Amides, sulfonamides and ureas--the effect of capping the distal basic piperidine nitrogen

This letter describes the further synthesis and SAR, developed through an iterative analog library approach, of analogs of the highly selective M1 allosteric agonist TBPB by deletion of the distal basic piperidine nitrogen by the formation of amides, sulfonamides and ureas. Despite the large change in basicity and topology, M1 selectivity was maintained.     

Aglycone exploration of C-arylglucoside inhibitors of renal sodium-dependent glucose transporter SGLT2

Inhibition of sodium-dependent glucose transporter 2 (SGLT2), the transporter that is responsible for renal re-uptake of glucose, leads to glucosuria in animals. SGLT-mediated glucosuria provides a mechanism to shed excess plasma glucose to ameliorate diabetes-related hyperglycemia and associated complications. The current study demonstrates that the proper relationship of a 4′-substituted benzyl group to a β-1C-phenylglucoside is important for potent and selective SGLT2 inhibition. The lead C-arylglucoside (7a) demonstrates superior metabolic stability to its O-arylglucoside counterpart (4) and it promotes glucosuria when administered in vivo.     

Synthesis and activity of N-cyanoguanidine-piperazine P2X7 antagonists

A novel series of cyanoguanidine-piperazine P2X₇ antagonists were identified and structure–activity relationship (SAR) studies described. Compounds were assayed for activity at human and rat P2X₇ receptors in addition to their ability to inhibit IL-1β release from stimulated human whole blood cultures. Compound 27 possesses potent activity (0.12 μM) in this latter assay and demonstrates moderate clearance in-vivo.     

Compatibility of magnetic imprinting and secular variation

Diverse ocean migrants, including some sea turtles, elephant seals, and salmon, begin life in particular reproductive areas along coastlines, disperse across vast expanses of sea, and then return as adults to their natal areas to reproduce [1], [2] and [3]. Little is known about how such marine animals guide themselves to the correct coastal region from hundreds or thousands of kilometers away and after absences ranging in duration from a few months to a decade or more. One hypothesis is that animals imprint on the magnetic field of their home area and use this information to return [1]. The Earth's field varies predictably across the globe, so different geographic areas are marked by distinctive magnetic fields that might, in principle, provide unique magnetic signatures for natal areas [4]. A potentially serious complication for this hypothesis is that the Earth's field changes gradually over time [1] and [4], causing the magnetic signatures that define natal areas to slowly drift. This secular variation could make natal homing via magnetic imprinting impossible if the magnetic signatures moved too far from the natal area [1], [5] and [6]. To investigate whether magnetic imprinting is compatible with secular variation, we sought a species with a life history that poses challenges for the hypothesis, reasoning that if magnetic imprinting is consistent with natal homing under unfavorable circumstances, then it would also be plausible in most other cases. We chose the Kemp's ridley sea turtle (Lepidochelys kempii), an endangered species that ranges widely over the Gulf of Mexico, northern Caribbean, and the eastern U.S. coast, but returns to nest along a single, limited region of coastline in northern Mexico [7]. This species requires approximately 10–15 years to reach sexual maturity [7] and is thus absent from its natal area for much longer than animals such as salmon and elephant seals [2] and [3]. Given this long absence, the Kemp's ridley appears to be particularly susceptible to effects of secular variation if it relies on magnetic imprinting. The modeling results we report here show that the magnetic imprinting hypothesis can account for how the Kemp's ridley turtle returns to its natal region even after absences of a decade or more.     

Identification of minimal sequence for HIV-1 fusion inhibitors

Emergence of multi-drug resistant HIV-1 is a serious problem for AIDS treatment. Recently, the virus-cell membrane fusion process has been identified as a promising target for the development of novel drugs against these resistant variants. In this study, we identified a 29-residue peptide fusion inhibitor, SC29EK, which shows activity comparable to the previously reported inhibitor SC35EK. Some residues in SC29EK not required for interaction with virus gp41 heptad repeat 1 (HR1) were replaced with a non-proteinogenic amino acid, 2-aminoisobutyric acid (Aib), to stabilize the alpha-helix structure and to provide resistance to peptidases.     

Growth chronologies of white sucker, Catostomuscommersoni, and lake trout, Salvelinusnamaycush: a comparison among lakes and between trophic levels

A growth chronology index was used to determine whether changes in ecosystem structure and function in lakes could be associated with fish growth histories. Growth chronologies were compared for white suckers, Catostomus commersoni, from Little Moose (oligotrophic), Oneida (eutrophic), and Cayuga (mesotrophic) lakes (New York) from opercular bone growth increments, and for lake trout, Salvelinus namaycush, from Little Moose Lake using otolith growth. The longevity of these species allowed the development of chronologies from 17 to 33 years in length using only contemporary collections. We used these chronologies to examine whether fish growth histories could be used as an index for ecosystem-scale changes. Specifically, we examined whether zebra mussel, Dreissena polymorpha, invasion in Oneida and Cayuga lakes in the early 1990s, and treatment of sewage effluent from dwellings around Little Moose Lake beginning during the late 1980s could be detected in white sucker and lake trout growth chronologies. White sucker growth in Oneida and Cayuga Lakes did not differ before and after zebra mussel invasions. Neither white sucker nor lake trout growth chronologies from Little Moose Lake reflect changes in growth expected with reduced productivity levels associated with improved sewage treatment. Growth chronologies of these two species did not detect the ecosystem-scale changes that occurred in the study lakes.