Bacillus subtilis strain 168 is commonly used as a host to produce recombinant proteins and as a chassis for bio-based chemicals production. However, its preferred nitrogen source is organic nitrogen, which greatly increases production costs. In this study, adaptive laboratory evolution (ALE) was used to improve B. subtilis 168 growth using NH4Cl as the sole nitrogen source. The cell density (OD600) of a mutant strain LJ-3 was 208.7% higher than that of the original strain. We also optimized the metal ions in the medium and this resulted in a further increase in growth rate by 151.3%. Reintroduction of the sfp+ gene into strain LJ-3 led to the LJ-31 clone, which restored LJ-3’s ability to synthesize surfactin. The fermentation system was optimized (C/N, aeration, pH) in a 5 L bioreactor. Dry cell weight of 7.4 g/L and surfactin concentration of 4.1 g/L were achieved using the optimized mineral salt medium after 22 h of batch fermentation with a YP/S value of 0.082 g/g and a YP/X of 0.55 g/g. HPLC analysis identified the surfactin isoforms produced by strain LJ-31 in the synthetic medium as C13-surfactin 13.3%, C14-surfactin 44.02%, and C15-surfactin 32.79%. Hence, the variant LJ-3 isolated by ALE is a promising engineering chassis for efficient and cost-effective production of a variety of metabolites.

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Bones at different anatomical locations vary dramatically in size. For example, human femurs are 20-fold longer than the phalanges in the fingers and toes. The mechanisms responsible for these size differences are poorly understood. Bone elongation occurs at the growth plates and advances rapidly in early life but then progressively slows due to a developmental program termed "growth plate senescence." This developmental program includes declines in cell proliferation and hypertrophy, depletion of cells in all growth plate zones, and extensive underlying changes in the expression of growth-regulating genes.Here, we show evidence that these functional, structural, and molecular senescent changes occur earlier in the growth plates of smaller bones (metacarpals, phalanges) than in the growth plates of larger bones (femurs, tibias) and that this differential aging contributes to the disparities in bone length. We also show evidence that the molecular mechanisms that underlie the differential aging between different bones involve modulation of critical paracrine regulatory pathways, including insulin-like growth factor (Igf), bone morphogenetic protein (Bmp), and Wingless and Int-1 (Wnt) signaling. Taken together, the findings reveal that the striking disparities in the lengths of different bones, which characterize normal mammalian skeletal proportions, is achieved in part by modulating the progression of growth plate senescence.

Cellular architectural proteins often participate in organ development and maintenance.Although functional decay of some of these proteins during aging is known, the cell-type specific developmental role and the cause and consequence of their subsequent decay remain to be established especially in mammals. By studying lamins, the nuclear structural proteins, we demonstrate that lamin-B1 functions specifically in the thymic epithelial cells (TECs) for proper thymus organogenesis. An upregulation of proinflammatory cytokines in the intra-thymic myeloid immune cells during aging accompanies a gradual reduction of adult TEC lamins-B1.These cytokines cause adult TEC senescence and lamin-B1 reduction. We identify 17 adult TEC subsets and show that TEC lamin-B1 maintains the composition of these TECs. Lamin-B1 supports the expression of TEC genes needed for maintaining adult thymic architecture and function. Thus, structural proteins involved in organ building and maintenance can undergo inflammation-driven decay which can in turn contribute to age-associated organ degeneration. mTECs. We mixed an adherent keratin-negative cell line (RAW264.7 from ATCC #TIB-71) applying FACS to isolate macrophages in thymuses dissected from 2, 6, 12, 16, and 20-mon-old 0 Chromium and then single-cell RNA-seq libraries were generated using the Chromium Single Cell 3' Reagent Kit (10X Genomics) by the core facility at the Embryology Department of Carnegie Institute for Science. Briefly, TEC single-cell suspension (~1,000 cells per 1 µl PBS) was mixed thoroughly with Single Cell 3' gel beads and partitioning oil into a Single Cell 3' Chip (10X genomics) following the recommended protocol for the Chromium Single Cell 3' Reagent Kit (v2 Chemistry). RNA transcripts of single cells were uniquely barcoded and reversetranscribed within the individual droplets. cDNA molecules were then pre-amplified and pooled together followed by the final library construction. Libraries were sequenced by paired-end 150bp reads on Illumina NextSeq500. Post-processing and quality control were performed by the same genomics core facility using the 10X Cell Ranger package (V2.