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1. Ultrafast laser surgery probe for sub-surface ablation to enable biomaterial injection in vocal folds

   https://doi.org/10.1038/s41598-022-24446-5  

   Andrus, Liam ; Jeon, Hamin ; Pawlowski, Michal ; Debord, Benoit ; Gerome, Frederic ; Benabid, Fetah ; Mau, Ted ; Tkaczyk, Tomasz ; Ben-Yakar, Adela ( November 2022 , Scientific Reports)

   Creation of sub-epithelial voids within scarred vocal folds via ultrafast laser ablation may help in localization of injectable therapeutic biomaterials towards an improved treatment for vocal fold scarring. Several ultrafast laser surgery probes have been developed for precise ablation of surface tissues; however, these probes lack the tight beam focusing required for sub-surface ablation in highly scattering tissues such as vocal folds. Here, we present a miniaturized ultrafast laser surgery probe designed to perform sub-epithelial ablation in vocal folds. The requirement of high numerical aperture for sub-surface ablation, in addition to the small form factor and side-firing architecture required for clinical use, made for a challenging optical design. An Inhibited Coupling guiding Kagome hollow core photonic crystal fiber delivered micro-Joule level ultrashort pulses from a high repetition rate fiber laser towards a custom-built miniaturized objective, producing a 1/e²focal beam radius of 1.12 ± 0.10 μm and covering a 46 × 46 μm²scan area. The probe could deliver up to 3.8 μJ pulses to the tissue surface at 40% transmission efficiency through the entire system, providing significantly higher fluences at the focal plane than were required for sub-epithelial ablation. To assess surgical performance, we performed ablation studies on freshly excised porcine hemi-larynges and found that largemore »area sub-epithelial voids could be created within vocal folds by mechanically translating the probe tip across the tissue surface using external stages. Finally, injection of a model biomaterial into a 1 × 2 mm²void created 114 ± 30 μm beneath the vocal fold epithelium surface indicated improved localization when compared to direct injection into the tissue without a void, suggesting that our probe may be useful for pre-clinical evaluation of injectable therapeutic biomaterials for vocal fold scarring therapy. With future developments, the surgical system presented here may enable treatment of vocal fold scarring in a clinical setting.

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2. Contributions of biomass-burning, urban, and biogenic emissions to the concentrations and light-absorbing properties of particulate matter in central Amazonia during the dry season

   https://doi.org/10.5194/acp-19-7973-2019  

   de Sá, Suzane S. ; Rizzo, Luciana V. ; Palm, Brett B. ; Campuzano-Jost, Pedro ; Day, Douglas A. ; Yee, Lindsay D. ; Wernis, Rebecca ; Isaacman-VanWertz, Gabriel ; Brito, Joel ; Carbone, Samara ; et al ( January 2019 , Atmospheric Chemistry and Physics)

   Abstract. Urbanization and deforestation have important impacts on atmosphericparticulate matter (PM) over Amazonia. This study presents observations andanalysis of PM₁ concentration, composition, and opticalproperties in central Amazonia during the dry season, focusing on theanthropogenic impacts. The primary study site was located 70 km downwind ofManaus, a city of over 2 million people in Brazil, as part of theGoAmazon2014/5 experiment. A high-resolution time-of-flight aerosol massspectrometer (AMS) provided data on PM₁ composition, and aethalometermeasurements were used to derive the absorption coefficient b_abs,BrC ofbrown carbon (BrC) at 370 nm. Non-refractory PM₁ mass concentrationsaveraged 12.2 µg m⁻³ at the primary study site, dominated byorganics (83 %), followed by sulfate (11 %). A decrease inb_abs,BrC was observed as the mass concentration of nitrogen-containingorganic compounds decreased and the organic PM₁ O:C ratio increased,suggesting atmospheric bleaching of the BrC components. The organic PM₁was separated into six different classes by positive-matrix factorization(PMF), and the mass absorption efficiency E_abs associated with eachfactor was estimated through multivariate linear regression ofb_abs,BrC on the factor loadings. The largest E_abs values wereassociated with urban (2.04±0.14 m² g⁻¹) and biomass-burning(0.82±0.04 to 1.50±0.07 m² g⁻¹) sources. Together, these sources contributed at least 80 % ofb_abs,BrCmore »while accounting for 30 % to 40 % of the organic PM₁ massconcentration. In addition, a comparison of organic PM₁ compositionbetween wet and dry seasons revealed that only part of the 9-foldincrease in mass concentration between the seasons can be attributed tobiomass burning. Biomass-burning factor loadings increased by 30-fold,elevating its relative contribution to organic PM₁ from about 10 % inthe wet season to 30 % in the dry season. However, most of the PM₁mass (>60 %) in both seasons was accounted for by biogenicsecondary organic sources, which in turn showed an 8-fold seasonalincrease in factor loadings. A combination of decreased wet deposition andincreased emissions and oxidant concentrations, as well as a positivefeedback on larger mass concentrations are thought to play a role in theobserved increases. Furthermore, fuzzy c-means clustering identified threeclusters, namely “baseline”, “event”, and “urban” to representdifferent pollution influences during the dry season. The baseline cluster,representing the dry season background, was associated with a mean massconcentration of 9±3 µg m⁻³. This concentration increasedon average by 3 µg m⁻³ for both the urban and the event clusters.The event cluster, representing an increased influence of biomass burningand long-range transport of African volcanic emissions, was characterized byremarkably high sulfate concentrations. The urban cluster, representing theinfluence of Manaus emissions on top of the baseline, was characterized byan organic PM₁ composition that differed from the other two clusters.The differences discussed suggest a shift in oxidation pathways as well asan accelerated oxidation cycle due to urban emissions, in agreement withfindings for the wet season.

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3. Elucidation of Sequence–Function Relationships for an Improved Biobutanol In Vivo Biosensor in E. coli

   Kim, Nancy M. ; Sinnott, Riley W. ; Rothschild, Lily N. ; Sandoval Nicholas R. ( October 2022 , Frontiers in bioengineering and biotechnology)

   Transcription factor (TF)–promoter pairs have been repurposed from native hosts to provide tools to measure intracellular biochemical production titer and dynamically control gene expression. Most often, native TF–promoter systems require rigorous screening to obtain desirable characteristics optimized for biotechnological applications. High-throughput techniques may provide a rational and less labor-intensive strategy to engineer user-defined TF–promoter pairs using fluorescence-activated cell sorting and deep sequencing methods (sort-seq). Based on the designed promoter library’s distribution characteristics, we elucidate sequence–function interactions between the TF and DNA. In this work, we use the sort-seq method to study the sequence–function relationship of a σ⁵⁴-dependent, butanol-responsive TF–promoter pair, BmoR-P_BMO derived from Thauera butanivorans, at the nucleotide level to improve biosensor characteristics, specifically an improved dynamic range. Activities of promoters from a mutagenized P_BMO library were sorted based on gfp expression and subsequently deep sequenced to correlate site-specific sequences with changes in dynamic range. We identified site-specific mutations that increase the sensor output. Double mutant and a single mutant, CA(129,130)TC and G(205)A, in P_BMO promoter increased dynamic ranges of 4-fold and 1.65-fold compared with the native system, respectively. In addition, sort-seq identified essential sites required for the proper function of the σ⁵⁴-dependent promoter biosensor in the context of themore »host. This work can enable high-throughput screening methods for strain development.« less
4. On the Impact of ²² Ne on the Pulsation Periods of Carbon–Oxygen White Dwarfs with Helium-dominated Atmospheres

   https://doi.org/10.3847/1538-4357/abdec4  

   Chidester, Morgan T. ; Timmes, F. X. ; Schwab, Josiah ; Townsend, Richard H. D. ; Farag, Ebraheem ; Thoul, Anne ; Fields, C. E. ; Bauer, Evan B. ; Montgomery, Michael H. ( March 2021 , The Astrophysical Journal)

   We explore changes in the adiabatic low-order g-mode pulsation periods of 0.526, 0.560, and 0.729M_⊙carbon–oxygen white dwarf models with helium-dominated envelopes due to the presence, absence, and enhancement of²²Ne in the interior. The observed g-mode pulsation periods of such white dwarfs are typically given to 6−7 significant figures of precision. Usually white dwarf models without²²Ne are fit to the observed periods and other properties. The rms residuals to the ≃150−400 s low-order g-mode periods are typically in the range ofσ_rms≲ 0.3 s, for a fit precision ofσ_rms/P≲ 0.3%. We find average relative period shifts of ΔP/P≃ ±0.5% for the low-order dipole and quadrupole g-mode pulsations within the observed effective temperature window, with the range of ΔP/Pdepending on the specific g-mode, abundance of²²Ne, effective temperature, and the mass of the white dwarf model. This finding suggests a systematic offset may be present in the fitting process of specific white dwarfs when²²Ne is absent. As part of the fitting processes involves adjusting the composition profiles of a white dwarf model, our study on the impact of²²Ne can provide new inferences on the derived interior mass fraction profiles. We encourage routinely including²²Ne mass fraction profiles, informed by stellar evolution models, to futuremore »generations of white dwarf model-fitting processes.

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5. Identification of SLAC1 anion channel residues required for CO ₂ /bicarbonate sensing and regulation of stomatal movements

   https://doi.org/10.1073/pnas.1807624115  

   Zhang, Jingbo ; Wang, Nuo ; Miao, Yinglong ; Hauser, Felix ; McCammon, J. Andrew ; Rappel, Wouter-Jan ; Schroeder, Julian I. ( October 2018 , Proceedings of the National Academy of Sciences)

   Increases in CO₂concentration in plant leaves due to respiration in the dark and the continuing atmospheric [CO₂] rise cause closing of stomatal pores, thus affecting plant–water relations globally. However, the underlying CO₂/bicarbonate (CO₂/HCO₃⁻) sensing mechanisms remain unknown. [CO₂] elevation in leaves triggers stomatal closure by anion efflux mediated via the SLAC1 anion channel localized in the plasma membrane of guard cells. Previous reconstitution analysis has suggested that intracellular bicarbonate ions might directly up-regulate SLAC1 channel activity. However, whether such a CO₂/HCO₃⁻regulation of SLAC1 is relevant for CO₂control of stomatal movements in planta remains unknown. Here, we computationally probe for candidate bicarbonate-interacting sites within the SLAC1 anion channel via long-timescale Gaussian accelerated molecular dynamics (GaMD) simulations. Mutations of two putative bicarbonate-interacting residues, R256 and R321, impaired the enhancement of the SLAC1 anion channel activity by CO₂/HCO₃⁻inXenopusoocytes. Mutations of the neighboring charged amino acid K255 and residue R432 and the predicted gate residue F450 did not affect HCO₃⁻regulation of SLAC1. Notably, gas-exchange experiments withslac1-transformed plants expressing mutated SLAC1 proteins revealed that the SLAC1 residue R256 is required for CO₂regulation of stomatal movements in planta, but not for abscisic acid (ABA)-induced stomatal closing. Patch clamp analyses of guard cells show that activation ofmore »S-type anion channels by CO₂/HCO₃⁻, but not by ABA, was impaired, indicating the relevance of R256 for CO₂signal transduction. Together, these analyses suggest that the SLAC1 anion channel is one of the physiologically relevant CO₂/HCO₃⁻sensors in guard cells.

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