High-Density Proximity Mapping Reveals the Subcellular Organization of mRNA-Associated Granules and Bodies

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A machine‐learning approach for extending classical wildlife resource selection analyses

Resource selection functions (RSFs) are tremendously valuable for ecologists and resource managers because they quantify spatial patterns in resource utilization by wildlife, thereby facilitating identification of critical habitat areas and characterizing specific habitat features that are selected or avoided. RSFs discriminate between known‐use resource units (e.g., telemetry locations) and available (or randomly selected) resource units based on an array of environmental features, and in their standard form are performed using logistic regression. As generalized linear models, standard RSFs have some notable limitations, such as difficulties in accommodating nonlinear (e.g., humped or threshold) relationships and complex interactions. Increasingly, ecologists are using flexible machine‐learning methods (e.g., random forests, neural networks) to overcome these limitations. Herein, we investigate the seasonal resource selection patterns of mule deer (Odocoileus hemionus) by comparing a logistic regression framework with random forest (RF), a popular machine‐learning algorithm. Random forest (RF) models detected nonlinear relationships (e.g., optimal ranges for slope and elevation) and complex interactions which would have been very challenging to discover and characterize using standard model‐based approaches. Compared with standard RSF models, RF models exhibited improved predictive skill, provided novel insights about resource selection patterns of mule deer, and, when projected across a relevant geographic space, manifested notable differences in predicted habitat suitability. We recommend that wildlife researchers harness the strengths of machine‐learning tools like RF in addition to “classical” tools (e.g., mixed‐effects logistic regression) for evaluating resource selection, especially in cases where extensive telemetry data sets are available.     

A 15.6 frames per second 1‐megapixel multiple exposure laser speckle contrast imaging setup

A multiple exposure laser speckle contrast imaging (MELSCI) setup for visualizing blood perfusion was developed using a field programmable gate array (FPGA), connected to a 1000 frames per second (fps) 1‐megapixel camera sensor. Multiple exposure time images at 1, 2, 4, 8, 16, 32 and 64 milliseconds were calculated by cumulative summation of 64 consecutive snapshot images. The local contrast was calculated for all exposure times using regions of 4 × 4 pixels. Averaging of multiple contrast images from the 64‐millisecond acquisition was done to improve the signal‐to‐noise ratio. The results show that with an effective implementation of the algorithm on an FPGA, contrast images at all exposure times can be calculated in only 28 milliseconds. The algorithm was applied to data recorded during a 5 minutes finger occlusion. Expected contrast changes were found during occlusion and the following hyperemia in the occluded finger, while unprovoked fingers showed constant contrast during the experiment. The developed setup is capable of massive data processing on an FPGA that enables processing of MELSCI data in 15.6 fps (1000/64 milliseconds). It also leads to improved frame rates, enhanced image quality and enables the calculation of improved microcirculatory perfusion estimates compared to single exposure time systems.      

Elongator—a tRNA modifying complex that promotes efficient translational decoding

Naturally occurring modifications of the nucleosides in the anticodon region of tRNAs influence their translational decoding properties. Uridines present at the wobble position in eukaryotic cytoplasmic tRNAs often contain a 5-carbamoylmethyl (ncm⁵) or 5-methoxycarbonylmethyl (mcm⁵) side-chain and sometimes also a 2-thio or 2′-O-methyl group. The first step in the formation of the ncm⁵ and mcm⁵ side-chains requires the conserved six-subunit Elongator complex. Although Elongator has been implicated in several different cellular processes, accumulating evidence suggests that its primary, and possibly only, cellular function is to promote modification of tRNAs. In this review, we discuss the biosynthesis and function of modified wobble uridines in eukaryotic cytoplasmic tRNAs, focusing on the in vivo role of Elongator-dependent modifications in Saccharomyces cerevisiae. This article is part of a Special Issue entitled: SI: Regulation of tRNA synthesis and modification in physiological conditions and disease edited by Dr. Boguta Magdalena.     

<Superscript>15</Superscript>N photo-CIDNP MAS NMR analysis of reaction centers of <Emphasis Type="Italic">Chloracidobacterium thermophilum</Emphasis>

Photochemically induced dynamic nuclear polarization (photo-CIDNP) has been observed in the homodimeric, type-1 photochemical reaction centers (RCs) of the acidobacterium, Chloracidobacterium (Cab.) thermophilum, by ¹⁵N magic-angle spinning (MAS) solid-state NMR under continuous white-light illumination. Three light-induced emissive (negative) signals are detected. In the RCs of Cab. thermophilum, three types of (bacterio)chlorophylls have previously been identified: bacteriochlorophyll a (BChl a), chlorophyll a (Chl a), and Zn-bacteriochlorophyll a′ (Zn-BChl a′) (Tsukatani et al. in J Biol Chem 287:5720–5732, 2012). Based upon experimental and quantum chemical ¹⁵N NMR data, we assign the observed signals to a Chl a cofactor. We exclude Zn-BChl because of its measured spectroscopic properties. We conclude that Chl a is the primary electron acceptor, which implies that the primary donor is most likely Zn-BChl a′. Chl a and 8¹-OH Chl a have been shown to be the primary electron acceptors in green sulfur bacteria and heliobacteria, respectively, and thus a Chl a molecule serves this role in all known homodimeric type-1 RCs.     

Probing the structure and function of the estrogen receptor ligand binding domain by analysis of mutants with altered transactivation characteristics.

We have developed a genetic screen for the yeast Saccharomyces cerevisiae to isolate estrogen receptor (ER) mutants with altered transactivation characteristics. Use of a "reverse" ER, in which the mutagenized ligand binding domain was placed at the N terminus of the receptor, eliminated the isolation of truncated constitutively active mutants. A library was screened with a low-affinity estrogen, 2-methoxyestrone (2ME), at concentrations 50-fold lower than those required for activation of the unmutagenized ER. Several mutants displaying enhanced sensitivity to 2ME were isolated. We further characterized a mutant carrying the substitution L536P, which was located immediately N terminal to the AF-2-activating domain of the receptor. Amino acid 536 corresponds to a ligand contact residue in retinoic acid receptor gamma, suggesting that key contact points are conserved among receptors. Introduction of L536P into the original ER cDNA isolate HE0, which contains the substitution G400V, rendered the receptor more sensitive to a variety of agonists. When introduced into the wild-type ER HEG0, L536P also rendered the receptor more sensitive to agonists, and, in addition, induced high levels of constitutive activity that could be inhibited by antiestrogens. Estrogens containing a keto substitution in the steroid D ring, but not those containing a hydroxyl group, were full agonists of L536P-HEG0. Limited proteolytic analysis suggested that the L536P substitution, which is located immediately N terminal to the AF-2 domain, induces a conformational change in the ER that partially mimics binding by hormone. Both HEG0 and L536P-HEG0 formed complexes with hsp90 in vitro, indicating a lack of correlation between interaction with hsp90 in vitro and hormonal regulation of ER transactivation in vivo. This supports the idea that a factor(s) acting downstream of hsp90 is important for controlling activity of the hormone-free receptor.     

Heterochromatin protein HP1Hsβ(p25β) and its localization with centromeres in mitosis

Some autoimmune sera containing anticentromere autoantibodies also recognize a doublet of Mr 23 000 (p23) and 25 000 (p25) in addition to CENP (centromere protein)-A (Mr 19 000), -B (Mr 80 000), and -C (Mr 140 000). A p25 antigen (HP1^Hsα) has been shown to be a human homolog of Drosophila HP1 (heterochromatin protein 1). We have isolated a cDNA clone encoding another form of p25 (HP1^Hsβor p25β) from a λZap HepG2 library using human autoimmune serum. The deduced amino acid sequence of the clone contained a conserved chromodomain (chromatin modifier domain) in the N-terminal region and a heterochromatin binding domain in the C-terminal region. In immunofluorescence experiments, only affinity purified antibodies reactive with the C-terminal (amino acids 70–185) domain showed nucleoplasmic and heterochromatin staining, whereas N-terminal (amino acids 1–115) specific antibodies were nonreactive. In metaphase chromosome spreads, the C-terminal domain antibody was also localized to the centromeric regions of chromosomes. Association with centromeres was most prominent at anaphase and changed to a more generalized association with whole chromosomes in telophase. The cooccurrence of autoantibodies to centromere proteins and HP1 in certain autoimmune diseases might be a reflection of coordinated immune responses to these closely associated sets of proteins. Received: 8 August 1996; in revised form: 4 December 1996 / Accepted: 17 December 1996     

Ultrastructural changes in gametophytes of Acrostichum aureum L. cultured in different sodium chloride concentrations

Gametophytes cultured in solutions containing 0.0 to 0.7 % NaCl exhibited no change in ultra structural organization of chloroplasts. In 1.0% NaCl-grown gametophytes, there were thinner granal stacks, relatively larger spaces between granal thylakoidal membranes and larger plastoglobuli in the chloroplasts. These changes were accompanied by a decrease in photosynthesis. Cup shape, horseshoe shape, ring shape, and amoeboid mitochondria were observed in gametophytes grown in 0.0 to 0.7% NaCl. Only round mitochondria were observed in the gametophytes grown in 1.0 % NaCl. Mitochondria seemed to be more resistant to salt stress compared to chloroplasts. There was no direct relationship between changes in respiration rate and changes in mitochondrial shape among gametophytes grown in different NaCl concentrations. This revised version was published online in July 2006 with corrections to the Cover Date.     

Quartz crystal microbalance investigation of the interaction of bacterial toxins with ganglioside containing solid supported membranes

The binding of cholera toxin, tetanus toxin and pertussis toxin to ganglioside containing solid supported membranes has been investigated by quartz crystal microbalance measurements. The bilayers were prepared by fusion of phospholipid-vesicles on a hydrophobic monolayer of octanethiol chemisorbed on one gold electrode placed on the 5 MHz AT-cut quartz crystal. The ability of the gangliosides G_M1, G_M3, G_D1a, G_D1b, G_T1b and asialo-G_M1 to act as suitable receptors for the different toxins was tested by measuring the changes of quartz resonance frequencies. To obtain the binding constants of each ligand-receptor-couple Langmuir-isotherms were successfully fitted to the experimental adsorption isotherms. Cholera toxin shows a high affinity for G_M1 (K_a = 1.8 ⋅ 10⁸M^–1), a lower one for asialo-G_M1 (K_a = 1.0 ⋅ 10⁷ M^–1) and no affinity for G_M3. The C-fragment of tetanus toxin binds to ganglioside G_D1a, G_D1b and G_T1b containing membranes with similar affinity (K_a∼10⁶ M^–1), while no binding was observed with G_M3. Pertussis toxin binds to membranes containing the ganglioside G_D1a with a binding constant of K_a = 1.6 ⋅ 10⁶ M^–1, but only if large amounts (40 mol%) of G_D1a are present. The maximum frequency shift caused by the protein adsorption depends strongly on the molecular structure of the receptor. This is clearly demonstrated by an observed maximum frequency decrease of 99 Hz for the adsorption of the C-fragment of tetanus toxin to G_D1b. In contrast to this large frequency decrease, which was unexpectedly high with respect to Sauerbrey's equation, implying pure mass loading, a maximum shift of only 28 Hz was detected after adsorption of the C-fragment of tetanus toxin to G_D1a. Received: 14 January 1997 / Accepted: 15 April 1997     

Proteomic investigation of metabolic shift in mammalian cell culture.

Mammalian cells, under typical cultivation conditions, produce large quantities of lactate and ammonia that affect cell growth adversely and result in low cell concentration. Controlled nutrient feeding to maintain low concentrations of glucose and glutamine reduces metabolite production drastically, altering the metabolism of the cells. This metabolic shift results in higher cell concentration in continuous cultures and does not affect the specific productivity of the cells. We have taken a proteomics approach to investigate the differential protein expression with metabolic shift. Using two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS), we have found at least eight differentially expressed spots; two proteins were down-regulated, and the others were up-regulated with metabolic shift. These included metabolic enzymes, the brain form of phosphoglycerate mutase, which was down-regulated, and the precursor of the 23 kDa subunit of NADH-ubiquinone oxidoreductase, which was up-regulated. Another enzyme, the L1 isozyme of ubiquitin carboxyl-terminal hydrolase, which is involved in protein turnover and degradation, was also up-regulated in the metabolically altered cells. The remaining down-regulated spot had been identified as two isoforms of cytoplasmic actins, while three of the up-regulated spots were viral GAG polyproteins from various murine viruses. An unidentified protein was also up-regulated in the cells with altered metabolic state. This study shows the potential of using a proteomics approach in deciphering the intracellular changes in cells with physiological changes such as metabolism shift. The new insight into cell metabolism afforded by this analysis will greatly facilitate process optimization of continuous cell cultures.