The regulatory role of the Aspergillus flavus core retromer complex in aflatoxin metabolism.

Aflatoxins are a series of highly toxic and carcinogenic secondary metabolites that are synthesized by Aspergillus species. The degradation of aflatoxin enzymes is an important regulatory mechanism which modulates mycotoxin producing. The retromer complex is responsible for the retrograde transport of specific biomolecules and the vacuolar fusion in the intracellular transport. Late endosomal-associated GTPase (Rab7) has been shown to be a downstream effector protein of the retromer complex. A deficiency in the retromer complex or Rab7 results in several cellular trafficking problems in yeast and humans, like protein abnormal accumulation. However, whether retromer dysfunction is involved in aflatoxin synthesis remains unclear. Here, we report that the core retromer complex, which comprises three vacuolar protein sorting-associated proteins (AflVps26-AflVps29-AflVps35), is essential for the development of dormant and resistant fungal forms such as conidia (asexual reproductive spore) and sclerotia (hardened fungal mycelium), as well as aflatoxin production and pathogenicity, in Aspergillus flavus. In particular, we show the AflVps26-AflVps29-AflVps35 complex is negatively correlated with aflatoxin exportation. Structural simulation, site-specific mutagenesis, and coimmunoprecipitation experiments showed that interactions among AflVps26, AflVps29, and AflVps35 played crucial roles in the retromer complex executing its core functions. We further found an intrinsic connection between AflRab7 and the retromer involved in vesicle-vacuole fusion, which in turn affected the accumulation of aflatoxin synthesis-associated enzymes, suggesting that they work together to regulate the production of toxins. Overall, these results provide mechanistic insights that contribute to our understanding of the regulatory role of the core retromer complex in aflatoxin metabolism.

Cellular Apoptosis Induced by Deoxynivalenol.

Deoxynivalenol (DON) is synthesized by Fusarium species that frequently infect crops during storage, and it's harm risk to human is reflected in the consumption of infected food crops or indirectly through foods of animal origin. In this study, Hela and Chang liver cells were used to research the cellular apoptosis induced by deoxynivalenol. Cells were treated by DON toxin with a series of concentration and incubated for different time. MTT, fluorescence microscope, flow cytometer and Western blot methods were used to analyze the effect of DON on the cell apoptosis in vitro and in vivo systematically. The results showed that DON was toxic to the cells tested. After being treated by DON, the morphology of Chang livers and Hela cells changed significantly. The DON promoted apoptosis in a dose- and time-dependent manner. The activity of Caspase 3 was significantly increased in DON-induced apoptosis. Moreover, endogenous Glutathione (GSH) level in these cell lines was gradually decreased. In the early apoptosis progress, oxidative stress was induced by DON. When DON reached 10 µg/mL, a markedly increased content of Malondialdehyde (MDA) was detected in both Hela and Chang liver cells. Furthermore, an in vivo test indicated that DON had toxicity to mice by causing weight loss and swollen spleen, and significantly increased expression of AST and ALT. In conclusion, the DON was toxic to mice and could induce the apoptosis of tested cells undergoing a Caspase-3 related pathway.

Effects of microbubble pretreatment on physiochemical and microbial properties of excess activated sludge.

Fast increased amount of excess activated sludge (EAS) from wastewater treatment plants has aroused universal concerns on its environmental risks and demands for appropriate treatments, while effective treatment is dependent upon proper pretreatment. In this study, air-supplied microbubbles (air-MBs) with generated size of 25.18 to 28.25 µm were used for EAS pretreatment. Different durations (30, 60, 90, and 120 s) yielded sludge with varied physiochemical conditions, and 60 s decreased sludge oxidation status and significantly increased adenosine triphosphate (ATP) content. Soluble, loosely-bound, and tightly-bound extracellular polymeric substances (SEPS, LB-EPS, and TB-EPS) were extracted from the sludge through a stepwise approach and examined through three-dimensional excitation-emission matrix (3D-EEM) and quantitative analysis. The results showed that 60- and 120-s treatments generated stronger fluorescence intensities on dissolved organic matters (DOMs) of protein-like and fulvic acid in LB-EPS and TB-EPS, which indicated the decrease of counterparts in EAS, and therefore facilitated sludge dewaterability and reduction. The dominant microbial communities in EAS, including Proteobacteria, Bacteroidota, Chloroflexi, and Actinobacteriota, were not significantly affected by MB pretreatment. The results collectively revealed the effects of MB pretreatment on EAS and indicated that MBs could be an effective pretreatment technique for EAS treatment process.

N-Myristoyltransferase, a Potential Antifungal Candidate Drug-Target for Aspergillus flavus.

The filamentous fungus Aspergillus flavus causes devastating diseases not only to cash crops but also to humans by secreting a series of secondary metabolites called aflatoxins. In the cotranslational or posttranslational process, N-myristoyltransferase (Nmt) is a crucial enzyme that catalyzes the myristate group from myristoyl-coenzyme A (myristoyl-CoA) to the N terminus or internal glycine residue of a protein by forming a covalent bond. Members of the Nmt family execute a diverse range of biological functions across a broad range of fungi. However, the underlying mechanism of AflNmt action in the A. flavus life cycle is unclear, particularly during the growth, development, and secondary metabolic synthesis stages. In the present study, AlfNmt was found to be essential for the development of spore and sclerotia, based on the regulation of the xylose-inducible promoter. AflNmt, located in the cytoplasm of A. flavus, is also involved in modulating aflatoxin (AFB1) in A. flavus, which has not previously been reported in Aspergillus spp. In addition, we purified, characterized, and crystallized the recombinant AflNmt protein (rAflNmt) from the Escherichia coli expression system. Interestingly, the crystal structure of rAlfNmt is moderately different from the models predicted by AlphaFold2 in the N-terminal region, indicating the limitations of machine-learning prediction. In conclusion, these results provide a molecular basis for the functional role of AflNmt in A. flavus and structural insights concerning protein prediction. IMPORTANCE As an opportunistic pathogen, A. flavus causes crop loss due to fungal growth and mycotoxin contamination. Investigating the role of virulence factors during infection and searching for novel drug targets have been popular scientific topics in the field of fungal control. Nmt has become a potential target in some organisms. However, whether Nmt is involved in the developmental stages of A. flavus and aflatoxin synthesis, and whether AlfNmt is an ideal target for structure-based drug design, remains unclear. This study systematically explored and identified the role of AlfNmt in the development of spore and sclerotia, especially in aflatoxin biosynthesis. Moreover, although there is not much difference between the AflNmt model predicted using the AlphaFold2 technique and the structure determined using the X-ray method, current AI prediction models may not be suitable for structure-based drug development. There is still room for further improvements in protein prediction.

Glutamine Synthetase Contributes to the Regulation of Growth, Conidiation, Sclerotia Development, and Resistance to Oxidative Stress in the Fungus Aspergillus flavus.

The basic biological function of glutamine synthetase (Gs) is to catalyze the conversion of ammonium and glutamate to glutamine. This synthetase also performs other biological functions. However, the roles of Gs in fungi, especially in filamentous fungi, are not fully understood. Here, we found that conditional disruption of glutamine synthetase (AflGsA) gene expression in Aspergillus flavus by using a xylose promoter leads to a complete glutamine deficiency. Supplementation of glutamine could restore the nutritional deficiency caused by AflGsA expression deficiency. Additionally, by using the xylose promoter for the downregulation of AflgsA expression, we found that AflGsA regulates spore and sclerotic development by regulating the transcriptional levels of sporulation genes abaA and brlA and the sclerotic generation genes nsdC and nsdD, respectively. In addition, AflGsA was found to maintain the balance of reactive oxygen species (ROS) and to aid in resisting oxidative stress. AflGsA is also involved in the regulation of light signals through the production of glutamine. The results also showed that the recombinant AflGsA had glutamine synthetase activity in vitro and required the assistance of metal ions. The inhibitor molecule L-α-aminoadipic acid suppressed the activity of rAflGsA in vitro and disrupted the morphogenesis of spores, sclerotia, and colonies in A. flavus. These results provide a mechanistic link between nutrition metabolism and glutamine synthetase in A. flavus and suggest a strategy for the prevention of fungal infection.

GTPase Rac Regulates Conidiation, AFB1 Production and Stress Response in Pathogenic Fungus Aspergillus flavus.

As a member of the Rho family, Rac plays important roles in many species, including proliferation, differentiation, apoptosis, DNA damage responses, metabolism, angiogenesis, and immunosuppression. In this study, by constructing Rac-deleted mutants in Aspergillus flavus, it was found that the deletion of Rac gene led to the decline of growth and development, conidia production, AFB1 toxin synthesis, and seed infection ability of A. flavus. The deletion of Rac gene also caused the disappearance of A. flavus sclerotium, indicating that Rac is required for sclerotium formation in A. flavus. The sensitivity of Rac-deficient strains responding to cell wall stress and osmotic pressure stress increased when compared to A.flavus WT. The Western blot result showed that mitogen-activated serine/threonine-protein kinase Slt2 and mitogen-activated protein kinase Hog1 proteins were no longer phosphorylated in Rac-deficient strains of A. flavus, showing that Rac may be used as a molecular switch to control the Slt2-MAPK cascade pathway and regulate the osmotic Hog-MAPK cascade pathway in A. flavus in response to external stress. Altogether, these results indicated that Rac was involved in regulating the growth and development, conidia formation and AFB1 synthesis, and response to cell wall stress and osmotic pressure stress in A. flavus.

A New Modified Hysteroscopic-Laparoscopic Surgery for Cesarean Scar Pregnancy of Stable Type III.

OBJECTIVE: To introduce a modified hysteroscopic-laparoscopic operation for cesarean scar pregnancy (CSP) of stable type III. PATIENTS AND METHODS: We retrospectively studied the case notes of 31 patients with stable type III cesarean scar pregnancy who underwent hysteroscopic-laparoscopic surgery in our hospital. Thirteen patients received the modified hysteroscopic-laparoscopic surgery (modified surgery group), and eighteen patients received traditional hysteroscopic-laparoscopic surgery (traditional surgery group). RESULTS: There was no significant difference in patients' age, gestational age, number of previous cesarean sections, the serum human chorionic gonadotropin (hCG) level before surgery, gestational sac diameter, myometrium thickness between the two groups. In the modified hysteroscopic-laparoscopic surgery, the mean surgical time was 50.45±24.45 mins, the mean length of stay in hospital was 4.50±0.50 days, which was significantly shorter than the traditional surgery group (84.75±33.28 mins and 5.50±0.75 days, respectively). And the intraoperative hemorrhage in the modified group was also less than that in the traditional group (40.50±12.25 mL vs 75.33±25.45mL). Whereas the time for hCG normalization, postoperative vaginal bleeding and menstrual recovery had no significant difference between the modified surgery group and the traditional surgery group. There was no recurrence of CSP in both groups. CONCLUSION: The modified hysteroscopic-laparoscopic surgery had shorter operation time, less blood loss, and sooner recovery time after surgery compared to traditional hysteroscopic-laparoscopic surgery, which could be more beneficial to our patients and should be applied in clinics generally.