Improved amber and opal suppressor tRNAs for incorporation of unnatural amino acids in vivo. Part 1: Minimizing misacylation

The incorporation of unnatural amino acids site-specifically is a valuable technique for structure–function studies, incorporation of biophysical probes, and determining protein–protein interactions. THG73 is an amber suppressor tRNA used extensively for the incorporation of >100 different residues in over 20 proteins, but under certain conditions THG73 is aminoacylated in vivo by endogenous aminoacyl-tRNA synthetase. Similar aminoacylation is seen with the Escherichia coli Asn amber suppressor tRNA, which has also been used to incorporate UAAs in many studies. We now find that the natural amino acid placed on THG73 is Gln. Since the E. coli GlnRS recognizes positions in the acceptor stem, we made several acceptor stem mutations in the second to fourth positions on THG73. All mutations reduce aminoacylation in vivo and allow for the selection of highly orthogonal tRNAs. To show the generality of these mutations, we created opal suppressor tRNAs that show less aminoacylation in Xenopus oocytes relative to THG73. We have created a library of Tetrahymena thermophila Gln amber suppressor tRNAs that will be useful for determining optimal suppressor tRNAs for use in other eukaryotic cells

Efficient non-degenerate two-photon excitation for fluorescence microscopy

Non-degenerate two-photon excitation (ND-TPE) has been explored in two-photon excitation microscopy. However, a systematic study of the efficiency of ND-TPE to guide the selection of fluorophore excitation wavelengths is missing. We measured the relative non-degenerate two-photon absorption cross-section (ND-TPACS) of several commonly used fluorophores (two fluorescent proteins and three small-molecule dyes) and generated 2-dimensional ND-TPACS spectra. We observed that the shape of a ND-TPACS spectrum follows that of the corresponding degenerate two-photon absorption cross-section (D-TPACS) spectrum, but is higher in magnitude. We found that the observed enhancements are higher than theoretical predictions.Published versio

The Role of Nonlinear Dynamics in Quantitative Atomic Force Microscopy

Various methods of force measurement with the Atomic Force Microscope (AFM) are compared for their ability to accurately determine the tip-surface force from analysis of the nonlinear cantilever motion. It is explained how intermodulation, or the frequency mixing of multiple drive tones by the nonlinear tip-surface force, can be used to concentrate the nonlinear motion in a narrow band of frequency near the cantilevers fundamental resonance, where accuracy and sensitivity of force measurement are greatest. Two different methods for reconstructing tip-surface forces from intermodulation spectra are explained. The reconstruction of both conservative and dissipative tip-surface interactions from intermodulation spectra are demonstrated on simulated data.Comment: 25 pages (preprint, double space) 7 figure

Phase imaging with intermodulation atomic force microscopy

Intermodulation atomic force microscopy (IMAFM) is a dynamic mode of atomic force microscopy (AFM) with two-tone excitation. The oscillating AFM cantilever in close proximity to a surface experiences the nonlinear tip-sample force which mixes the drive tones and generates new frequency components in the cantilever response known as intermodulation products (IMPs). We present a procedure for extracting the phase at each IMP and demonstrate phase images made by recording this phase while scanning. Amplitude and phase images at intermodulation frequencies exhibit enhanced topographic and material contrast.Comment: 6 pages, 6 page

Nocturnal position in the Panamanian Golden Frog, Atelopus zeteki (Anura, Bufonidae), with notes on fluorescent pigment tracking

La rana dorada de Panamá, Atelopus zeteki, una especie en peligro de extinción, habita en los ríos de los bosques lluviosos de mediana elevación del istmo panameño. Con el propósito de conocer mejor ésta especie para su conservación, decidimos determinar los sitios de descanso nocturno de estos animales, los cuales se caracterizan por ser activos durante el día. Suponíamos que los machos adultos y los juveniles podrían ocupar micro hábitat distintos durante la noche, basándose en las diferencias en tamaños y patrones de coloración. Los resultados presentados aquí demuestran que, al caer la noche los machos adultos trepan significativamente más alto que los juveniles y que la distancia de estos movimientos desde el punto inicial hasta la posición final de descanso es también significativamente diferente. Estos cambios de posición durante el día y la noche de los machos adultos podrían estar relacionados con una conducta antipredatoria. Finalmente este estudio demuestra que con el uso de pigmentos fluorescentes es posible rastrear anfibios en los bosques lluviosos con habitates húmedos a mojados a través de distancias cortas a moderadas de manera exitosa.The endangered Panamanian golden frog, Atelopus zeteki, is a stream dweller of middle elevation rain forests of the Panamanian isthmus. In order to better understand this species for conservation, we set out to determine the nocturnal whereabouts of this diurnally active animal. It was expected that adult males and juveniles might occupy different nocturnal microhabitats based on differences in size, coloration and patterning. Findings presented here demonstrate that adult males climb significantly higher than juveniles at night and that movement distances to final resting positions also significantly differed. This change in diurnal and nocturnal position in adult males may be related to predator vigilance and avoidance. Lastly, this study demonstrated that individual rain forest amphibians can be successfully tracked over short to moderate distances in humid to wet environments using fluorescent pigments

Single-Molecule Imaging of a Fluorescent Unnatural Amino Acid Incorporated Into Nicotinic Receptors

We report on the first, to our knowledge, successful detection of a fluorescent unnatural amino acid (fUAA), Lys(BODIPYFL), incorporated into a membrane protein (the muscle nicotinic acetylcholine receptor, nAChR) in a living cell. Xenopus oocytes were injected with a frameshift-suppressor tRNA, amino-acylated with Lys(BODIPYFL) and nAChR (α/β19′GGGU/γ/δ) mRNAs. We measured fluorescence from oocytes expressing nAChR β19′Lys(BODIPYFL), using time-resolved total internal reflection fluorescence microscopy. Under conditions of relatively low receptor density (<0.1 receptors/μm^2), we observed puncta with diffraction-limited profiles that were consistent with the point-spread function of our microscope. Furthermore, diffraction-limited puncta displayed step decreases in fluorescence intensity, consistent with single-molecule photobleaching. The puncta densities agreed with macroscopic ACh-induced current densities, showing that the fUAA was incorporated, and that receptors were functional. Dose-response relations for the nAChR β19′Lys(BODIPYFL) receptors were similar to those for wild-type receptors. We also studied nAChR β19′Lys(BODIPYFL) receptors labeled with α-bungarotoxin monoconjugated with Alexa488 (αBtxAlexa488). The nAChR has two αBtx binding sites, and puncta containing the Lys(BODIPYFL) labeled with αBtxAlexa488 yielded the expected three discrete photobleaching steps. We also performed positive control experiments with a nAChR containing enhanced green fluorescent protein in the γ-subunit M3-M4 loop, which confirmed our nAChR β19′Lys(BODIPYFL) measurements. Thus, we report on the cell-based single-molecule detection of nAChR β19′Lys(BODIPYFL)

Microvascular cutaneous coverage in wounds that expose the Achilles tendon: case report

Achilles tendon rupture, being one of the main tendon ruptures present, surgery being the most favourable option even taking into account complications such as infections and skin necrosis, it is necessary to develop techniques which help to reduce complications and increase benefits. The methods follow-up of 2 cases of patients with skin defects in the region of the Achilles tendon in patients who were treated at the "Luis Guillermo lbarra lbarra" national rehabilitation institute during the period 2020-2021 in the Traumatology service, treated with radial free flap forearm, using the description of the most used flaps in the literature. Results in both cases integrity of the free radial forearm flap in its entirety, functional recovery for walking, ability to put shoes back on, and without the need for reoperation, as well as a high level of satisfaction by patients. Conclusions if we are talking about an Achilles tendon rupture with a significant skin defect, the free radial forearm flap is an adequate option, if we are talking about a complete defect, the composite flap is the best option, remember that the decision on what type of flap will be used will depend on of the size of the lesion

Multiple plant traits shape the genetic basis of herbivore community assembly

1. Community genetics research has posited a genetic basis to the assembly of ecological communities. For arthropod herbivores in particular, there is strong support that genetic variation in host plants is a key factor shaping their diversity and composition. However, the specific plant phenotypes underlying herbivore responses remain poorly explored for most systems. 2. We address this knowledge gap by examining the influence of both genetic and phenotypic variation in a dominant host-plant species, Salix hookeriana, on its associated arthropod herbivore community in a common garden experiment. Specifically, we surveyed herbivore responses among five different arthropod feeding guilds to 26 distinct S. hookeriana genotypes. Moreover, we quantified the heritability of a suite of plant traits that determine leaf quality (e.g. phenolic compounds, trichomes, specific leaf area, C : N) and whole-plant architecture, to identify which traits best accounted for herbivore community responses to S. hookeriana genotype. 3. We found that total herbivore abundance and community composition differed considerably among S. hookeriana genotypes, with strong and independent responses of several species and feeding guilds driving these patterns. We also found that leaf phenolic chemistry displayed extensive heritable variation, whereas leaf physiology and plant architecture tended to be less heritable. Of these traits, herbivore responses were primarily associated with leaf phenolics and plant architecture; however, different herbivore species and feeding guilds were associated with different sets of traits. Despite our thorough trait survey, plant genotype remained a significant predictor of herbivore responses in most trait association analyses, suggesting that unmeasured host-plant characteristics and/or interspecific interactions were also contributing factors. 4. Taken together, our results support that the genetic basis of herbivore community assembly occurs through a suite of plant traits for different herbivore species and feeding guilds. Still, identifying these phenotypic mechanisms requires measuring a broad range of plant traits and likely further consideration of how these traits affect interspecific interactions.Fil: Barbour, Matthew A.. University Of British Columbia; CanadáFil: Rodriguez Cabal, Mariano Alberto. University Of British Columbia; Canadá. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Patagonia Norte. Instituto de Investigación en Biodiversidad y Medioambiente; Argentina. Universidad Nacional del Comahue; ArgentinaFil: Wu, Elizabeth T.. Humboldt State University; Estados UnidosFil: Julkunen Tiitto, Riitta. University of Eastern Finland; FinlandiaFil: Ritland, Carol E.. University Of British Columbia; CanadáFil: Miscampbell, Allyson E.. University Of British Columbia; CanadáFil: Jules, Erik S.. Humboldt State University; Estados UnidosFil: Crutsinger, Gregory M.. University Of British Columbia; Canad

C-BERST: Defining subnuclear proteomic landscapes at genomic elements with dCas9-APEX2 [preprint]

Mapping proteomic composition at distinct genomic loci and subnuclear landmarks in living cells has been a long-standing challenge. Here we report that dCas9-APEX2 Biotinylation at genomic Elements by Restricted Spatial Tagging (C-BERST) allows the rapid, unbiased mapping of proteomes near defined genomic loci, as demonstrated for telomeres and centromeres. By combining the spatially restricted enzymatic tagging enabled by APEX2 with programmable DNA targeting by dCas9, C-BERST has successfully identified nearly 50% of known telomere-associated factors and many known centromere-associated factors. We also identified and validated SLX4IP and RPA3 as telomeric factors, confirming C-BERST\u27s utility as a discovery platform. C-BERST enables the rapid, high-throughput identification of proteins associated with specific sequences, facilitating annotation of these factors and their roles in nuclear and chromosome biology