<i>Lactobacillus acidophilus</i> attenuated TBI-mediated increase of MLCP.

<p>The interaction of <i>Lactobacillus acidophilus</i> and time on MLCP protein concentrations was not significant. (A) ELISA analysis showed that the concentrations of MLCP in intestinal smooth muscle were significantly increased after TBI, **<i>P</i> < 0.01 compared with control. <i>Lactobacillus acidophilus</i> significantly attenuated TBI-mediated increase of MLCP, #<i>P</i> < 0.05 compared with TBI. (B) Immunohistochemistry analysis showed that marked increases in MLCP were observed in intestinal smooth muscle after TBI, and treatment with <i>Lactobacillus acidophilus</i> significantly attenuated TBI-mediated increases of MLCP.</p

Nitrogen Isotope Composition of Thermally Produced NO_<i>x</i> from Various Fossil-Fuel Combustion Sources

The nitrogen stable isotope composition of NO_<i>x</i> (δ¹⁵N-NO_<i>x</i>) may be a useful indicator for NO_<i>x</i> source partitioning, which would help constrain NO_<i>x</i> source contributions in nitrogen deposition studies. However, there is large uncertainty in the δ¹⁵N-NO_<i>x</i> values for anthropogenic sources other than on-road vehicles and coal-fired energy generating units. To this end, this study presents a broad analysis of δ¹⁵N-NO_<i>x</i> from several fossil-fuel combustion sources that includes: airplanes, gasoline-powered vehicles not equipped with a three-way catalytic converter, lawn equipment, utility vehicles, urban buses, semitrucks, residential gas furnaces, and natural-gas-fired power plants. A relatively large range of δ¹⁵N-NO_<i>x</i> values was measured from −28.1‰ to 8.5‰ for individual exhaust/flue samples that generally tended to be negative due to the kinetic isotope effect associated with thermal NO_<i>x</i> production. A negative correlation between NO_<i>x</i> concentrations and δ¹⁵N-NO_<i>x</i> for fossil-fuel combustion sources equipped with selective catalytic reducers was observed, suggesting that the catalytic reduction of NO_<i>x</i> increases δ¹⁵N-NO_<i>x</i> values relative to the NO_<i>x</i> produced through fossil-fuel combustion processes. Combining the δ¹⁵N-NO_<i>x</i> measured in this study with previous published values, a δ¹⁵N-NO_<i>x</i> regional and seasonal isoscape was constructed for the contiguous U.S., which demonstrates seasonal and regional importance of various NO_<i>x</i> sources

<i>P</i>-values of a two-way ANOVA for the effects of <i>Lactobacillus acidophilus</i>, time, and their interactions on the ten variables (N = 90).

<p>Note: MLCK: myosin light chain kinase; MLCP: myosin light chain phosphatase; PKC: protein kinase C; ICC numbers: the numbers of interstitial cells of Cajal.</p><p>*indicates a significant difference (<i>P</i> < 0.05)</p><p>**indicates a highly significant difference (<i>P</i> < 0.01).</p><p><i>P</i>-values of a two-way ANOVA for the effects of <i>Lactobacillus acidophilus</i>, time, and their interactions on the ten variables (N = 90).</p

<i>Lactobacillus acidophilus</i> restored the impaired ICC networks mediated by TBI.

<p>The ICC networks and numbers were detected under fluorescence microscope (400×) by immunofluorescence in ileal tissue sections. The interaction of <i>Lactobacillus acidophilus</i> and time on ICC numbers was not significant. (A) Low-density of ICC networks was observed after TBI compared with control. <i>Lactobacillus acidophilus</i> significantly restored TBI-mediated disruption of ICC networks. (B) The reduction of ICC numbers was also observed after TBI, **<i>P</i> < 0.01 compared with control. <i>Lactobacillus acidophilus</i> significantly restored TBI-mediated reduction of ICC numbers, #<i>P</i> < 0.05 compared with TBI.</p

Standardized Iterative Genome Editing Method for Escherichia coli Based on CRISPR-Cas9

The introduction of complex biosynthetic pathways into the hosts’ chromosomes is gaining attention with the development of synthetic biology. While CRISPR-Cas9 has been widely employed for gene knock-in, the process of multigene insertion remains cumbersome due to laborious and empirical gene cloning procedures. To address this, we devised a standardized iterative genome editing system for Escherichia coli, harnessing the power of CRISPR-Cas9 and MetClo assembly. This comprehensive toolkit comprises two fundamental elements based on the Golden Gate standard for modular assembly of sgRNA or CRISPR arrays and donor DNAs. We achieved a gene insertion efficiency of up to 100%, targeting a single locus. Expression of tracrRNA using a strong promoter enhances multiplex genomic insertion efficiency to 7.3%, compared with 0.76% when a native promoter is used. To demonstrate the robust capabilities of this genome editing toolbox, we successfully integrated 5–10 genes from the coenzyme B12 biosynthetic pathway ranging from 5.3 to 8 Kb in length into the chromosome of E. coli chassis cells, resulting in 14 antibiotic-free, plasmid-free producers. Following an extensive screening process involving genes from diverse sources, cistronic design modifications, and chromosome repositioning, we obtained a recombinant strain yielding 1.49 mg L–1 coenzyme B12, the highest known titer achieved by using E. coli as the producer. Illuminating its user-friendliness, this genome editing system is an exceedingly versatile tool for expediently integrating complex biosynthetic pathway genes into hosts’ genomes, thus facilitating pathway optimization for chemical production

<i>Lactobacillus acidophilus</i> improved the morphology of villus.

<p>The intestinal sections from control mice presented intact structures with complete intestinal mucosae and villi (3A, 3D and 3G). TBI caused abnormal intestinal wall morphology and damaged the intestinal villi and structural integrity (3B, 3E and 3H). <i>Lactobacillus acidophilus</i> recovered intestinal mucosae and restructured villi (3C, 3F and 3I).</p

<i>Lactobacillus acidophilus</i> attenuated TBI-mediated decrease in levels of phospho-MYPT1 and increases in PKC protein concentrations.

<p>The interaction of <i>Lactobacillus acidophilus</i> and time on phospho-MYPT1 concentrations was not significant. (A and B) A marked decreases in phospho-MYPT1 were observed in intestinal smooth muscle after TBI, **<i>P</i> < 0.01 compared with control. Treatment with <i>Lactobacillus acidophilus</i> significantly attenuated TBI-mediated inhibition of MYPT1 phosphorylation, ##<i>P</i> < 0.01 compared with TBI. The interaction of <i>Lactobacillus acidophilus</i> and time on PKC protein concentrations was significant. (C) ELISA analysis showed that the concentrations of PKC were increased in intestinal smooth muscle after TBI, *<i>P</i> < 0.05 compared with control. <i>Lactobacillus acidophilus</i> significantly attenuated TBI-mediated induction of PKC protein concentrations, #<i>P</i> < 0.05 compared with TBI.</p

<i>Lactobacillus acidophilus</i> improved the contractile activity of intestinal smooth muscle impaired by TBI.

<p>Ninety C57BL/6 mice were randomly divided into three groups including control, TBI and TBI + <i>Lactobacillus acidophilus</i> groups. The interaction of <i>Lactobacillus acidophilus</i> and time on intestinal contractile activity was not significant. (A) The contractile activity was determined by histologic and physiologic analyses. (B) The average contractile amplitude was decreased after TBI, **<i>P</i> < 0.01 compared with control. <i>Lactobacillus acidophilus</i> could increase in contractile amplitude, #<i>P</i> < 0.05 compared with TBI. (C and D) The contractile frequency and tension were also decreased after TBI, **<i>P</i> < 0.01 compared with control. <i>Lactobacillus acidophilus</i> significantly attenuated TBI-mediated decreases of contractile frequency and tension, #<i>P</i> < 0.05 compared with TBI. (E) The intestinal transit rate was decreased after TBI, **<i>P</i> < 0.01 compared with control. <i>Lactobacillus acidophilus</i> significantly attenuated TBI-mediated decrease of intestinal transit rate, ##<i>P</i> < 0.01 compared with TBI.</p

<i>Lactobacillus acidophilus</i> attenuated TBI-mediated reduction of MLCK.

<p>The interaction of <i>Lactobacillus acidophilus</i> and time on MLCK protein concentrations was not significant. (A) ELISA analysis showed that the concentrations of MLCK in intestinal smooth muscle were significantly decreased after TBI, *<i>P</i> < 0.05 compared with control. <i>Lactobacillus acidophilus</i> significantly attenuated TBI-mediated reduction of MLCK concentrations, ##<i>P</i> < 0.01 compared with TBI. (B) Immunohistochemistry analysis showed that marked decreases in MLCK were observed in intestinal smooth muscle after TBI, and treatment with <i>Lactobacillus acidophilus</i> significantly attenuated TBI-mediated inhibition of MLCK.</p

<i>Lactobacillus acidophilus</i> could increase the MLC₂₀ phosphorylation.

<p>The interaction of <i>Lactobacillus acidophilus</i> and time on MLC₂₀ phosphorylation was not significant. (A and B) The levels of phospho-MLC₂₀ in intestinal smooth muscle were significantly decreased after TBI, *<i>P</i> < 0.05 compared with control. Treating with <i>Lactobacillus acidophilus</i> attenuated TBI-mediated inhibition of MLC₂₀ phosphorylation, #<i>P</i> < 0.05 compared with TBI. (C) The marked decreases in immunoreactivity for phospho-MLC₂₀ were observed in intestinal smooth muscle after TBI, and treatment with <i>Lactobacillus acidophilus</i> significantly attenuated TBI-mediated inhibition of MLC₂₀ phosphorylation.</p