Engineering luminopsins with improved coupling efficiencies.

Significance: Luminopsins (LMOs) are bioluminescent-optogenetic tools with a luciferase fused to an opsin that allow bimodal control of neurons by providing both optogenetic and chemogenetic access. Determining which design features contribute to the efficacy of LMOs will be beneficial for further improving LMOs for use in research. Aim: We investigated the relative impact of luciferase brightness, opsin sensitivity, pairing of emission and absorption wavelength, and arrangement of moieties on the function of LMOs. Approach: We quantified efficacy of LMOs through whole cell patch clamp recordings in HEK293 cells by determining coupling efficiency, the percentage of maximum LED induced photocurrent achieved with bioluminescent activation of an opsin. We confirmed key results by multielectrode array recordings in primary neurons. Results: Luciferase brightness and opsin sensitivity had the most impact on the efficacy of LMOs, and N-terminal fusions of luciferases to opsins performed better than C-terminal and multi-terminal fusions. Precise paring of luciferase emission and opsin absorption spectra appeared to be less critical. Conclusions: Whole cell patch clamp recordings allowed us to quantify the impact of different characteristics of LMOs on their function. Our results suggest that coupling brighter bioluminescent sources to more sensitive opsins will improve LMO function. As bioluminescent activation of opsins is most likely based on Förster resonance energy transfer, the most effective strategy for improving LMOs further will be molecular evolution of luciferase-fluorescent protein-opsin fusions.

Efficient opto- and chemogenetic control in a single molecule driven by FRET-modified bioluminescence.

Significance: Bioluminescent optogenetics (BL-OG) offers a unique and powerful approach to manipulate neural activity both opto- and chemogenetically using a single actuator molecule (a LuMinOpsin, LMO). Aim: To further enhance the utility of BL-OG by improving the efficacy of chemogenetic (bioluminescence-driven) LMO activation. Approach: We developed novel luciferases optimized for Förster resonance energy transfer when fused to the fluorescent protein mNeonGreen, generating bright bioluminescent (BL) emitters spectrally tuned to Volvox Channelrhodopsin 1 (VChR1). Results: A new LMO generated from this approach (LMO7) showed significantly stronger BL-driven opsin activation compared to previous and other new variants. We extensively benchmarked LMO7 against LMO3 (current standard) and found significantly stronger neuronal activity modulation ex vivo and in vivo, and efficient modulation of behavior. Conclusions: We report a robust new option for achieving multiple modes of control in a single actuator and a promising engineering strategy for continued improvement of BL-OG.

Engineering luminopsins with improved coupling efficiencies.

Significance: Luminopsins (LMOs) are bioluminescent-optogenetic tools with a luciferase fused to an opsin that allow bimodal control of neurons by providing both optogenetic and chemogenetic access. Determining which design features contribute to the efficacy of LMOs will be beneficial for further improving LMOs for use in research. Aim: We investigated the relative impact of luciferase brightness, opsin sensitivity, pairing of emission and absorption wavelength, and arrangement of moieties on the function of LMOs. Approach: We quantified efficacy of LMOs through whole cell patch clamp recordings in HEK293 cells by determining coupling efficiency, the percentage of maximum LED induced photocurrent achieved with bioluminescent activation of an opsin. We confirmed key results by multielectrode array (MEAs) recordings in primary neurons. Results: Luciferase brightness and opsin sensitivity had the most impact on the efficacy of LMOs, and N-terminal fusions of luciferases to opsins performed better than C-terminal and multi-terminal fusions. Precise paring of luciferase emission and opsin absorption spectra appeared to be less critical. Conclusions: Whole cell patch clamp recordings allowed us to quantify the impact of different characteristics of LMOs on their function. Our results suggest that coupling brighter bioluminescent sources to more sensitive opsins will improve LMO function. As bioluminescent activation of opsins is most likely based on Förster resonance energy transfer (FRET), the most effective strategy for improving LMOs further will be molecular evolution of luciferase-fluorescent protein-opsin fusions.

A New Highly Efficient Molecule for Both Optogenetic and Chemogenetic Control Driven by FRET Amplification of BioLuminescence.

SIGNIFICANCE: Bioluminescent optogenetics (BL-OG) offers a unique and powerful approach to manipulate neural activity both opto- and chemogenetically using a single actuator molecule (a LuMinOpsin, LMO). AIM: To further enhance the utility of BL-OG by improving the efficacy of chemogenetic (bioluminescence-driven) LMO activation. APPROACH: We developed novel luciferases optimized for Forster resonance energy transfer (FRET) when fused to the fluorescent protein mNeonGreen, generating bright bioluminescent (BL) emitters spectrally tuned to Volvox Channelrhodopsin 1 (VChR1). RESULTS: A new LMO generated from this approach (LMO7) showed significantly stronger BL-driven opsin activation compared to previous and other new variants. We extensively benchmarked LMO7 against LMO3 (current standard), and found significantly stronger neuronal activity modulation ex vivo and in vivo, and efficient modulation of behavior. CONCLUSIONS: We report a robust new option for achieving multiple modes of control in a single actuator, and a promising engineering strategy for continued improvement of BL-OG.

Improved Locomotor Recovery in a Rat Model of Spinal Cord Injury by BioLuminescent-OptoGenetic (BL-OG) Stimulation with an Enhanced Luminopsin.

Irrespective of the many strategies focused on dealing with spinal cord injury (SCI), there is still no way to restore motor function efficiently or an adequate regenerative therapy. One promising method that could potentially prove highly beneficial for rehabilitation in patients is to re-engage specific neuronal populations of the spinal cord following SCI. Targeted activation may maintain and strengthen existing neuronal connections and/or facilitate the reorganization and development of new connections. BioLuminescent-OptoGenetics (BL-OG) presents an avenue to non-invasively and specifically stimulate neurons; genetically targeted neurons express luminopsins (LMOs), light-emitting luciferases tethered to light-sensitive channelrhodopsins that are activated by adding the luciferase substrate coelenterazine (CTZ). This approach employs ion channels for current conduction while activating the channels through treatment with the small molecule CTZ, thus allowing non-invasive stimulation of all targeted neurons. We previously showed the efficacy of this approach for improving locomotor recovery following severe spinal cord contusion injury in rats expressing the excitatory luminopsin 3 (LMO3) under control of a pan-neuronal and motor-neuron-specific promoter with CTZ applied through a lateral ventricle cannula. The goal of the present study was to test a new generation of LMOs based on opsins with higher light sensitivity which will allow for peripheral delivery of the CTZ. In this construct, the slow-burn Gaussia luciferase variant (sbGLuc) is fused to the opsin CheRiff, creating LMO3.2. Taking advantage of the high light sensitivity of this opsin, we stimulated transduced lumbar neurons after thoracic SCI by intraperitoneal application of CTZ, allowing for a less invasive treatment. The efficacy of this non-invasive BioLuminescent-OptoGenetic approach was confirmed by improved locomotor function. This study demonstrates that peripheral delivery of the luciferin CTZ can be used to activate LMOs expressed in spinal cord neurons that employ an opsin with increased light sensitivity.

Biochar-based compost: a bibliometric and visualization analysis.

The co-application of biochar compost as organic amendment for crop production and soil remediation has gained momentum due to their positive effect on plant growth and soil quality improvement. The application of biochar and compost which are green and cost-effective soil remediators would promote the availability and distribution of food, planetary conservation, alleviate poverty, and enhance the attainment of Sustainable Millennium Development Goals (SDGs). A bibliometric analysis was conducted to overview research on biochar compost from 2011 to 2021. Two hundred and fifty-four research papers were retrieved from the Scopus database and analyzed using VOS viewer. Analysis revealed that 217 (85.43%) were articles, 21 (8.27%) were conference papers, and 12 (4.72%) were review papers. The results showed an exponential increase in the number of publications. The most productive countries in the investigated subject were China (49), followed by USA (36), Australia (29), Italy (28), Germany (25), and Indonesia (20). After the search terms, 'soil,' which had links with keywords like 'soil fertility,' 'soil quality,' 'soil pollution,' 'phosphorus,' 'nitrogen,' 'maize,' 'greenhouse gas,' etc., had the highest occurrences (94). From the results of the current hotspot research in the field, the effect of biochar-compost mixture and co-composted biochar on soil remediation is currently being studied by several researchers. Biochar and compost incorporation in soil reduce the uptake of pollutants by plants which consequently increase essential nutrients for plant and soil productivity.

● Biochar-compost research has been increasing for the past eleven years● China is most productive country in biochar and compost application●Biochar-compost and co-composted biochar are effective elixirs for soil amendment and nutritional enrichment.

Synthesis and Structural Studies of Manganese Ferrite and Zinc Ferrite Nanocomposites and Their Use as Photoadsorbents for Indigo Carmine and Methylene Blue Dyes.

Solar-moderated adsorptions of indigo carmine and methylene blue dyes were investigated using manganese and zinc ferrite capped with biochar prepared from the root of Chromolaena odorata. TEM micrograph of the as-prepared manganese ferrite nanocomposites (MnFe2O4@BC) revealed octagonally shaped particles with an average size of 42.64 nm while the zinc ferrite nanocomposite (ZnFe2O4@BC) micrograph revealed mixtures of rod- and cone-shaped particles with an average size of 43.82 nm. Biochar capping of MnFe2O4@BC reduced the band gap from 3.63 to 2.08 eV. The nanocomposite surface areas were 197.64 and 92.14 m2/g for MnFe2O4@BC and ZnFe2O4@BC, respectively. Photoadsorption of the as-prepared nanocomposites showed that 10 mg of ZnFe2O4@BC effectively removed 69.07 and 98.60% of 70 mg/L indigo carmine and methylene blue dyes while MnFe2O4@BC removed 77.65 and 94.83% of indigo carmine and methylene blue dyes after 2 h of equilibration under visible light irradiation, respectively. The nonlinear form of the Langmuir isotherm had a better approximation to the experimental solid-phase concentration (q e) for the adsorption of indigo carmine dye using both nanocomposites. In contrast, the linear form gave a better goodness-of-fit for the adsorptions of methylene blue dye. The manganese ferrite (MnFe2O4@BC) and zinc ferrite (ZnFe2O4@BC) nanocomposites showed no inhibition of Escherichia coli and Staphylococcus aureus, which indicates that they could be used for both biological and environmental applications.

Multifunctional Magnetic Oxide Nanoparticle (MNP) Core-Shell: Review of Synthesis, Structural Studies and Application for Wastewater Treatment.

The demand for water is predicted to increase significantly over the coming decades; thus, there is a need to develop an inclusive wastewater decontaminator for the effective management and conservation of water. Magnetic oxide nanocomposites have great potentials as global and novel remediators for wastewater treatment, with robust environmental and economic gains. Environment-responsive nanocomposites would offer wide flexibility to harvest and utilize massive untapped natural energy sources to drive a green economy in tandem with the United Nations Sustainable Development Goals. Recent attempts to engineer smart magnetic oxide nanocomposites for wastewater treatment has been reported by several researchers. However, the magnetic properties of superparamagnetic nanocomposite materials and their adsorption properties nexus as fundamental to the design of recyclable nanomaterials are desirable for industrial application. The potentials of facile magnetic recovery, ease of functionalization, reusability, solar responsiveness, biocompatibility and ergonomic design promote the application of magnetic oxide nanocomposites in wastewater treatment. The review makes a holistic attempt to explore magnetic oxide nanocomposites for wastewater treatment; futuristic smart magnetic oxides as an elixir to global water scarcity is expounded. Desirable adsorption parameters and properties of magnetic oxides nanocomposites are explored while considering their fate in biological and environmental media.

Data for experimental and calculated values of the adsorption of Pb(II) and Cr(VI) on APTES functionalized magnetite biochar using Langmuir, Freundlich and Temkin equations.

Adsorption isotherms are indispensable tools for the description of sorption processes of pollutants on adsorbents. The closeness of the equilibrium concentration (qe) to the calculated solid phase concentration (qecal) of the adsorbate, together with the co-efficient of determination (R2) and associated errors are important in determining the best goodness-of-fit model. In this work we have investigated the adsorption of Pb(II) and Cr(VI) on a nanocomposite that was prepared using magnetite nanoparticles capped with locally prepared biochar and functionalized using 3-(aminopropyl) triethoxysilane (APTES) at 3 different temperatures. Detailed discussion of data can be found in DOI:10.1016/j.micromeso.2020.110573. The sorption processes were equally analyzed utilizing both the linear and non-linear forms of Langmuir, Freundlich and Temkin equations.

Intra- and interspecific chromosome polymorphisms in cultivated Cichorium L. species (Asteraceae).

Endive (Cichorium endivia L.) and chicory (C. intybus L.) both have 2n = 18, but until now, there has been no detailed karyomorphological characterization. The present work evaluated five accessions of each species using FISH with rDNA probes and fluorochrome staining with CMA and DAPI. Both species presented distinct banding patterns after fluorochrome staining: while endive had proximal CMA(++)/DAPI(-) bands in the short arms of pairs 1, 2 and 3, chicory had proximal CMA-positive bands in chromosomes 1 and 3 and interstitial in the short arm of chromosome 8. Among endive accessions, FISH procedures revealed conserved position and number of 5S and 45S rDNA sites (two and three pairs, respectively), associated with the CMA-positive bands. Notwithstanding, polymorphisms were detected within chicory accessions regarding the number and the distribution of rDNA sites in relation to the most frequent karyotype (two pairs with 45S and one with 5S rDNA). The karyological markers developed allowed karyotypic differentiation between both species, uncovering peculiarities in the number and position of rDNA sites, which suggest chromosome rearrangements, such as translocations in chicory cultivars. The interspecific and intraspecific polymorphisms observed emphasize the potential of karyomorphological evaluations, helping our understanding of the relationships and evolution of the group.

Panitumumab combined with irinotecan for patients with KRAS wild-type metastatic colorectal cancer refractory to standard chemotherapy: a GERCOR efficacy, tolerance, and translational molecular study.

BACKGROUND: The purpose of this study was to evaluate the combination of panitumumab and irinotecan in patients with KRAS wild-type metastatic colorectal cancer refractory to standard chemotherapy (oxaliplatin, fluoropyrimidines-irinotecan and bevacizumab). PATIENTS AND METHODS: KRAS status was first determined locally but subsequent validation of KRAS status and additional screenings (rare KRAS, NRAS, BRAF mutations and EGFR copy number) were centrally assessed. Patients received panitumumab (6 mg/kg) and irinotecan (180 mg/m²) every 2 weeks. RESULTS: Sixty-five eligible patients were analyzed. The objective response rate (ORR) was 29.2% [95% confidence interval (95% CI) 18.2-40.3]. Median progression-free and overall survivals were 5.5 and 9.7 months, respectively. Most frequent grade 3/4 toxic effects were skin 32.3%, diarrhea 15.4% and neutropenia 12.3%. Tissue samples were available for 60 patients. For the confirmed KRAS wild-type population codon 12 or 13 mutation (n = 54), ORR was 35.2% (95% CI 22.4.1-47.9). Thirteen patients had a NRAS, a BRAF or a rare KRAS mutation, and no tumor response was observed in this subgroup when compared with 46.3% (95% CI 31.1-61.6) ORR in the subgroup of 41 patients with no identified mutation. CONCLUSION: Panitumumab and irinotecan is an active third-line regimen in a well-defined population based on biomarkers. ClinicalTrials.gov Identifier NCT00655499.