Organelle-dependent polyprotein designs enable stoichiometric expression of nitrogen fixation components targeted to mitochondria.

Introducing nitrogen fixation (nif  ) genes into eukaryotic genomes and targeting Nif components to mitochondria or chloroplasts is a promising strategy for engineering nitrogen-fixing plants. A prerequisite for achieving nitrogen fixation in crops is stable and stoichiometric expression of each component in organelles. Previously, we designed a polyprotein-based nitrogenase system depending on Tobacco Etch Virus protease (TEVp) to release functional Nif components from five polyproteins. Although this system satisfies the demand for specific expression ratios of Nif components in Escherichia coli, we encountered issues with TEVp cleavage of polyproteins targeted to yeast mitochondria. To overcome this obstacle, a version of the Nif polyprotein system was constructed by replacing TEVp cleavage sites with minimal peptide sequences, identified by knowledge-based engineering, that are susceptible to cleavage by the endogenous mitochondrial-processing peptidase. This replacement not only further reduces the number of genes required, but also prevents potential precleavage of polyproteins outside the target organelle. This version of the polyprotein-based nitrogenase system achieved levels of nitrogenase activity in E. coli, comparable to those observed with the TEVp-based polyprotein nitrogenase system. When applied to yeast mitochondria, stable and balanced expression of Nif components was realized. This strategy has potential advantages, not only for transferring nitrogen fixation to eukaryotic cells, but also for the engineering of other metabolic pathways that require mitochondrial compartmentalization.

Low Pneumoperitoneum Pressure Reduces Gas Embolism During Laparoscopic Liver Resection: A Randomized Controlled Trial.

OBJECTIVE: To compare the effect of low and standard pneumoperitoneal pressure (PP) on the occurrence of gas embolism during laparoscopic liver resection (LLR). BACKGROUND: LLR has an increased risk of gas embolism. Although animal studies have shown that low PP reduces the occurrence of gas embolism, clinical evidence is lacking. METHODS: This parallel, dual-arm, double-blind, randomized controlled trial included 141 patients undergoing elective LLR. Patients were randomized into standard ("S," 15 mm Hg; n = 70) or low ("L," 10 mm Hg; n = 71) PP groups. Severe gas embolism (≥ grade 3, based on the Schmandra microbubble method) was detected using transesophageal echocardiography and recorded as the primary outcome. Intraoperative vital signs and postoperative recovery profiles were also evaluated. RESULTS: Fewer severe gas embolism cases (n = 29, 40.8% vs n = 47, 67.1%, P = 0.003), fewer abrupt decreases in end-tidal carbon dioxide partial pressure, shorter severe gas embolism duration, less peripheral oxygen saturation reduction, and fewer increases in heart rate and lactate during gas embolization episodes was found in group L than in group S. Moreover, a higher arterial partial pressure of oxygen and peripheral oxygen saturation were observed, and fewer fluids and vasoactive drugs were administered in group L than in group S. In both groups, the distensibility index of the inferior vena cava negatively correlated with central venous pressure throughout LLR, and a comparable quality of recovery was observed. CONCLUSIONS: Low PP reduced the incidence and duration of severe gas embolism and achieved steadier hemodynamics and vital signs during LLR. Therefore, a low PP strategy can be considered a valuable choice for the future LLR.

Using synthetic biology to overcome barriers to stable expression of nitrogenase in eukaryotic organelles.

Engineering biological nitrogen fixation in eukaryotic cells by direct introduction of nif genes requires elegant synthetic biology approaches to ensure that components required for the biosynthesis of active nitrogenase are stable and expressed in the appropriate stoichiometry. Previously, the NifD subunits of nitrogenase MoFe protein from Azotobacter vinelandii and Klebsiella oxytoca were found to be unstable in yeast and plant mitochondria, respectively, presenting a bottleneck to the assembly of active MoFe protein in eukaryotic cells. In this study, we have delineated the region and subsequently a key residue, NifD-R98, from K. oxytoca that confers susceptibility to protease-mediated degradation in mitochondria. The effect observed is pervasive, as R98 is conserved among all NifD proteins analyzed. NifD proteins from four representative diazotrophs, but not their R98 variants, were observed to be unstable in yeast mitochondria. Furthermore, by reconstituting mitochondrial-processing peptidases (MPPs) from yeast, Oryza sativa, Nicotiana tabacum, and Arabidopsis thaliana in Escherichia coli, we demonstrated that MPPs are responsible for cleavage of NifD. These results indicate a pervasive effect on the stability of NifD proteins in mitochondria resulting from cleavage by MPPs. NifD-R98 variants that retained high levels of nitrogenase activity were obtained, with the potential to stably target active MoFe protein to mitochondria. This reconstitution approach could help preevaluate the stability of Nif proteins for plant expression and paves the way for engineering active nitrogenase in plant organelles.

Boosting Evaporative Cooling Performance with Microporous Aerogel.

Hydrogel-based evaporative cooling with a low carbon footprint is regarded as a promising technology for thermal regulation. Yet, the efficiency of hydrogel regeneration at night generally mismatches with vapor evaporation during the day, resulting in a limited cooling time span, especially in arid regions. In this work, we propose an efficient approach to improve hydrogel cooling performance, especially the cooling time span, with a bilayer structure, which comprises a bottom hydrogel layer and an upper aerogel layer. The microporous aerogel layer can reduce the saturation vapor density at the hydrogel surface by employing daytime radiative cooling, together with increased convective heat transfer resistance by thermal insulation, thus boosting the duration of evaporative cooling. Specifically, the microstructure of porous aerogel for efficient radiative cooling and vapor transfer is synergistically optimized with a cooling performance model. Results reveal that the proposed structure with a 2-mm-thick SiO2 aerogel can reduce the temperature by 1.4 °C, meanwhile extending the evaporative cooling time span by 11 times compared to a single hydrogel layer.

Application of RNA subcellular fraction estimation method to explore RNA localization regulation.

RNA localization is involved in multiple biological processes. Recent advances in subcellular fractionation-based sequencing approaches uncovered localization pattern on a global scale. Most of existing methods adopt relative localization ratios (such as ratios of separately normalized transcripts per millions of different subcellular fractions without considering the difference in total RNA abundances in different fractions), however, absolute ratios may yield different results on the preference to different cellular compartment. Experimentally, adding external Spike-in RNAs to different fractionation can be used to obtain absolute ratios. In addition, a spike-in independent computational approach based on multiple linear regression model can also be used. However, currently, no custom tool is available. To solve this problem, we developed a method called subcellular fraction abundance estimator to correctly estimate relative RNA abundances of different subcellular fractionations. The ratios estimated by our method were consistent with existing reports. By applying the estimated ratios for different fractions, we explored the RNA localization pattern in cell lines and also predicted RBP motifs that were associated with different localization patterns. In addition, we showed that different isoforms of same genes could exhibit distinct localization patterns. To conclude, we believed our tool will facilitate future subcellular fractionation-related sequencing study to explore the function of RNA localization in various biological problems.

Effects of MFHAS1 on cognitive impairment and dendritic pathology in the hippocampus of septic rats.

AIMS: To investigate the effects of malignant fibrous histiocytoma amplified sequence 1 (MFHAS1) on cognitive dysfunction, the expression of tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß and amyloid ß peptide (Aß) in the hippocampus, as well as dendritic pathology in the hippocampal CA1 region in sepsis-associated encephalopathy (SAE) rats. MAIN METHODS: The rats were randomly divided into four groups: 1) control group (subjected to sham surgery), 2) control plus Mfhas1 siRNA group (rats received intracerebroventricular injection of Mfhas1 siRNA after sham surgery), 3) CLP plus control siRNA group (rats received intracerebroventricular injection of control siRNA after cecal ligation and puncture (CLP)), 4) CLP plus Mfhas1 siRNA group (rats received intracerebroventricular injection of Mfhas1 siRNA after CLP). The learning and memory capabilities of the rats were examined by means of fear conditioning and Barnes maze test. The concentration of TNF-α and IL-1ß was determined by enzyme-linked immunosorbent assay. The efficiency of siRNA transfection, MFHAS1 and Aß expression were detected by Western blotting. Total branch lengths of pyramidal dendrites of the CA1 basilar trees and spine density were determined by Golgi staining. KEY FINDINGS: We observed that MFHAS1 knock-down by Mfhas1 siRNA intracerebroventricular injection could improve cognitive impairment, reduce the expression of TNF-α, IL-1ß and Aß in the hippocampus induced by CLP, and alleviate the dendritic spinal loss of the pyramidal neurons, as well as increase the dendritic branching of the CA1 basilar trees of septic rats. SIGNIFICANCE: MFHAS1 knock-down can alleviate cognitive impairment, neuroinflammation and dendritic spinal loss in SAE rats.