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Cells May 2023Controversial reports have suggested that SARS-CoV E and 3a proteins are plasma membrane viroporins. Here, we aimed at better characterizing the cellular responses...
Controversial reports have suggested that SARS-CoV E and 3a proteins are plasma membrane viroporins. Here, we aimed at better characterizing the cellular responses induced by these proteins. First, we show that expression of SARS-CoV-2 E or 3a protein in CHO cells gives rise to cells with newly acquired round shapes that detach from the Petri dish. This suggests that cell death is induced upon expression of E or 3a protein. We confirmed this by using flow cytometry. In adhering cells expressing E or 3a protein, the whole-cell currents were not different from those of the control, suggesting that E and 3a proteins are not plasma membrane viroporins. In contrast, recording the currents on detached cells uncovered outwardly rectifying currents much larger than those observed in the control. We illustrate for the first time that carbenoxolone and probenecid block these outwardly rectifying currents; thus, these currents are most probably conducted by pannexin channels that are activated by cell morphology changes and also potentially by cell death. The truncation of C-terminal PDZ binding motifs reduces the proportion of dying cells but does not prevent these outwardly rectifying currents. This suggests distinct pathways for the induction of these cellular events by the two proteins. We conclude that SARS-CoV-2 E and 3a proteins are not viroporins expressed at the plasma membrane.
Topics: Cricetinae; Animals; SARS-CoV-2; Cricetulus; COVID-19; Cell Membrane; CHO Cells
PubMed: 37296595
DOI: 10.3390/cells12111474 -
PloS One 2023It has been proposed that the composition of the culture medium, especially its amino acids, is an important part of getting microspore androgenesis to occur in some...
It has been proposed that the composition of the culture medium, especially its amino acids, is an important part of getting microspore androgenesis to occur in some plants. However, there have been far fewer studies done on the Solanaceae family. In this study, we studied what happened to eggplant microspore culture when we mixed casein hydrolysate (0 and 100 mg L-1) with four amino acids: proline (0, 100, 500, and 900 mg L-1), glutamine (0 and 800 mg L-1), serine (0 and 100 mg L-1), and alanine (0 and 100 mg L-1). The results showed that a combination of 800 mg L-1 of glutamine, 100 mg L-1 of serine, 100 mg L-1 of casein hydrolysate, and 500 mg L-1 of proline produced the maximum number of calli per Petri dish (938). Calli had a globular shape and a compact appearance when formed in media containing 500 mg L-1 of proline (alone or combined with serine, alanine, and/or casein hydrolysate). Most of these structures were observed in a medium with 500 mg L-1 of proline, 100 mg L-1 of casein hydrolysate, and 100 mg L-1 of serine. We also investigated what happened when gum arabic (2400, 2600, 3600, 4600, and 5600 mg L-1) was combined with proline (0 and 500 mg L-1), casein hydrolysate (0 and 100 mg L-1), and glutamine (0, 400, and 800 mg L-1). The findings demonstrated the involvement of proline in the increase of calli. Overall, the results give us new information about how amino acids work in eggplant microspore culture and suggest that proline can move this plant's microspore androgenesis pathway forward.
Topics: Amino Acids; Solanum melongena; Glutamine; Proline; Alanine; Serine
PubMed: 37289731
DOI: 10.1371/journal.pone.0286809 -
Environmental Pollution (Barking, Essex... Aug 2023Microplastics (MPs) in soil undergo different aging processes such as photoaging, mechanical abrasion and biodegradation, leading to alterations in the surface...
Microplastics (MPs) in soil undergo different aging processes such as photoaging, mechanical abrasion and biodegradation, leading to alterations in the surface properties of MPs. In this study, we investigated the adsorption-desorption of chlorpyrifos (CPF) on pristine and UV light-aged low-density polyethylene (LDPE) and biodegradable (Bio) MPs that were derived from plastic mulch films. We also tested the bioconcentration of pristine and aged MPs (LDPE- and Bio-MPs aged under UV light and LDPE-MPs aged in three different soils) associated with CPF by earthworms (Lumbricus terrestris). The results showed that UV-aged MPs showed higher CPF adsorption capacities than pristine MPs, with the adsorption capacities at 184.9 ± 5.3, 200.5 ± 1.8, 193.0 ± 8.7, and 215.9 ± 1.1 μg g for pristine LDPE-, UV-aged LDPE-, pristine Bio- and UV-aged Bio-MPs, respectively. The desorption rate of CPF from UV-aged LDPE-MPs within 48 h was lower than the desorption from pristine ones (28.8 ± 7.7% vs. 40.0 ± 3.9%), while both pristine and UV-aged Bio-MPs showed very low CPF desorption rates. A 4-day Petri dish experiment showed that UV-aged MPs were significantly less concentrated in earthworm casts than pristine counterparts (52% and 36% lower for UV-aged LDPE- and Bio-MPs), while UV-aged MPs with adsorbed CPF were concentrated significantly more than UV-aged MPs without CPF. Interestingly, LDPE-MPs aged in soil with a high carbon, nitrogen, and carbon-to-nitrogen ratio were significantly more concentrated in earthworm casts than pristine LDPE-MPs. In conclusion, UV-aged MPs acted as stronger vectors for CPF than pristine MPs. The bioconcentration of MPs differed significantly due to microplastic aging, as well as the combined effect with CPF. Moreover, LDPE-MPs aged in soil with enriched carbon and nitrogen were significantly concentrated in earthworm casts. Further studies on the environmental behaviours of aged MPs associated with other pollutants in soil, especially soils high in carbon and nitrogen, are needed.
Topics: Animals; Microplastics; Plastics; Chlorpyrifos; Polyethylene; Adsorption; Bioaccumulation; Soil; Oligochaeta
PubMed: 37247767
DOI: 10.1016/j.envpol.2023.121910 -
International Journal of Molecular... May 2023is a necrotrophic fungus characterized mainly by its wide host range of infected plants. The deletion of the gene (), which encodes for a blue-light...
is a necrotrophic fungus characterized mainly by its wide host range of infected plants. The deletion of the gene (), which encodes for a blue-light receptor/transcription factor, causes a decrease in virulence, particularly when assays are conducted in the presence of light or photocycles. However, despite ample characterization, the extent of the light-modulated transcriptional responses regulated by BcWCL1 remains unknown. In this study, pathogen and pathogen:host RNA-seq analyses, conducted during non-infective in vitro plate growth and when infecting leaves, respectively, informed on the global gene expression patterns after a 60 min light pulse on the wild-type B05.10 or ∆ strains. The results revealed a complex fungal photobiology, where the mutant did not react to the light pulse during its interaction with the plant. Indeed, when infecting , no photoreceptor-encoding genes were upregulated upon the light pulse in the ∆ mutant. Differentially expressed genes (DEGs) in under non-infecting conditions were predominantly related to decreased energy production in response to the light pulse. In contrast, DEGs during infection significantly differ in the B05.10 strain and the ∆ mutant. Upon illumination at 24 h post-infection in planta, a decrease in the virulence-associated transcripts was observed. Accordingly, after a light pulse, biological functions associated with plant defense appear enriched among light-repressed genes in fungus-infected plants. Taken together, our results show the main transcriptomic differences between wild-type B05.10 and ∆ after a 60 min light pulse when growing saprophytically on a Petri dish and necrotrophically over .
Topics: Photobiology; Arabidopsis; Botrytis; Gene Expression; Plant Diseases; Gene Expression Regulation, Plant
PubMed: 37240051
DOI: 10.3390/ijms24108705 -
Plant Cell, Tissue and Organ Culture 2023This manuscript describes a unique protocol for the rapid transformation of A17 cell suspension cultures mediated by . Medicago cells were collected on day 7 of the...
This manuscript describes a unique protocol for the rapid transformation of A17 cell suspension cultures mediated by . Medicago cells were collected on day 7 of the growth curve, which corresponded to the beginning of the exponential phase. They were then co-cultured with Agrobacterium for 3 days before being spread onto a petri dish with appropriate antibiotic selection. The Receptor Binding Domain of the Spike protein of SARS-CoV-2 was used as a model to develop this protocol. The presence of the transgene was assessed using PCR, and the integrity of the product was evaluated by SDS-PAGE and Western-blotting.
PubMed: 37197004
DOI: 10.1007/s11240-023-02495-6 -
Biology Open May 2023Head and neck cancer (HNC) differs at anatomical sites and hypopharyngeal cancer (HPC) is a type of HNC. The non-surgical treatment option for advanced cases of HPC is...
Head and neck cancer (HNC) differs at anatomical sites and hypopharyngeal cancer (HPC) is a type of HNC. The non-surgical treatment option for advanced cases of HPC is radiotherapy (RT) with or without chemotherapy but survival is poor. Thus, new treatment approaches in combination with RT are essential. Yet, obtaining post-RT treated tumour specimens and lack of animal models with identical anatomical sites are the major translational research barriers. To overcome these barriers, for the first time, we have developed a tumour-stroma based in vitro three-dimensional (3D)-tumouroid co-culture model of HPC by growing FaDu and HS-5 cells together to mimic the complex tumour-microenvironment in a Petri dish. Before growing the cells together, imaging flow cytometry revealed distinct epithelial and non-epithelial characteristics of the cells. Growth rate of the 3D-tumouroid co-culture was significantly higher compared to the tumouroid monoculture of FaDu. Histology and morphometric analysis were done for the characterisation as well as the development of hypoxia was measured by CAIX immunostaining in this 3D-tumouroid co-culture. Taken together, this innovative in vitro 3D model of HPC resembles many features of the original tumour. The wider application of this pre-clinical research tool is in understanding newer combination (e.g. immunotherapy) treatment approaches with RT in HPC and beyond.
Topics: Animals; Coculture Techniques; Hypopharyngeal Neoplasms; Bioengineering; Tumor Microenvironment
PubMed: 37194999
DOI: 10.1242/bio.059949 -
Plants (Basel, Switzerland) Apr 2023Seed germination and seedling growth are highly sensitive to deficit moisture and temperature stress. This study was designed to investigate barley ( L.) seeds'...
Seed germination and seedling growth are highly sensitive to deficit moisture and temperature stress. This study was designed to investigate barley ( L.) seeds' germination and seedling growth under conditions of abiotic stresses. Constant temperature levels of 5, 10, 15, 20, 25, 30, and 35 °C were used for the germination test. Drought and waterlogging stresses using 30 different water levels were examined using two methods: either based at 1 milliliter intervals or, on the other hand, as percentages of thousand kernel weight (TKW). Seedling density in a petri dish and antifungal application techniques were also investigated. Temperature significantly impacted germination time and seedling development with an ideal range of 15-20 °C, with a more comprehensive range to 10 °C. Higher temperatures reversely affected germination percentage, and the lower ones affected the germination and seedling growth rate. Germination commenced at 130% water of the TKW, and the ideal water range for seedling development was greater and more extensive than the range for germination, which means there is a difference between the starting point for germination and the seedling development. Seed size define germination water requirements and provides an objective and more precise basis suggesting an optimal range supply of 720% and 1080% of TKW for barley seedling development. A total of 10 seeds per 9 cm petri dish may be preferable over greater densities. The techniques of priming seeds with an antifungal solution (Bordóilé or Hypo) or antifungal application at even 5 ppm in the media significantly prevented fungal growth. This study is novel regarding the levels and types of abiotic stresses, the crop, the experimental and measurement techniques, and in comparison to the previous studies.
PubMed: 37176849
DOI: 10.3390/plants12091792 -
Plants (Basel, Switzerland) Apr 2023Following the novel weapon hypothesis, the invasiveness of non-native species, such as common ragweed ( L.) can result from a loss of natural competitors due to the...
Following the novel weapon hypothesis, the invasiveness of non-native species, such as common ragweed ( L.) can result from a loss of natural competitors due to the production of chemical compounds, which negatively affect native communities. Particularly the genus Ambrosia produces several types of organic compounds, which have the potential to inhibit germination and growth of other plants. Subsequent to an assessment of the chemical content of three different ragweed extracts (aqueous shoot and root extracts, as well as essential oil), two different trials on the effects of different concentrations of these extracts, as well as ragweed residues, were conducted on two different mediums (Petri dish vs. soil). In addition, we investigated the impact on the infection potential of on soybean roots in three different soil types (arable soil, potting soil, and sand). The results showed that the exposure to common ragweed extracts and residues induced changes in the biomass and root production of crops and ragweed itself. Even though crops and ragweed differed in their response behavior, the strongest negative impact on all crops and ragweed was observed with ragweed residues, leading to reductions in biomass and root growth of up to 90%. Furthermore, we found a decrease in the number of rhizobial nodules of up to 48% when soybean was exposed to ragweed root extract.
PubMed: 37176826
DOI: 10.3390/plants12091768 -
Plant Disease May 2023In July of 2020, a hop (Humulus lupulus L.) grower in Berrien County, Michigan submitted 'Chinook' leaf samples to MSU Plant & Pest Diagnostics. The leaves were covered...
In July of 2020, a hop (Humulus lupulus L.) grower in Berrien County, Michigan submitted 'Chinook' leaf samples to MSU Plant & Pest Diagnostics. The leaves were covered in small, tan colored lesions, with a small chlorotic halo with an approximate diameter of 5 mm. The grower reported that foliar lesions were in the lower 2 m of the fully developed hop canopy. Disease incidence and severity were estimated at approximately 20% and 5 to 10%, respectively. After incubation at 100% relative humidity, acervuli with orange spore masses and a few setae were present. A pure culture was obtained from these sporulating lesions using water agar. The isolate was hyphal tipped onto potato dextrose agar (PDA) and stored in a glycerol-salt solution at -80o C (isolate CL001) (Miles et al. 2011). On PDA, cultures displayed gray growth on the top of the colony and a red color on the underside of the Petri dish. After 14 days, acervuli with no setae appeared exuding orange conidial masses on the surface of the culture. Conidia were hyaline, aseptate, smooth-walled and rounded at the ends and measured on average 15.89 μm (13.81 to 16.91 μm) × 7.26 (6.82 to 8.41 μm) (n = 20). The color and size of the conidia matched other descriptions of C. acutatum sensu lato (Damm et al. 2012). Four loci (ITS/515 bp - OQ026167, GAPDH/238 bp - OQ230832, CHS1/228 bp -OQ230830, and TUB2/491 bp - OQ230831) were amplified from isolate CL001 (using the primers ITS1/ITS4, GDF1/GDR1, CSH-79f/CHS-354R, and T1/Bt-2b, respectively) and had 100% pairwise identity with C. fioriniae 125396 (JQ948299, JQ948629, JQ948960, JQ949950, respectively, Damm et al. 2012). The GAPDH, CSH1, and TUB2 sequences from isolate CL001 were trimmed, concatenated and aligned with 31 different members of Colletotrichum acutatum sensu lato and C. gloesporioides 356878 (Damm et al. 2012; Kennedy et al. 2022). The alignment was then used to produce a maximum likehood phylogenetic tree using Geneious Prime (Biomatters Ltd.) with the PHYML add on using the HKY + G model (G = 0.34) (Guindon et al. 2010). Isolate CL001 had the closest similarity to C. fioriniae with a bootstrap value of 100. Pathogenicity tests were performed on 2 month-old 'Chinook' hop plants. Twelve plants were inoculated with 50 ml of a conidial suspension (7.95 x 106 conidia/ml) of isolate CL001 (n = 6) or water (n = 6) using a spray bottle until runoff. Inoculated plants were sealed in clear plastic bags and grown in a greenhouse at 21o C with a photoperiod of 14 h. After 7 days, lesions appeared on the hop plants inoculated with CL001, but no symptoms appeared on the water inoculated hop plants. Lesions with a chlorotic halo were observed but they were smaller than field lesions and no setae were present (approx. 1 mm in diam.). Leaves were surface sterilized (0.3% sodium hypochlorite solution for 15 s and then rinsed three times) and the leading margin of the lesions or healthy tissue (water control) were placed on 1% ampicillin amended PDA. Fungal isolates on PDA morphologically matched C. fioriniae were recovered from all CL001-inoculated plants. No C. fioriniae isolates were recovered from the water-inoculated plants. Based on conidial morphology, the four loci, and the phylogenetic tree, isolate CL001 was identified as C. fioriniae. This is the first report of Colletotrichum fioriniae (syn = Glomerella acutata var. fioriniae Marcelino & Gouli) infecting common hop and further investigation is needed to determine if management is needed for this pathogen.
PubMed: 37172977
DOI: 10.1094/PDIS-01-23-0195-PDN -
Plant Disease May 2023Strawberry (Fragaria × ananassa Duch.) is a widely cultivated economic crop in China. In April 2022, an unusual wilt disease was observed on strawberry plants (6...
Strawberry (Fragaria × ananassa Duch.) is a widely cultivated economic crop in China. In April 2022, an unusual wilt disease was observed on strawberry plants (6 months' old) in Chenzui town, Wuqing district, Tianjin, China (117°1'E, 39°17'N). The incidence across the greenhouses (≈0.34 ha) was approximately 50 to 75%. The first wilt symptoms were observed on the outer leaves, then the whole seedlings wilted and died. The rhizome of the diseased seedlings changed color and became necrotic and rotted. Symptomatic roots were surface disinfected with 75% ethanol for 30 s, washed with sterile distilled water for three times, and then cut into 3 mm2 pieces (four pieces per seedling) and placed on petri dish with potato dextrose agar (PDA) containing 50 mg/L of streptomycin sulfate and incubated at 26℃ in the dark. After 6 days' incubation, hyphal tips of the growing colonies were transferred onto PDA. Eighty-four isolates belong to five fungal species were obtained from 20 diseased root samples based on their morphological characteristics. To confirm their pathogenicity, ten two-month-old healthy seedlings of strawberry (cv Red Face) planted in sterilized nutrient soil, were inoculated by pouring 50 mL of conidial suspension (107 conidia/mL) (Cai et al. 2021). Another ten seedlings poured with sterile distilled water were used as controls. Each treatment was repeated three times in a greenhouse at 25 to 28℃ and 75% relative humidity under a 12-h photoperiod. After 15 days,only seedlings inoculated with Plectosphaerella (an original percentage of 35.71%) exhibited similar symptoms to those of diseased seedlings originally observed in the field. Seedlings had no symptoms in the control and other fungi inoculation treatments. To fulfill the Koch's postulates, Plectosphaerella isolates were reisolated from each inoculated, symptomatic seedling with a percentage of 100%, but were not recovered from any of the control seedlings. The experiments were repeated twice with similar results. The results indicated that the genus Plectosphaerella was the pathogen causing strawberry wilt. Colonies of the genus Plectosphaerella isolates on PDA were white to cream, and then gradually became salmon pink, with few aerial hyphae and slimy surfaces. Colonies produced numerous hyphal coils with conidiophores. Conidia were 4.56 to 10.07 μm × 1.11 to 4.54 μm (avg. 7.10 × 2.56 μm, n=100), septate or aseptate, ellipsoidal, hyaline and smooth. Such morphological characteristics were identical to those of Plectosphaerella spp. (Palm et al. 1995). For species identification, the ITS region and D1/D2 domain of the 28S rRNA gene of representative isolates (CM2, CM3, CM4, CM5 and CM6) were amplified and sequenced with the primer pair ITS1/ITS4 and NL1/NL4, respectively (White et al. 1990; O'Donnell and Gray 1993). By BLASTn analysis, the obtained sequences of ITS amplicon (ON629742, ON629743, ON629744, ON629745, and ON629746) and D1/D2 domain amplicon (OQ519896, OQ519897, OQ519898, OQ519899, and OQ519900) showed 99.14% to 99.81% identity to sequences of P. cucumerina (MW320463.1 and HQ239025.1) in the NCBI database. A multilocus phylogenetic tree performed by the UPGMA analysis showed that the representative isolates were assigned to the group of P. cucumerina. To our knowledge, this is the first report of P. cucumerina causing strawberry wilt worldwide. This disease may induce serious economic losses in strawberry production, thus effective management strategies should be taken.
PubMed: 37157096
DOI: 10.1094/PDIS-03-23-0544-PDN