Enrichment Extraction and Activity Study of the Different Varieties of Hericium erinaceus against HCT-8 Colon Cancer Cells.

Hericium erinaceus (HE), a widely utilized natural remedy and dietary source, has garnered significant attention for its therapeutic potential in various diseases. In this study, we employed supercritical fluid extraction (SFE) technology to isolate the bioactive compounds from HE's fruiting body. Comprehensive assessments of the antioxidant and antibacterial activities were conducted, along with in vitro investigations on the human colon cancer cell line (HCT-8). The SFE rate served as the evaluation metric, while the variables of extraction time, pressure, and temperature were systematically examined. By integrating the response surface center composite design, we successfully optimized the extraction process, yielding optimal parameters of 80 min, 30 MPa, and 35 °C, thus resulting in an extraction rate of 2.51%. These optimized conditions exhibited considerable antioxidant capacity, anticancer activity, and antibacterial potential. Furthermore, we employed graded alcohol extraction to refine the crude extracts, thereby confirming superior anticancer effects under a 70% alcohol precipitation. To elucidate the composition, Fourier-transform infrared spectroscopy (FT-IR) and gas chromatography-mass spectrometry (GC-MS) were employed to analyze the crude extracts and isolates of HE, facilitating a comparative analysis of six HE varieties. Our findings suggest that sterol derivatives hold promise as the active component against the colon cancer HCT-8 cell line. In conclusion, this study underscores the potential of HE SFE in the development of functional foods or alternative drugs for colon cancer treatment, thus opening new avenues for therapeutic interventions.

Escherichia coli phage phi2013: genomic analysis and receptor identification.

Escherichia coli is an important foodborne pathogen that can cause severe human disease. Here, we report the isolation and characterization of the lytic virus phi2013, which is specific for Escherichia coli laboratory strains. Transmission electron microscopy showed that phage phi2013 has an icosahedral head and a long, fragile, noncontractile tail, exhibiting the typical form of a siphovirus. Evidence revealed that the phi2013 genome is a linear double-stranded DNA molecule of 49,833 bp with 79 predicted genes without any known antibiotic resistance genes, virulence factor genes, or integrase genes. Moreover, the conserved outer membrane protein FhuA, which is present in members of several genera of the family Enterobacteriaceae, was identified as the receptor of phage phi2013. To evaluate the potential of phage phi2013 as a biocontrol agent for controlling E. coli contamination, it was tested in several foods, including sterilized milk, ready-to-eat beef, and crisphead lettuce. The data showed that phage phi2013 can efficiently inhibit E. coli growth in the tested foods at 4°C and 25°C. We therefore conclude that phage phi2013 or cocktails containing phi2013 may be used as an antimicrobial agent in extending the shelf-life of food products by effectively controlling the growth of E. coli.

Dicer promotes Atg8 expression through RNAi independent mechanism in Cryptococcus neoformans.

ATG8 is one of the critical genes that participate in several essential autophagic steps. The expression of ATG8 must be exquisitely regulated to avoid physiological disorder and even cell death. However, the mechanisms of regulating ATG8 expression remain to be fully uncovered. In this investigation, we found that Dicer homologs in Cryptococcus neoformans could activate the expression of ATG8 independent of RNAi. Deletion of two Dicer homologs (DCR1 and DCR2) from C. neoformans, especially DCR2, led to significantly reduced Atg8 protein level, but deletion of other RNAi components did not result in the same phenotype. The autophagic flux, the numbers of autophagic bodies and the tolerance to glucose starvation of dcr2∆ were also significantly reduced. Further investigation showed that Dcr2 activates the expression of ATG8 through the promoter region, not the Open Reading Frame or 3' Untranslated Region. We also found that a similar phenomenon exists in mammalian cells, as DCR1 instead of AGO2 knockdown also reduced the expression of LC3, indicating that this mechanism may be conservative in eukaryotic cells. Therefore, a novel transcription activation mechanism was revealed in this paper.

Identification of a Unique Azaphilone Produced by Chaetomium globosum Isolated from Polygonatum sibiricum.

A new azaphilone, chaephilone E, eight azaphilone derivatives, and three chaetoglobosins were isolated from endophytic fungi Chaetomium globosum. The structures of the compounds were elucidated by 1D and 2D NMR as well as HR-ESI-MS data, and the absolute configuration of chaephilone E was established on the basis of electronic circular dichroism and NOESY spectrum. The activity of chaephilone E was evaluated via the cytotoxic assay (human hepatoma cell lines HepG-2) and brine shrimp (Artemia salina) bioassay.

Characterization of a novel lytic myophage, phiA8-29, infecting Aeromonas strains.

Members of the bacterial genus Aeromonas are important aquatic pathogens that cause severe fish diseases. Here, we characterize a novel lytic phage, Aeromonas virus phiA8-29, isolated from the alimentary tract of a freshwater fish. Transmission electron microscopy showed that phiA8-29 has a long contractile tail and thus can be classified as a member of the family Myoviridae. The phage genome was identified as a double-stranded DNA molecule of 144,974 bp containing 185 open reading frames and nine tRNA-encoding genes. Comparative genomic analysis revealed that the phiA8-29 genome has little similarity to any of the currently sequenced Aeromonas phage genomes. Our data indicate that phiA8-29 is a novel lytic Myoviridae phage that does not belong to any of the known genera.

Removal of nitrogen by heterotrophic nitrification-aerobic denitrification of a novel halotolerant bacterium Pseudomonas mendocina TJPU04.

Excess inorganic nitrogen in water poses a severe threat to enviroment. Removal of inorganic nitrogen by heterotrophic nitrifying-aerobic denitrifying microorganism is supposed to be a promising and applicable technology only if the removal rate can be maintained sufficiently high in real wastewater under various conditions, such as high concentration of salt and wide range of different nitrogen concentrations. Here, a new heterotrophic nitrifying-aerobic denitrifying bacterium was isolated and named as Pseudomonas mendocina TJPU04, which removes NH4+-N, NO3--N and NO2--N with average rate of 4.69, 5.60, 4.99 mg/L/h, respectively. It also maintains high nitrogen removal efficiency over a wide range of nitrogen concentrations. When concentration of NH4+-N, NO3--N and NO2--N was up to 150, 150 and 50 mg/L, 98%, 93%, and 100% removal efficiency could be obtained, respectively, after 30-h incubation under sterile condition. When it was applied under non-sterile condition, the ammonia removal efficiency was slightly lower than that under sterile condition. However, the nitrate and nitrite removal efficiencies under non-sterile condition were significantly higher than those under sterile condition. Strain TJPU04 also showed efficient nitrogen removal performance in the presence of high concentration of salt and nitrogen. In addition, the removal efficiencies of NH4+-N, NO3--N and TN in real wastewater were 91%, 52%, and 75%, respectively. These results suggest that strain TJPU04 is a promising candidate for efficient removal of inorganic nitrogen in wastewater treatment.

Efficient degradation of Azo dyes by a newly isolated fungus Trichoderma tomentosum under non-sterile conditions.

A fast-growing fungus with remarkable ability to degrade several azo dyes under non-sterile conditions was isolated and identified. This fungus was identified as Trichoderma tomentosum. Textile effluent of ten-fold dilution could be decolorized by 94.9% within 72h before optimization. Acid Red 3R model wastewater with a concentration of 85.5mgL-1 could be decolorized by 99.2% within the same time after optimization. High-level of manganese peroxidase and low-level of lignin peroxidase activities were detected during the process of decolorization from the culture supernatant, indicating the possible involvement of two enzymes in azo dye decolorization. No aromatic amine products were detected from the degradation products of Acid Red 3R by gas chromatography-mass spectrometry (GC/MS) analysis, indicating the possible involvement of a special symmetrical oxidative degradation pathway. Phytotoxicity assay confirmed the lower toxicity toward the test plant seeds of the degradation products when compared to the original dye.

Improved pestalotiollide B production by deleting competing polyketide synthase genes in Pestalotiopsis microspora.

Pestalotiollide B, an analog of dibenzodioxocinones which are inhibitors of cholesterol ester transfer proteins, is produced by Pestalotiopsis microspora NK17. To increase the production of pestalotiollide B, we attempted to eliminate competing polyketide products by deleting the genes responsible for their biosynthesis. We successfully deleted 41 out of 48 putative polyketide synthases (PKSs) in the genome of NK17. Nine of the 41 PKS deleted strains had significant increased production of pestalotiollide B (P < 0.05). For instance, deletion of pks35, led to an increase of pestalotiollide B by 887%. We inferred that these nine PKSs possibly lead to branch pathways that compete for precursors with pestalotiollide B, or that convert the product. Deletion of some other PKS genes such as pks8 led to a significant decrease of pestalotiollide B, suggesting they are responsible for its biosynthesis. Our data demonstrated that improvement of pestalotiollide B production can be achieved by eliminating competing polyketides.

[Identification, cloning and functional verification of U6 promoter from Cryptococcus neoformans].

Objective: To identify and clone the polymerase Ⅲ U6 promoter from Cryptococcus neoformans (CnU6 promoter), and verify if CnU6 promoter can effectively transcribe shRNA and gRNA of CRISPR/Cas9 system. Methods: Combining the C. neoformans genome information published in GenBank database and RNA-seq library data from our laboratory, we obtained the U6 RNA sequence with high transcriptional level by bioinformatics analysis. The putative CnU6 promoter was ligated upstream of shRNA and gRNA by EasyGeno and overlapping PCR respectively. Based on shRNA-mediated target gene silence phenotype by RNAi and gene mutation by gRNA-guided Cas9 nuclease mediated target sites editing by CRISPR/Cas9 system, we could identify if CnU6 promoter could drive the transcription of short RNA. Results: CnU6 promoter could drive the transcribtion of shRNA, which could silence the target gene, and gRNA, which could guide Cas9 nuclease to cut the target site. Conclusion: The CnU6 promoter from C. neoformans was successfully identified and cloned, which could drive the transcription of shRNA and gRNA efficiently.

Effects of CTR4 deletion on virulence and stress response in Cryptococcus neoformans.

Roles of the high-affinity copper transporter Ctr4 in the virulence of Cryptococcus neoformans remain to be fully determined. Here we demonstrate that Ctr4 plays a necessary role in virulence and tolerance to a number of stress conditions. We first observed, with the method of flame atomic absorption spectrometry, that deletion of CTR4 resulted in a significant decrease in intracellular copper level, confirming the role of Ctr4 as a copper transporter in C. neoformans. Furthermore, CTR4 was critical for the yeast to survive at both elevated and low temperatures, as the growth rate of the ctr4Δ mutant at 4 and 37 °C was significantly decreased. The mutant ctr4Δ also exhibited hypersensitivity to osmotic stress imposed by 2 M NaCl or KCl, indicating the possible crosstalk of Ctr4 with the HOG signalling pathway. Moreover, cell wall and plasma membrane integrity appeared to be impaired in the ctr4Δ strain. The virulence of ctr4Δ in two mouse cryptococcosis models was remarkably reduced either via an intranasal or intravenous inoculation. Our work confirms the roles of Ctr4 in virulence and copper homeostasis as well as other additional novel functions.

[Snf1/AMPK affects cell wall integrity through regulating the transcription of cell wall as sembly-related genesin Saccharomyces cerevisiae].

Objective: To study the role of Snf1/AMPK kinase in cell wall integrity in Saccharomyces cerevisiae JY102. Methods: SNF1 deletion mutant of S. cerevisiae was created by homologous recombination. Congo red and Calcofluor white were applied to evaluate the cell wall integrity for the mutant strain. qRT-PCR was used to analyze the transcription of cell wall-related genes. Results: The SNF1 disruption mutant was severely sensitive to Congo red and Calcofluor white, manifesting the impairment of cell wall integrity. The mutant exhibited apparently growth defect at high temperature. The results of qRT-PCR revealed down-regulated transcription of several genes involved in ß-glucan biosynthesis. Conclusion: The deletion of Snf1 impairs the cell wall integrity by reducing the transcription of ß-glucan-related genes, suggesting a new role of Snf1 in the activation of cell wall synthesis.

Cryptococcal phosphoglucose isomerase is required for virulence factor production, cell wall integrity and stress resistance.

Regulation of virulence factor production in the pathogen Cryptococcus neoformans remains to be fully illustrated. We present here a finding that a gene, encoding the glycolysis enzyme phosphoglucose isomerase (Pgi1), is critical for the biosynthesis of melanin and capsule, cell wall integrity and resistance to stress conditions. A leaky mutant of the yeast, LZM19, resulted from an insertion of T-DNA in the PGI1 promoter region, expressed PGI1 at a level only 1.9% of the wild type. LZM19 could synthesize the pigment melanin in the presence of 2% glucose, suggesting a status of LAC1 derepression. Phenotypically, capsule biosynthesis in LZM19 was remarkably reduced. Integrity of the cell wall and plasma membrane of LZM19 were impaired based on its sensitivity to Congo red and SDS. Also, LZM19 exhibited hypersensitivity to osmotic stress generated by 2 M NaCl or 1 M KCl, indicating possible impairment in the HOG signaling pathway. Furthermore, LZM19 failed to utilize mannose and fructose, suggesting a possible involvement of Pgi1 in the breakdown of these two sugars. Our results revealed a crucial role of PGI1 in coordination of the production of virulence factors, cell wall integrity and stress response in C. neoformans.

GWT1 encoding an inositol acyltransferase homolog is required for laccase repression and stress resistance in the basidiomycete Cryptococcus neoformans.

The transcriptional expression of laccase, which has been confirmed to contribute to the virulence of Cryptococcus neoformans, is often repressed by a high concentration of glucose in many fungi, including C. neoformans. The underlying mechanism of the repression remains largely unknown. In this study, we found that a GWT1 gene that encodes a glycosylphosphatidylinositol (GPI) anchor biosynthesis-related protein is required for laccase repression by glucose in the basidiomycete C. neoformans. Disruption of GWT1 with the Agrobacterium tumefaciens-mediated T-DNA random insertional mutagenesis (ATMT) method resulted in constitutive expression of the laccase gene LAC1 and constant melanin formation. The loss of GWT1 also dramatically affected the cell membrane integrity and stress resistance. Our results revealed a GPI-dependent glucose repression mechanism in C. neoformans, and it may be helpful for understanding the virulence of C. neoformans.

A novel cleaning process for industrial production of xylose in pilot scale from corncob by using screw-steam-explosive extruder.

Steam explosion is the most promising technology to replace conventional acid hydrolysis of lignocellulose for biomass pretreatment. In this paper, a new screw-steam-explosive extruder was designed and explored for xylose production and lignocellulose biorefinery at the pilot scale. We investigated the effect of different chemicals on xylose yield in the screw-steam-explosive extrusion process, and the xylose production process was optimized as followings: After pre-impregnation with sulfuric acid at 80 °C for 3 h, corncob was treated at 1.55 MPa with 9 mg sulfuric acid/g dry corncob (DC) for 5.5 min, followed by countercurrent extraction (3 recycles), decoloration (activated carbon dosage 0.07 g/g sugar, 75 °C for 40 min), and ion exchange (2 batches). Using this process, 3.575 kg of crystal xylose was produced from 22 kg corncob, almost 90 % of hemicellulose was released as monomeric sugar, and only a small amount of by-products was released (formic acid, acetic acid, fural, 5-hydroxymethylfurfural, and phenolic compounds were 0.17, 1.14, 0.53, 0.19, and 1.75 g/100 g DC, respectively). All results indicated that the screw-steam-explosive extrusion provides a more effective way to convert hemicellulose into xylose and could be an alternative method to traditional sulfuric acid hydrolysis process for lignocellulose biorefinery.

A Novel Preservative Film with a Pleated Surface Structure and Dual Bioactivity Properties for Application in Strawberry Preservation due to Its Efficient Apoptosis of Pathogenic Fungal Cells.

Botrytis cinerea (B. cinerea) and Colletotrichum gloeosporioides (C. gloeosporioides) were isolated from the decaying strawberry tissue. The antifungal properties of Monarda didyma essential oil (MEO) and its nanoemulsion were confirmed, demonstrating complete inhibition of the pathogens at concentrations of 0.45 µL/mL (0.37 mg/mL) and 10 µL/mL, respectively. Thymol, a primary component of MEO, was determined as an antimicrobial agent with IC50 values of 34.51 (B. cinerea) and 53.40 (C. gloeosporioides) µg/mL. Hippophae rhamnoides oil (HEO) was confirmed as a potent antioxidant, leading to the development of a thymol-HEO-chitosan film designed to act as an antistaling agent. The disease index and weight loss rate can be reduced by 90 and 60%, respectively, with nutrients also being well-preserved, offering an innovative approach to preservative development. Studies on the antifungal mechanism revealed that thymol could bind to FKS1 to disrupt the cell wall, causing the collapse of mitochondrial membrane potential and a burst of reactive oxygen species.

Kaempferide-7-O-(4"-O-acetylrhamnosyl)-3-O-rutinoside reduces myocardial infarction size after coronary artery ligation in rats.

Kaempferide-7-O-(4"-O-acetylrhamnosyl)-3-O-rutinoside (A-F-B) is a novel flavonoid extracted from the leaves of Actinidia kolomikta. We recently reported that A-F-B administration could improve lipid profiles. A-F-B actions are associated with regulating the activities of PAP and HMG-CoA reductase in hepatic tissue. This study evaluated the effects of A-F-B on acute myocardial infarction (AMI) in rats. An AMI model was established by ligating the left anterior descending coronary artery. The myocardial infarct size (MIS), creatine kinase (CK-MB) activity, troponin T level, endothelial nitric oxide synthase (eNOS) activity, superoxide dismutase (SOD) activity, catalase activity, malondialdehyde (MDA) content, nitric oxide (NO) content were measured. The results showed that the groups treated with A-F-B showed a dose-dependent reduction in MIS. A-F-B markedly inhibited the elevation of the activity of CK-MB, troponin T level, and the content of MDA induced by AMI. A-F-B also showed a capacity to increase the activities of SOD, catalase, and eNOS. The NO content in A-F-B-treated groups also augmented. The findings suggest that A-F-B exerted cardioprotective effects against acute myocardial ischemic injury by regulating antioxidative enzymes activity and endothelial nitric oxide synthase activity.

Myasthenia-like paraneoplastic syndrome with multiple cranial nerve tumor infiltration: A case report and literature review.

RATIONALE: Approximately 0.001% of patients with cancer have paraneoplastic nerve system syndrome, which can affect the central nervous system, neuromuscular junction, or peripheral nervous system. Although myasthenia gravis (MG) may exist as a thymic paraneoplastic syndrome (PNPS), its association with primary lung cancer remains unknown. PATIENT CONCERNS: A 55-year-old female presented with slurred speech, weakness in chewing, sporadic difficulty in swallowing, and weakness in both lower limbs for half a year. DIAGNOSES: Based on cerebrospinal fluid and electromyography findings, we present the case of a female patient diagnosed with overlapping multicranial nerve tumor infiltration and MG-like neurological PNPS secondary to lung adenocarcinoma. INTERVENTIONS: The patient received intrathecal injections of pemetrexed and neurotrophic (vitamin B) therapy before ceasing chemoradiotherapy and chose cabozantinib on her own. OUTCOMES: Weakness of the proximal limbs, choking cough, and chewing problems did not improve significantly. LESSONS: Although it is unclear why MG coexists with lung cancer, it is probable that MG is a paraneoplastic condition. Cerebrospinal fluid testing should be carried out along with electrophysiological, serological, and pharmacological procedures pertinent to the diagnosis of MG to thoroughly examine if people simultaneously experience MG-like PNPS and tumor growth. Starting immunotherapy and anticancer medication at the same time that tumor development and MG-like syndrome are discovered is crucial.

A palisade-shaped membrane reservoir is required for rapid ring cell inflation in Drechslerella dactyloides.

Fusion of individual vesicles carrying membrane-building materials with the plasma membrane (PM) enables gradual cell expansion and shape change. Constricting ring (CR) cells of carnivorous fungi triple in size within 0.1-1 s to capture passing nematodes. Here, we investigated how a carnivorous fungus, Drechslerella dactyloides, executes rapid and irreversible PM expansion during CR inflation. During CR maturation, vesicles carrying membrane-building materials accumulate and fuse, forming a structure named the Palisade-shaped Membrane-building Structure (PMS) around the rumen side of ring cells. After CR inflation, the PMS disappears, with partially inflated cells displaying wavy PM and fully inflated cells exhibiting smooth PM, suggesting that the PMS serves as the reservoir for membrane-building materials to enable rapid and extensive PM expansion. The DdSnc1, a v-SNARE protein, accumulates at the inner side of ring cells and is necessary for PMS formation and CR inflation. This study elucidates the unique cellular mechanisms underpinning rapid CR inflation.

Nano-emulsification essential oil of Monarda didyma L. to improve its preservation effect on postharvest blueberry.

The reduction in blueberry harvest due to pathogen infection was reported to reach 80%. Essential oil (EO) can provide a new way to preserve blueberry. Here, in search for plants volatiles with preservation ability, a novel device was designed for the screening of aromatic plants led to the discovery of hit plant Monarda didyma L. Consequently, antifungi activity of M. didyma EO (MEO) and its nano-emulsion (MNE) were tested. 2 species of pathogenic fungi were isolated from blueberries, namely Alternaria sp. and Colletotrichum sp. were used as the target strains. In the in vitro activity test, the pathogenic were completely inhibited when the EO was 4 µL or 1.0 µL/mL. Compared with EO, MNE exhibited superior antimicrobial activity. Moreover, MNE can cause serious morphological changes and result in a decrease in the rot and weightlessness rate of blueberry. Hence, NME represents a promising agent for the preservation of postharvest blueberry.