Phenazine, 2-methoxy

Rotifers are microinvertebrate models to study the phylogenetically based mechanisms of aging. Our study aimed to develop a physiological system with electron deprivation via a chemical electron carrier/acceptor pair together with extreme caloric restriction (ECR). Middle-aged Philodina acuticornis rotifers were treated with combinations of phenazine methosulfate (PMS, electron carrier) and 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt (XTT, electron acceptor) for a period of 72 hours under total food deprivation (preselection). The ability of XTT to be reduced was confirmed both in vitro (with NADH) and in vivo (with live rotifers). Subsequently, the respective electron acceptor alone at a lower dose was administered in combination with ECR for several months on preselected survivors. We found that the longevity of rotifers markedly increased (4×) after PMS/XTT/total food deprivation preselection followed by XTT/ECR treatment. Ascorbic acid in equivalent concentrations caused similar but less pronounced tendencies. The synergistic effect of chemical electron deprivation and ECR caused delayed aging and the development of an outstanding phenotype that we refer to as "super rotifers," characterized by increased longevity and retained reproductive ability compared with normal middle-aged individuals. The presented model provides new insights into the connection between redox modulation and age-related features in vivo.

Objective: To observe the effects of New Dayuan powder (NDYP) on Methicillin-resistant Staphylococcus aureus (MRSA) biofilms and the embedded bacteria in vitro. Methods: 2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assays were used to study the effects of NDYP on developing MRSA biofilms: 100 μL of bacterial culture and 100 μL drug solution were added to wells of 96-well plates. After 24 h of incubation, the plates were washed and XTT-phenazine methyl sulfate (PMS) was added to enable counting of the number of live bacteria in biofilms using a microplate reader. XTT assays were also used to explore the effects of NDYP on mature MRSA biofilms: 100 μL of bacterial culture were added to wells of 96-well plates. Bacteria were cultured in the plates for 24 h, and then drug solution was added. The plates were cultured for another 24 h, and then XTT-PMS was added to detect the number of live bacteria in the biofilms. Scanning electron microscopy (SEM) was used to observe the effects of NDYP on mature MRSA biofilms: washed and sterilized glass coverslips were added to 24-well plates. Bacterial culture was added. After 24 h of incubation, drug solution was added. After another 24 h of incubation, the samples were observed by SEM. Results: XTT assays showed that the number of live bacteria in both developing and mature MRSA biofilms decreased significantly (P < 0.01) after the administration of NDYP. SEM images showed that NDYP could destroy the structure of the bacteria and resulted in uneven thickness of MRSA biofilms. Conclusion: In vitro, NDYP has obvious inhibitory effects on the formation of MRSA biofilms and on mature biofilms.

Osteoblast and adipocyte are differentiated from mesenchymal stem cells and dysregulation of the differentiation might result in disease, such as osteoporosis and diabetes. To find small compounds that induce osteoblast differentiation, we screened an in-house natural compounds library with mouse preosteoblastic MC3T3-E1 cells using alkaline phosphatase (ALP) expression as an early osteoblast marker. We found that phenazine-1-carboxylic acid (PCA), one of the major phenazine derivatives produced by Pseudomonas, induced osteoblast differentiation in the cells at micromolar concentrations. PCA acted synergistically with an agonist of hedgehog signaling in inducing ALP activity in the cells. We also found that 2-hydroxy-PCA (2H-PCA) induced osteoblast differentiation in the cells but 2-methoxy-PCA and 1-hydroxy-phenazine did not. Unexpectedly, treatment of mouse pluripotent mesenchymal C3H10T1/2 cells with PCA or 2H-PCA induced an obvious morphological change. Oil Red O staining and real-time reverse-transcription PCR analysis revealed that PCA induced not osteoblast differentiation but adipocyte differentiation in C3H10T1/2 cells. These compounds could allow us to investigate the mechanism of osteoblast and adipocyte differentiation in the two model cell systems through a chemical biology approach.