Melanin depletion affects Aspergillus flavus conidial surface proteins, architecture, and virulence.

Melanin is an Aspergillus flavus cell wall component that provides chemical and physical protection to the organism. However, the molecular and biological mechanisms modulating melanin-mediated host-pathogen interaction in A. flavus keratitis are not well understood. This work aimed to compare the morphology, surface proteome profile, and virulence of melanized conidia (MC) and non-melanized conidia (NMC) of A. flavus. Kojic acid treatment inhibited melanin synthesis in A. flavus, and the conidial surface protein profile was significantly different in kojic acid-treated non-melanized conidia. Several cell wall-associated proteins and proteins responsible for oxidative stress, carbohydrate, and chitin metabolic pathways were found only in the formic acid extracts of NMC. Scanning electron microscopy (SEM) analysis showed the conidial surface morphology difference between the NMC and MC, indicating the role of melanin in the structural integrity of the conidial cell wall. The levels of calcofluor white staining efficiency were different, but there was no microscopic morphology difference in lactophenol cotton blue staining between MC and NMC. Evaluation of the virulence of MC and NMC in the Galleria mellonella model showed NMC was less virulent compared to MC. Our findings showed that the integrity of the conidial surface is controlled by the melanin layer. The alteration in the surface protein profile indicated that many surface proteins are masked by the melanin layer, and hence, melanin can modulate the host response by preventing the exposure of fungal proteins to the host immune defense system. The G. mellonella virulence assay also confirmed that the NMC were susceptible to host defense as in other Aspergillus pathogens. KEY POINTS: • l-DOPA melanin production was inhibited in A. flavus isolates by kojic acid, and for the first time, scanning electron microscopy (SEM) analysis revealed morphological differences between MC and NMC of A. flavus strains • Proteome profile of non-melanized conidia showed more conidial surface proteins and these proteins were mainly involved in the virulence, oxidative stress, and metabolism pathways • Non-melanized conidia of A. flavus strains were shown to be less virulent than melanised conidia in an in vivo virulence experiment with the G. melonella model.

Functional Attributes of Myco-Synthesized Silver Nanoparticles from Endophytic Fungi: A New Implication in Biomedical Applications.

To develop a benign nanomaterial from biogenic sources, we have attempted to formulate and fabricate silver nanoparticles synthesized from the culture filtrate of an endophytic fungus Penicillium oxalicum strain LA-1 (PoAgNPs). The synthesized PoAgNPs were exclusively characterized through UV-vis absorption spectroscopy, Fourier Transform Infra-Red spectroscopy (FT-IR), X-ray powder diffraction (XRD), and Transmission Electron Microscopy (TEM) with energy dispersive X-ray spectroscopy (EDX). The synthesized nanoparticles showed strong absorbance around 430 nm with surface plasmon resonance (SPR) and exhibited a face-centered cubic crystalline nature in XRD analysis. Proteins presented in the culture filtrate acted as reducing, capping, and stabilization agents to form PoAgNPs. TEM analysis revealed the generation of polydispersed spherical PoAgNPs with an average size of 52.26 nm. The PoAgNPs showed excellent antibacterial activity against bacterial pathogens. The PoAgNPs induced a dose-dependent cytotoxic activity against human adenocarcinoma breast cancer cell lines (MDA-MB-231), and apoptotic morphological changes were observed by dual staining. Additionally, PoAgNPs demonstrated better larvicidal activity against the larvae of Culex quinquefasciatus. Moreover, the hemolytic test indicated that the as-synthesized PoAgNPs are a safe and biocompatible nanomaterial with versatile bio-applications.

Calponin 3 promotes invasion and drug resistance of colon cancer cells.

BACKGROUND: Calponin 3 (CNN3) is an actin-binding protein expressed in smooth muscle and non-smooth muscle cells. It is required for cytoskeletal rearrangement and wound healing. AIM: To dissect the role of CNN3 in carcinogenesis with a focus on colon cancer. METHODS: A total of 20 cancer cell lines (8 breast, 11 colon, and HeLa cervical cancer cell as a positive control for mesenchymal phenotype) and 57 formalin-fixed, paraffin-embedded sections from archived sporadic colorectal carcinomas were included in this study. CNN3 expression analysis by western blot or immunohistochemistry was followed by functional analyses. The CNN3 gene was silenced by specific small interfering RNA (commonly known as siRNA), followed by confirmation of the silencing efficiency by western blotting. Then, the silenced cells and control siRNA-transfected cells were analyzed for changes in epithelial and mesenchymal markers, invasion, and response to 5-fluoruracil treatment. We also performed proteomics analysis using a phospho-kinase array-based panel of 45 proteins. RESULTS: CNN3 showed positive expression in 6/8 breast and 9/11 colon cancer lines and in HeLa cells. Interestingly, the colorectal adenocarcinoma line SW480 was negative, while the cell line developed from its matching lymph node metastasis (SW620) was positive for CNN3. CNN3 expression was fairly consistent with the metastatic phenotype in colon cancer because it was absent in one other colon cell line from a primary site and expressed in all others. We selected SW620 for subsequent functional analyses. CNN3-silenced SW620 cells showed a reduction in collagen invasion and loss of mesenchymal markers. CNN3 silencing caused an increase in the SW620 colon cancer cell sensitivity to 5-fluorouracil. Phospho-kinase array-based proteomics analysis showed that CNN3 silencing in SW620 reduced extracellular signal-regulated kinase, ß-Catenin, mutant p53, c-Jun, and heat shock protein 60 activities but increased that of checkpoint kinase 2. CNN3 was expressed in 20/57 (35%) colon cancer cases as shown by immunohistochemistry. CNN3 was associated with a decrease in overall survival in colon cancer in silico. CONCLUSION: These results show the involvement of CNN3 in lymph node metastasis and resistance to chemotherapy in colon cancer and suggest that significant oncogenic pathways are involved in these CNN3-related actions.

Molecular characterization of pathogenesis involving the GAS 1 gene from Entomopathogenic fungus Lecanicillium lecanii and its virulence against the insect host Diaphorina citri.

The entmopathogenic fungus Lecaniicillium lecanii is a naturally available biological control and it is considered to be one of the best mycoinsecticide agents against the destructive insect pest Diaphorina citri Kuwayama. The present study aimed to extract and characterize the toxic insecticidal protein from L. lecanii and to assess the toxicity level against the Asian citrus psyllid the vector of Huanglongbing disease (HLB), also called citrus greening. Extracts of a toxic substance from submerged batch culture examined by sodium dodecyl sulfate-poly-acrylamide (SDS-PAGE), had a molecular weight of 45 kDa. The most abundant toxic metabolite was subjected to HPLC to purify and identified it by mass spectrometry. Subsequently, metabolite toxicity was tested against D. citri at three different concentrations (1%, 2%, and 3%). The results showed that the highest concentration had a significant maximum mortality at 120 h post application. Furthermore, we investigated the expression of the GAS1 gene which was previously identified to have a role in pathogenicity in in vivo studies in adult insect psyllids. Results of this study indicated that expression of the virulence factor gene was present at three concentrations of the fungal suspension post inoculation. This is the first study to provide this novel approach for the characterization of fungal mediated synthesis of a cuticle degrading soluble protein against the insect D. citri. The present results provide strong information on the in vivo expression of the GAS1 gene involved in fungal virulence pertaining to penetration of the insect cuticle, but not to inhibiting the growth of the host.

Synthesis of mycotoxin protein IF8 by the entomopathogenic fungus Isaria fumosorosea and its toxic effect against adult Diaphorina citri.

Entomopathogenic fungi based microbial insecticides are considered as safe alternatives to chemical pesticides, which secretes several bioactive compounds to kill the host insects. In this study, we report a new approach for the synthesis and characterization of insecticide toxic protein IF8 produced by the Isaria fumosorosea 08, and to evaluate the mycotoxin level against the vector of Huanglongbing (HLB) or citrus greening disease, the Asian citrus psyllid, Diaphorina citri. Soluble toxic metabolites extracted from I. fumosorosea 08 through submerged liquid state culture had a molecular weight of 43 kDa when subjected by to sodium dodecyl sulfate-poly-acrylamide (SDS-PAGE) gel electrophoresis. The most abundant of toxic protein IF8 was determined by High-performance liquid chromatography (HPLC) and liquid chromatography electrospray ionization-mass spectroscopy (LC-ESI-MS) for the analysis of its molecular mass weight and purity. Further Matrix-assisted laser desorption ionization-time of flight (MALDI-TOFF) analysis confirmed the presence of toxic metabolites in liquid culture. Subsequently, mycotoxic effect of toxic protein IF8 was tested against D. citri at three different concentrations (1%, 2%, and 3%). The results showed the insecticidal activity of >80% when administered at three different concentrations at 48-120 hour post-application. Additionally, we also investigated the physicochemical properties and stability of IF8 by using computational biological tools. This is the first study to report the characterization of fungal mediated synthesis of the protein IF8 toxic to the insect D. citri. These results suggest the mycotoxin control of D. citri and prevention of HLB transmission by using a natural toxic compound which is eco-friendly and can be potentially used for the integrated management of D. citri.

Isolation and characterization of Metarhizium anisopliae TK29 and its mycoinsecticide effects against subterranean termite Coptotermes formosanus.

The entomopathogenic fungus Metarhizium anisopliae is widely used as biocontrol agent against many insect pests. In the present study, the potential isolate of M. anisopliae TK29 was isolated from the agricultural soils in Thekkady, India. The taxonomic identity of the isolate was confirmed based on its morphology and 18S rDNA gene sequence homology. Phylogenetic analysis confirmed that the isolated strains were related to the same species. A potential isolate (TK29) was optimized for mass cultivation and conidial spore production was enhanced using three different raw substrates (Rice, Maize, black gram) by solid-state fermentation. The results showed higher conidial spore yield from rice (2.6 ±â€¯0.32%) compared to black gram (2.1 ±â€¯0.28%) and maize (1.9 ±â€¯0.23%) substrates. Dry green conidia were applied against Formosan subterranean termite, Coptotermes formosanus at three different concentrations (1 × 106, 1 × 107, and 1 × 108 conidia/ml-1). The highest mortality rate was obtained from 1 × 108 conidia/ml-1 at 120 h post-treatment. Our study indicated that M. anisopliae TK29 had desirable attributes for the development of a mycoinsecticide against C. formosanus.

Carica papaya (Papaya) latex: a new paradigm to combat against dengue and filariasis vectors Aedes aegypti and Culex quinquefasciatus (Diptera: Culicidae).

This study manifests the larvicidal efficacy of Carica papaya latex extract and silver nanoparticles (CPAgNPs) synthesized using latex, against developing immature juveniles of Aedes aegypti and Culex quinquefasciatus. Briefly, the latex was collected and fractioned with different solvents such as chloroform, methanol and aqueously. The obtained crude extracts were subjected to larvicidal activity in the dose-dependent method. After 24 h, the mortality rate was calculated and statistically analyzed. From the results, it was demonstrated that the chloroform extract displayed prominent activity in IInd and IIIrd instar larvae of A. aegypti and C. quinquefasciatus with better LC50 values followed by methanol and aqueous extract. Subsequently, we profiled the qualitative analysis of a chloroform extract through biochemical tests; Fourier transform infrared spectroscopy and gas chromatography-mass spectrometry. Moreover, we authenticated the major secondary metabolites and activated larvicidal compound present in the extract. Further, we synthesized CPAgNPs using aqueous latex extract and challenged with IInd and IIIrd instar larvae of A. aegypti and C. quinquefasciatus. Noticeably, the synthesized nanoproducts were showed 100% mortality in a 24-h treatment with significant LC50 values. Hence, this study has opened up new vistas in the field of parasitological research to develop Carica papaya latex as a new stratagem in the insect vector management program.

Isolation of limonoid compound (Hamisonine) from endophytic fungi Penicillium oxalicum LA-1 (KX622790) of Limonia acidissima L. for its larvicidal efficacy against LF vector, Culex quinquefasciatus (Diptera: Culicidae).

Upon screening for novel and potential biocompounds with larvicidal activities, we successfully isolated hamisonine (HMSN) a limonoid compound from endophytic fungi Penicillium oxalicum LA-1 of Limonia acidissima. The extracted compound structure was elucidated by spectral studies such as UV-vis spectroscopy, thin-layer chromatography, FTIR, LC-ESI-MS, 1H NMR, and 13C NMR upon comparing with the spectral data available in the literature. Further, the isolated HMSN was tested against III and IV instar Culex quinquefasciatus larvae. The outcome of this study clearly emphasize that the extracted compound HMSN possesses a stupendous larvicidal activity in a dose-dependent manner with the LC50 and LC90 values of 1.779 and 7.685 ppm against III instar larvae and 3.031 and 28.498 ppm against IV instar larvae of C. quinquefasciatus, respectively. Interestingly, the histological studies evidently showing the damage of peritrophic membrane and epithelial cells of testing mosquito larvae.

Molecular docking of protease from Metarhizium anisopliae and their toxic effect against model insect Galleria mellonella.

Fungal virulence has been mostly associated with cuticle-degrading enzymes, which form the first formidable barrier to pathogens and pass through certain discrete stages before breaching the insect cuticle. The present study was conducted to extract and purify the extracellular protease enzyme from three isolates from Metarhizium anisopliae. The molecular weight of protease enzyme from each isolate was identified using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and found to be 35-40kDa. The partially purified enzymes were tested to identify its toxic effects against the developmental stages of IVth instar larvae of Galleria mellonella and the mortality of larvae among the three isolates was observed. The Tk6 isolate showed an ascending effect after 48h of exposure, with highest mortality at 120h post inoculation. It also showed more virulence against the model insect compared to other strains. Tk6 isolate's active protein band was analyzed by MALDI-TOF and docking study was carried out to find the interaction between the fungal and insect proteins.

Xenorhabdus stockiae KT835471-mediated feasible biosynthesis of metal nanoparticles for their antibacterial and cytotoxic activities.

In this study, extracellular metabolites of symbiotic bacteria Xenorhabdus stockiae (KT835471) was employed for the synthesis of silver (XsAgNPs) and gold nanoparticles (XsAuNPs). Synthesized NPs were characterized using high throughput instrumentation which confirms the generation of stable, crystalline XsAgNPs and XsAuNPs with the mean size of 14 ± 6 and 14 ± 5, respectively. Further, the NPs exhibits an excellent bactericidal effect against six different pathogens. On the other hand, NPs displayed an outstanding anticancer activity against human lung adenocarcinoma epithelial cells (A549). Therefore, the present study strongly suggests that metal NPs encrusted with functional bio-moieties can be used for different biomedical applications.