Balamuthia mandrillaris is an environmentally derived, free-living amoeba that causes fatal meningoencephalitis. We previously isolated B. mandrillaris from soil in the Aomori Prefecture and attempted to culture the cell-free amoeba using liquid medium; however, this was difficult to achieve because of contamination of the medium with endogenous bacteria. The aim of this study was to determine the presence of endogenous bacteria in environmentally derived B. mandrillaris and identify bacteria. Two new environmentally derived B. mandrillaris strains were isolated from soil samples collected throughout Japan. Environmentally derived B. mandrillaris was cultured under nutrient-free conditions for 60 days, and the induced cysts contained large amounts of viable bacteria. The sequence of the endophytic bacteria revealed that the genus Chitinophaga was common between the two strains of B. mandrillaris. The opportunistic pathogens Inquilinus and Brevundimonas were also detected. All of these bacteria were pigment-producing species. Bacterial pigment production helps protect organisms from extremes of heat and cold, increases the virulence of pathogenic strains, and protects organisms from protein and DNA damage caused by UV light and ionizing radiation. This suggests that B. mandrillaris preserving bacteria in a viable state for a long time under severe conditions with no nutrition may be the ability of the bacteria to produce pigments.

Perchlorate (ClO4 -) is a type of novel persistent inorganic pollutant that has gained increasing attention because of its high solubility, poor degradability, and widespread distribution. However, the impacts of perchlorate on aquatic autotrophs such cyanobacterium are still unclear. Herein, Synechocystis sp. PCC6803 (Synechocystis) was used to investigate the response mechanisms of perchlorate on cyanobacterium by integrating physiological and transcriptome analyses. Physiological results showed that perchlorate mainly damaged the photosystem of Synechocystis, and the inhibition degree of photosystem II (PSII) was severer than that of photosystem I (PSI). When the exposed cells were moved to a clean medium, the photosynthetic activities were slightly repaired but still lower than in the control group, indicating irreversible damage. Furthermore, perchlorate also destroyed the cellular ultrastructure and induced oxidative stress in Synechocystis. The antioxidant glutathione (GSH) content and the superoxide dismutase (SOD) enzyme activity were enhanced to scavenge harmful reactive oxygen (ROS) in Synechocystis. Transcriptome analysis revealed that the genes associated with photosynthesis and electron transport were significantly regulated. For instance, most genes related to PSI (e.g., psaf, psaf) and the electron transport chain were upregulated, whereas most genes related to PSII (e.g., psbA3, psbD1, psbB, and psbC) were downregulated. Additionally, perchlorate also induced the expression of genes related to the antioxidant system (sod2, gpx, gst, katG, and gshB) to reduce oxidative damage. Overall, this study is the first to investigate the impacts and mechanisms of cyanobacterium under perchlorate stress, which is conducive to assessing the risk of perchlorate in aquatic environments. (c) 2024 The Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences. Published by Elsevier B.V.

The earthworm -based vermiremediation facilitated with benign chemicals such as nano zero-valent iron (nZVI) is a promising approach for the remediation of a variety of soil contaminants including cyanotoxins. As the most toxic cyanotoxin, microcystin-LR (MC-LR) enter soil via runoff, irrigated surface water and sewage, and the application of cyanobacterial biofertilizers as part of the sustainable agricultural practice. Earthworms in such remediation systems must sustain the potential risk from both nZVI and MC-LR. In the present study, earthworms (Eisenia fetida) were exposed up to 14 days to MC-LR and nZVI (individually and in mixture), and the toxicity was investigated at both the organismal and metabolic levels, including growth, tissue damage, oxidative stress, metabolic response and gut microbiota. Results showed that co -exposure of MC-LR and nZVI is less potent to earthworms than that of separate exposure. Histological observations in the co -exposure group revealed only minor epidermal brokenness, and KEGG enrichment analysis showed that co -exposure induced earthworms to regulate glutathione biosynthesis for detoxification and reduced adverse effects from MC-LR. The combined use of nZVI promoted the growth and reproduction of soil and earthworm gut bacteria (e.g., Sphingobacterium and Acinetobacter) responsible for the degradation of MC-LR, which might explain the observed antagonism between nZVI and MC-LR in earthworm microcosm. Our study suggests the beneficial use of nZVI to detoxify pollutants in earthworm -based vermiremediation systems where freshwater containing cyanobacterial blooms is frequently used to irrigate soil and supply water for the growth and metabolism of earthworms. (c) 2024 The Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences. Published by Elsevier B.V.

Cyst nematodes are among the major plant-parasitic nematodes worldwide, and they cause significant damage to Brassicaceae crops, including Kimchi-cabbage, in Korea. To survey the incidence of cyst nematodes in Kimchi-cabbage fields, 469 soil samples were collected from the main producing areas between 2018 and 2021. Only cyst nematodes belonging to the genus Heterodera were investigated, and the overall nematode incidence was found to be 40%. Regionally, the highest incidence was observed in Taebaek, reaching 89%, with mean densities of cysts and eggs per 500 cm3 of soil recorded at 522 and 49,734, respectively. Based on Bayesian analysis of the mitochondrial DNA cytochrome c oxidase subunit I gene sequence, the cyst nematodes were identified as four species: clover cyst nematode (Heterodera schachtii, 4%), and white soybean cyst nematode (H. sojae, HSo, 2%). Mixed infestations were found in some fields, with HT + HG (4%) and HG + HSo (1%). These results indicate that HT is the dominant species in the main Kimchi-cabbage producing areas in Korea. In conclusion, implementing effective HT management strategies is critical to minimize economic losses in Kimchi-cabbage production in Korea.

Cyst nematodes, some of the most important plantparasitic nematodes globally, cause major damage to Chinese cabbage and soybean plants in Korea. Cysts are commonly used for cyst nematode bioassays because many eggs are included inside cyst. Traditionally, cysts are extracted from the soil using the paper strip method or the centrifugal flotation method (CFM) combined with sieving. The specific gravity of sugar solution (SGSS) is often used in the CFM; however, the efficiency of cyst extraction and egg hatching in the CFM has not been studied. In this study, we assessed the effects of SGSS in a specific gravity range of 1.15 to 1.30 in the CFM on the cyst extraction and egg hatching of clover cyst nematode (Heterodera trifolii) and sugar beet cyst nematode (H. schachtii). High SGSS in the CFM within the range of 1.15 to 1.30 was positively correlated with the extraction of more cysts. Egg-hatching rates were not different between SGSSs, indicating that SGSS did not directly affect egg-hatching rates. These results showed that the cysts of cyst nematodes can be efficiently extracted with high SGSS in the CFM.

PurposeAcanthamoeba species are eucaryotic protozoa found predominantly in soil and water. They cause ulceration and vision loss in the cornea (Acanthamoeba keratitis) and central nervous system (CNS) infection involving the lungs (granulomatous amoebic encephalitis). Antiparasitic drugs currently used in the treatment of infections caused by Acanthamoeba species are not effective at the desired level in some anatomical regions such as the eye and CNS. The existence of an agent effective against both cysts and trophozoites has not yet been proven. Drugs used for treatment of Acanthamoeba infrections are still limited.MethodThe present study investigates amoebicidal activites of various concentrations of ethanolic fruit extract of E. umbellata (EU) (40, 20, 10, 5, 2.5, 1.25, 0.625 mM/mL), silver nanoparticles (AgNP) that are synthesized from EU and confirmed with characterization tests (20, 10, 5, 1, 0.5 mM/mL), and lauric acid (LA) in EU detected with gas chromatography-mass spectrometry (GC-MS) against A. castellanii trophozoites. In addition, DNA-preserving activities of EU, AgNP and LA were studied on pBR322 plasmid DNA, following damage induced with hydroxyl radical (-OH). Cytotoxicity of EU over HeLa cells was examined with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Furthermore, the effects over the expression of SOD and CAT genes, which are coding oxidative stress enzymes in trophozoites, and expression of genes responsible for pseudocyst and cyst formation (CSII and CSP21, respectively) were investigated following methanol-induced stress, with reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR).ResultsAt highest concentrations, EU, AgNP and LA showed lethal effects against majority of trophozites at 24 th h and against all trophozoites at 48th hour. EU at 5 mg/mL concentration and LA at 1, 0.8, 0.6, 0.4 mM/mL concentrations prevented DNA damage. A dose-dependent decrease in cell viability was observed, EU was found to be non-cytotoxic for 53.82% of HeLa cells at 72 nd h even at 40 mg/mL concentration. Greatest inhibitory effects were found with EU, AgNP and LA on CSII, EU on CAT, LA on CSP21, and hydrogen peroxide (H2O2) on SOD genes.ConclusionThe findings of this study show that EU, LA and AgNPs can be used in a controlled manner to combat A. castellanii infections by reducing or blocking the activity of the parasite's antioxidant enzymes (SOD and CAT), without giving the parasite a chance to initiate the process of pseudocyst or proper cyst formation.

Lead (Pb2+) ions give an imminent danger since they have been known to cause persistent damage to humans, plants, and animals, even at low concentrations, and cysteine (Cys) elevated levels are critical indicators for many diseases. Therefore, their detection is critical in pharmaceutical and environmental samples. This study tailored an innovative fluorescence switch off-on assay to detect Pb2+ and Cys based on the amplification of G-quadruplex (G-4) to N-methylmesoporphyrin IX (NMM). This assay operates on the fluorescence of NMM serving as a signal reporter which could be enhanced by an adenine-guanine-rich probes G-4. Initially, the fluorescence of NMM was increased after binding with G-4 and Pb2+ and effectively quenching fluorescence without altering the structure of G-4. As it was proved by Circular dichroism (CD). The number of binding sites for Pb2+ per NMM was determined to be 0.80 with a binding constant of 1.9 x 10(4) mol /L. The presence of Cys may disrupt the interaction between Pb2+ and G-4/NMM due to its stronger binding affinity towards Pb2+ leading to high fluorescence recovery.The assay demonstrated the capability to detect Pb2+ within a concentration range of 0.4 to 1.6 mu M, achieving a high correlation coefficient (R-2 = 0.985). with the detection limit of 0.45 mu M was established. Similarly, Cys was effectively detected across a range of 1 to 6 mu M, possessing correlation (R-2 = 0.973) with a detection limit of 1.51 mu M, further confirming that the detection limit is not influenced by the starting point of the linear range. The assay detected these compounds among various other amino acids and heavy metals. Our approach is simple and innovative, enabling the accurate determination of Pb2+ and Cys concentrations in soil and medicinal samples, highlighting its potential in practical diagnostic and environmental applications.

The soybean cyst nematode (SCN; Heterodera glycines Ichinohe) is a significant agricultural pest that causes extensive damage to soybean production worldwide. Second-stage juveniles (J2s) of the SCN migrate through the soil and infest the roots of host plants in response to certain chemical substances secreted from the host roots. Therefore, controlling SCN chemotaxis could be an effective strategy for its management. In the present study, we identified the Hg-gpa-3d gene, which encodes the G protein alpha subunit, as a key regulator of SCN chemotaxis. Gene silencing of Hg-gpa-3d reduced the attraction of SCN J2s to host roots, as well as to nitrate ions, a chemoattractant recognized through a mechanism different from that of host recognition. However, silencing of Hg-gpa-3d did not affect avoidance behavior towards unpleasant temperatures or stylet protrusion. These results suggest that Hg-gpa-3d is a crucial gene in the regulation of SCN chemotaxis and provide new insights into the chemotactic mechanisms of the SCN.

In recent years, the increase in environmental pollutants has been one of the most important factors threatening human and environmental health. Arsenic, a naturally occurring element found in soil, water, and air, easily enters the human body and leads to many metabolic disorders. In this study, we focused on the possible protective effects of N-acetylcysteine (NAC) against sodium arsenite (As)-induced toxic effects on embryonic fibroblast cells. The effects of As and NAC treatment on cells were evaluated, including cytotoxicity, oxidative stress, and apoptosis. Embryonic fibroblast cells were exposed to As (ranging from 0.01 mu M to 10 mu M) and NAC (at a concentration of 2 mM) for 24 h. The assessment of cytotoxicity markers, such as cell viability and lactate dehydrogenase (LDH), showed that As significantly reduced cell viability and increased LDH levels. Furthermore, we observed that As increased the amount of reactive oxygen species (ROS) in the cell, decreased the activity of antioxidant enzymes, and triggered apoptosis in cells. Additionally, our research revealed that the administration of NAC mitigates the detrimental effects of As. The results showed that As exerted hazardous effects on embryonic fibroblast cells through the induction of oxidative stress and apoptosis. In this context, our study provides evidence that NAC may have a protective effect against the toxicity of As in embryonic fibroblast cells.

Phytophthora, one among the most devastating phytopathogenic genus representing the oomycetes inflicts substantial damage to a broad spectrum of economically important horticultural crops worldwide. The rapid dissemination of Phytophthora in agro-ecosystems is mediated through bi-flagellated zoospores and their homing response towards the host is profoundly influenced by chemo-electrotactic mechanisms leading to encystment and subsequent colonization. Though different procedures are reported to isolate Phytophthora from infested rhizospheric soils, studies addressing zoospore encystment-based method using different baits for isolation and subsequent retrieval of cultures (in case of bacterial contamination) are meagre in Phytophthora-black pepper host-pathosystem. In the present study, Phytophthora was isolated from infested rhizospheric soils of black pepper using different baits viz., thermocol, impregnated paper discs, leaves of Albizzia falcataria and green gram roots. Modified cabin-sequestering methods were subsequently adopted to salvage the cultures in case of bacterial contamination, if any. The zoospore encystment-based method is cost effective and consumes lesser time for isolation of Phytophthora from infested rhizospheric soils of black pepper. Further, the two approaches of cabin sequestering method which were found promising in obtaining pure cultures of Phytophthora devoid of bacterial contamination, can also be employed to retrieve bacterial contaminated cultures of research significance.