Creating an efficient antimicrobial photodynamic therapy (PDT) method while reducing the difficulty of preparation and operation is much anticipated but challenging. In this research, we facilely developed glycol chitosan (GC)/protoporphyrin IX (PpIX) nano-micelles for effective photodynamic inactivation of Gram-positive bacteria. The mixture of glycol chitosan (GC) and protoporphyrin IX (PpIX) self-assembled into positively charged GC/PpIX nano-micelles through hydrophobic interaction and electrostatic interaction. GC/PpIX nano-micelles significantly improved singlet oxygen generation, and notably enhanced the antibacterial PDT effect toward Gram-positive bacteria in a preincubation-free manner without developing drug resistance. Upon binding to the bacterial cells through electrostatic interaction, GC/PpIX nano-micelles were disassembled, leaving GC on the bacterial surface, inducing bacterial cell aggregation and acting like a glue to coat the bacterial surface and release PpIX into the cells, generating singlet oxygen under light irradiation to impair DNA. Collectively, the easy construction of GC/PpIX nano-micelles offers a modality for effective antibacterial PDT with simple operation and without drug resistance development, benefiting the treatment of Gram-positive bacterial infections in clinic.

Background Cellulase synthesized by fungi can environment-friendly and sustainably degrades cellulose to fermentable sugars for producing cellulosic biofuels, biobased medicine and fine chemicals. Great efforts have been made to study the regulation mechanism of cellulase biosynthesis in fungi with the focus on the carbon sources, while little attention has been paid to the impact and regulation mechanism of nitrogen sources on cellulase production. Results Glutamine displayed the strongest inhibition effect on cellulase biosynthesis in Trichoderma reesei, followed by yeast extract, urea, tryptone, ammonium sulfate and l-glutamate. Cellulase production, cell growth and sporulation in T. reesei RUT-C30 grown on cellulose were all inhibited with the addition of glutamine (a preferred nitrogen source) with no change for mycelium morphology. This inhibition effect was attributed to both l-glutamine itself and the nitrogen excess induced by its presence. In agreement with the reduced cellulase production, the mRNA levels of 44 genes related to the cellulase production were decreased severely in the presence of glutamine. The transcriptional levels of genes involved in other nitrogen transport, ribosomal biogenesis and glutamine biosynthesis were decreased notably by glutamine, while the expression of genes relevant to glutamate biosynthesis, amino acid catabolism, and glutamine catabolism were increased noticeably. Moreover, the transcriptional level of cellulose signaling related proteins ooc1 and ooc2, and the cellular receptor of rapamycin trFKBP12 was increased remarkably, whose deletion exacerbated the cellulase depression influence of glutamine. Conclusion Glutamine may well be the metabolite effector in nitrogen repression of cellulase synthesis, like the role of glucose plays in carbon catabolite repression. Glutamine under excess nitrogen condition repressed cellulase biosynthesis significantly as well as cell growth and sporulation in T. reesei RUT-C30. More importantly, the presence of glutamine notably impacted the transport and metabolism of nitrogen. Genes ooc1, ooc2, and trFKBP12 are associated with the cellulase repression impact of glutamine. These findings advance our understanding of nitrogen regulation of cellulase production in filamentous fungi, which would aid in the rational design of strains and fermentation strategies for cellulase production in industry.

Background A total of 11 β-glucosidases are predicted in the genome of Trichoderma reesei, which are of great importance for regulating cellulase biosynthesis. Nevertheless, the relevant function and regulation mechanism of each β-glucosidase remained unknown. Results We evidenced that overexpression of cel1b dramatically decreased cellulase synthesis in T. reesei RUT-C30 both at the protein level and the mRNA level. In contrast, the deletion of cel1b did not noticeably affect cellulase production. Protein CEL1B was identified to be intracellular, being located in vacuole and cell membrane. The overexpression of cel1b reduced the intracellular pNPGase activity and intracellular/extracellular glucose concentration without inducing carbon catabolite repression. On the other hand, RNA-sequencing analysis showed the transmembrane transport process and endoplasmic reticulum function were affected noticeably by overexpressing cel1b. In particular, some important sugar transporters were notably downregulated, leading to a compromised cellular uptake of sugars including glucose and cellobiose. Conclusions Our data suggests that the cellulase inhibition by cel1b overexpression was not due to the β-glucosidase activity, but probably the dysfunction of the cellular transport process (particularly sugar transport) and endoplasmic reticulum (ER). These findings advance the knowledge of regulation mechanism of cellulase synthesis in filamentous fungi, which is the basis for rationally engineering T. reesei strains to improve cellulase production in industry.

Real-time live-cell monitoring of Fe3+ and adenosine triphosphate (ATP) in mitochondria important for cellular functions and related to different diseases is highly desired, yet lack study. Herein, carbon dots, green bean (GB)-carbon dots (CDs), were made from GB through easy one-step hydrothermal treatment. GB-CDs were in spherical form with a diameter of 13 ± 3 nm, negatively charged, and green emissive. GB-CDs contained amino groups, which enabled their binding to Fe3+, resulting in their fluorescence quenching. Based on this, GB-CDs were employed to detect Fe3+ with high selectivity, showing a linear detection range of 10–70 μM and a detection limit of 3.6 nM. Furthermore, the addition of ATP can recover the fluorescence quenching of GB-CDs by Fe3+. Therefore, the GB-CD + Fe3+ system was deployed to assay ATP with a linear detection range of 50–600 μM and a detection limit of 60 nM, exhibiting high sensitivity and specificity. GB-CDs and GB-CDs + Fe3+ were individually harnessed to assess Fe3+ in tap water and ATP in fetal bovine serum. More importantly, GB-CDs can enter cells to localize in mitochondria for mitochondrial imaging with high biocompatibility and superior photostability. As a result, GB-CDs and GB-CD + Fe3+ system were implemented to track Fe3+ and ATP in mitochondria of A549, respectively. This study provides a potent tool to monitor Fe3+ and ATP in mitochondria, expanding the application of CDs as biosensors for biological compounds in subcellular organelles.

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