Enhanced-contrast two-photon optogenetic pH sensing and pH-resolved brain imaging

We present experiments on cell cultures and brain slices that demonstrate two-photon optogenetic pH sensing and pH-resolved brain imaging using a laser driver whose spectrum is carefully tailored to provide the maximum contrast of a ratiometric two-photon fluorescence readout from a high-brightness genetically encoded yellow-fluorescent-protein-based sensor, SypHer3s. Two spectrally isolated components of this laser field are set to induce two-photon-excited fluorescence (2PEF) by driving SypHer3s through one of two excitation pathways—via either the protonated or deprotonated states of its chromophore. With the spectrum of the laser field accurately adjusted for a maximum contrast of these two 2PEF signals, the ratio of their intensities is shown to provide a remarkably broad dynamic range for pH measurements, enabling high-contrast optogenetic deep-brain pH sensing and pH-resolved 2PEF imaging within a vast class of biological systems, ranging from cell cultures to the living brain.     

The odyssey of new production

In studies of primary production of the open ocean, the measurement of new production is often considered a measure of the degree of eutrophication. Because of new research this is questionable, which in our opinion, calls for a refinement to the original concept. We believe that the measure of variable fluorescence is pivotal to a new understanding. Our research enforces the growing conviction that the measurement of Fv/Fm can be interpreted as an analogue for nutrient stress. We measured variable fluorescence in axenic cultures at the CCMP at the Bigelow Laboratory. The Fv/Fm of cultures, upon transfer to new media reached a maximum followed by a decline after approximately 30 days. The rate of decline does not appear to be species specific. Most of the clones remained relatively high after 30 days. The high Fv/Fm values observed in nutrient-replete cultures are not characteristic of the oligotrophic surface waters off Florida and the Bahamas, but are approached in the eutrophic waters of the Gulf of Maine. In contrast, Fv/Fm measurements of attached macroalgae and coral zooxanthellae are characteristically much higher than microalgae of either oligotrophic or eutrophic regions. Accordingly, we advance the case for interpretation of Fv/Fm in terms of nutrient stress in ecological studies and advocate that the old concept of new production should be modified.     

Electronic structure and polarizabilities of some heterocycles: I. Hydroxypyrazoles and related systems

The (hyper)polarizabilities of different tautomer forms of hydroxypyrazoles and pyrazolones have been calculated by the finite-field procedure in the MNDO approximation and the sum of states formalism in the PPP approximation, with all singly- and doubly-excited electronic configurations in the CI method. It was shown that while in the ground electronic state the values of the (hyper) polarizabilities are not essentially different, in the first excited singlet Franck-Condon state an increase of the molecular polarizabilities of some tautomers is observed. This increase is attributed to a specific change in the electronic structure of the excited state, demonstrated by the localization of the electronic transition in the different pyrazolone tautomers. The electron-donor capabilities of phenyl-substituted hydroxypyrazoles and pyrazolones are discussed.     

Patterns and impacts of inorganic tripton in Cayuga Lake

Inorganic tripton in the upper waters of Cayuga Lake, NY, is characterized, quantified and contrasted for shallow (<6 m) and deep (>100 m) water regions based on analyses with scanning electron microscopy equipped with automated image analysis and X-ray energy spectroscopy (SAX) of samples collected in 1999 and 2000. The impacts of this material on water clarity and the phosphorus (P) pool are evaluated based on paired measurements of turbidity (T _n), Secchi disc transparency (SD), chlorophyll, and four fractions of particulate P (PP) determined according to selective extraction protocols. The major components of inorganic tripton in the lake are reported to be clay minerals and quartz, received from the watershed, and calcium carbonate (CaCO₃), that is produced internally. Clay minerals are the dominant component in a shallow region that receives tributary inflows. CaCO₃, precipitated during a mid-summer whiting event, is reported to be the dominant component of inorganic tripton for the deep water region for a low runoff year, while clays can become dominant in these areas after prolonged intervals of high runoff. It is demonstrated that inorganic tripton, rather than phytoplankton, is the primary regulator of T _n and SD, it represents most of the PP, and is primarily responsible for the higher T _n, lower SD, and higher TP in the shallow area compared to the deep water region. Efforts to improve clarity in this shallow region of the lake should consider the feasibility of controlling the input of terrigenous suspensoids rather than reductions in P loading.     

Optical plasticity of mammalian cells

Transparency is widespread in nature, ranging from transparent insect wings to ocular tissues that enable you to read this text, and transparent marine vertebrates. And yet, cells and tissue models in biology are usually strongly light scattering and optically opaque, precluding deep optical microscopy. Here we describe the directed evolution of cultured mammalian cells toward increased transparency. We find that mutations greatly diversify the optical phenotype of Chinese Hamster Ovary cells, a cultured mammalian cell line. Furthermore, only three rounds of high-throughput optical selection and competitive growth are required to yield fit cells with greatly improved transparency. Based on 15 monoclonal cell lines derived from this directed evolution experiment, we find that the evolved transparency frequently goes along with a reduction of nuclear granularity and physiological shifts in gene expression profiles. In the future this optical plasticity of mammalian cells may facilitate genetic clearance of living tissues for in vivo microscopy.     

Heteroarylamide smoothened inhibitors: Discovery of N-[2,4-dimethyl-5-(1-methylimidazol-4-yl)phenyl]-4-(2-pyridylmethoxy)benzamide (AZD8542) and N-[5-(1H-imidazol-2-yl)-2,4-dimethyl-phenyl]-4-(2- pyridylmethoxy)benzamide (AZD7254)

Aberrant hedgehog (Hh) pathway signaling is implicated in multiple cancer types and targeting the Smoothened (SMO) receptor, a key protein of the Hh pathway, has proven effective in treating metastasized basal cell carcinoma. Our lead optimization effort focused on a series of heteroarylamides. We observed that a methyl substitution ortho to the heteroaryl groups on an aniline core significantly improved the potency of this series of compounds. These findings predated the availability of SMO crystal structure in 2013. Here we retrospectively applied quantum mechanics calculations to demonstrate the o-Me substitution favors the bioactive conformation by inducing a dihedral twist between the heteroaryl rings and the core aniline. The o-Me also makes favorable hydrophobic interactions with key residue side chains in the binding pocket. From this effort, two compounds (AZD8542 and AZD7254) showed excellent pharmacokinetics across multiple preclinical species and demonstrated in vivo activity in abrogating the Hh paracrine pathway as well as anti- tumor effects.     

模拟大气硫酸铵污染对香樟幼苗生长及光合特性的影响

通过室内盆栽实验研究了大气颗粒污染物硫酸铵对香樟幼苗生长及光合特性的影响。结果表明,香樟幼苗叶片涂抹硫酸铵处理对植物生长无显著影响;低浓度硫酸铵(2 g·L^-1)提高了叶片叶绿素含量,而高浓度(4 g·L^-1)却降低了叶片叶绿素含量;与对照相比,低浓度处理的香樟叶片净光合速率、气孔导度、胞间二氧化碳浓度与蒸腾速率无显著差异;高浓度处理的香樟叶片净光合速率与蒸腾速率高于对照,而气孔导度与胞间二氧化碳浓度与对照无显著差异。机理分析表明,硫酸铵颗粒物主要通过影响叶片气孔导度来影响植物光合特性。     

Determination and correction of aberrations in full field optical coherence tomography using phase gradient autofocus by maximizing the likelihood function

A method for numerical estimation and correction of aberrations of the eye in fundus imaging with optical coherence tomography (OCT) is presented. Aberrations are determined statistically by using the estimate based on likelihood function maximization. The method can be considered as an extension of the phase gradient autofocusing algorithm in synthetic aperture radar imaging to 2D optical aberration correction. The efficacy of the proposed method has been demonstrated in OCT fundus imaging with 6λ aberrations. After correction, single photoreceptors were resolved. It is also shown that wave front distortions with high spatial frequencies can be determined and corrected.