Disruption of signaling pathways such as those mediated by Shh or Pdgf causes craniofacial disease, including cleft palate. The role that microRNAs play in modulating palatogenesis, however, is completely unknown. We show, in zebrafish, that the microRNA Mirn140 negatively regulates Pdgf signaling during palatal development and we provide a mechanism for how disruption of Pdgf signaling causes palatal clefting. The pdgf-receptor alpha (pdgfra) 3' UTR contains a Mirn140 binding site functioning in the negative regulation of Pdgfra protein levels in vivo. Both pdgfra mutants and Mirn140-injected embryos share panoply of facial defects including clefting of the crestderived cartilages that develop in the roof of the larval mouth. Concomitantly, the oral ectoderm beneath where these cartilages develop loses pitx2 and shha expression. Mirn140 modulates Pdgfmediated attraction of cranial neural crest cells to the oral ectoderm, where crest-derived signals are necessary for oral ectodermal gene expression. Both Mirn140 loss-of-function and pdgfra overexpression alters palatal shape and causes neural crest cells to accumulate around the optic stalk, a source of the ligand Pdgfaa. Conserved molecular genetics and expression patterns of mirn140 and pdgfra suggest that their regulatory interactions are ancient methods of palatogenesis that provide a candidate mechanism for cleft palate.Cleft palate and other craniofacial diseases are common in humans and have complex cellular and genetic etiologies. In amniotes, the palate serves to separate the nasal and oral cavities and is generated through an intricate series of morphogenic events that include early neural crest cell migration and cell-cell signaling during the formation of facial prominences, as well as later generation and fusion of palatal shelves. While later events involving palatal shelves have not been described in zebrafish, palatal precursors migrate both rostral and caudal to the eye to condense upon the oral ectoderm in amniotes 1 as well as zebrafish 2,3 and evidence continues to accumulate that the early signaling environment governing palatogenesis is also largely equivalent [3][4][5][6] . For instance, Hh signaling is crucial for palatogenesis in humans and zebrafish 3,4,7 . Zebrafish and amniotes also share expression patterns of palatogenic genes such as Shh 4,8 , Fgf8 4,9,10 and Pdgf receptor alpha (Pdgfra) [11][12][13] .In mouse, the Pdgf family consists of four soluble ligands, Pdgfa, Pdgfb, Pdgfc, and Pdgfd as well as two receptor tyrosine kinases, Pdgfra and Pdgfrb 14 . Pdgf signaling regulates a myriad of biological processes as demonstrated by analyses of mouse Pdgf ligand and receptor mutants 14 . Mice null for Pdgfra have a facial clefting phenotype that includes cleft palate 12,13 . This facial phenotype is fully recapitulated in mice doubly mutant for Pdgfa and Pdgfc 15. Most Pdgfc mutants have cleft palate 15 MicroRNAs (miRNAs) provide a unique mechanism for modulating signaling pathways [17][18][19][20] . Skeletogenic, including...
Flavins regulate the rate and direction of extracellular electron transfer (EET) in Shewanella oneidensis. However, low concentration of endogenously secreted flavins by the wild-type S. oneidensis MR-1 limits its EET efficiency in bioelectrochemical systems (BES). Herein, a synthetic flavin biosynthesis pathway from Bacillus subtilis was heterologously expressed in S. oneidensis MR-1, resulting in ∼25.7 times' increase in secreted flavin concentration. This synthetic flavin module enabled enhanced bidirectional EET rate of MR-1, in which its maximum power output in microbial fuel cells increased ∼13.2 times (from 16.4 to 233.0 mW/m(2)), and the inward current increased ∼15.5 times (from 15.5 to 255.3 μA/cm(2)).
Electroactive biofilms play essential roles in determining the power output of microbial fuel cells (MFCs). To engineer the electroactive biofilm formation of Shewanella oneidensis MR-1, a model exoelectrogen, we herein heterologously overexpressed a c-di-GMP biosynthesis gene ydeH in S. oneidensis MR-1, constructing a mutant strain in which the expression of ydeH is under the control of IPTG-inducible promoter, and a strain in which ydeH is under the control of a constitutive promoter. Such engineered Shewanella strains had significantly enhanced biofilm formation and bioelectricity generation. The MFCs inoculated with these engineered strains accomplished a maximum power density of 167.6 ± 3.6 mW/m(2) , which was ∼ 2.8 times of that achieved by the wild-type MR-1 (61.0 ± 1.9 mW/m(2) ). In addition, the engineered strains in the bioelectrochemical system at poised potential of 0.2 V vs. saturated calomel electrode (SCE) generated a stable current density of 1100 mA/m(2) , ∼ 3.4 times of that by wild-type MR-1 (320 mA/m(2) ).
The slow rate of extracellular electron transfer (EET) of electroactive microorganisms remains a primary bottleneck that restricts the practical applications of bioelectrochemical systems. Intracellular NAD(H/+) (i.e., the total level of NADH and NAD+) is a crucial source of the intracellular electron pool from which intracellular electrons are transferred to extracellular electron acceptors via EET pathways. However, how the total level of intracellular NAD(H/+) impacts the EET rate in Shewanella oneidensis has not been established. Here, we use a modular synthetic biology strategy to redirect metabolic flux towards NAD+ biosynthesis via three modules: de novo, salvage, and universal biosynthesis modules in S. oneidensis MR-1. The results demonstrate that an increase in intracellular NAD(H/+) results in the transfer of more electrons from the increased oxidation of the electron donor to the EET pathways of S. oneidensis, thereby enhancing intracellular electron flux and the EET rate.
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