Search Results (73)

Background/Objectives: Vaccines targeting melanoma antigens can elicit CD8⁺ T cell responses, but a growing body of work suggests CD4⁺ T cells also play a role in tumor control. Induction of CD4⁺ cells may also support B cells in producing tumor antigen-specific antibodies (Abs). We investigated Abs induced by vaccination with a cocktail of six class II MHC-restricted melanoma peptides (6MHP) and the effect of adjuvant type on Ab isotypes. We hypothesized that the vaccines would induce Abs that respond to different epitopes on individual peptides and that IgG isotype distribution varies with different vaccine adjuvants. Methods: Sera from patients who received a 6MHP vaccine were evaluated with enzyme-linked immunosorbent assays to map epitopes for polyclonal Ab responses to synthetic melanoma peptides. IgG isotypes of Ab responses to 6MHP were assessed in patients who received one of four adjuvants (Incomplete Freund’s Adjuvant (IFA) alone, IFA + polyICLC, IFA + systemic metronomic cyclophosphamide (mCy), or IFA + polyICLC + systemic mCy) to characterize IgG isotype distribution. Results: Epitope mapping revealed that at least 50% of patients had responses to two or more epitopes on the same peptide, suggesting polyclonal Ab responses. Serum evaluation for IgG isotypes showed predominant induction of IgG1 and IgG3. Mean total IgG was highest when IFA and polyICLC were used in combination. Patients who received TLR3 agonist polyICLC had significantly higher concentrations of total IgG, IgG1, and IgG3 compared to patients who did not receive polyICLC. Conclusions: Vaccine-induced Abs may respond to multiple epitopes within the same peptide, warranting further studies into their ability to facilitate antigen uptake and presentation through the formation of large immune complexes. The findings also show that adding polyICLC to IFA can significantly enhance Ab responses. Collectively, this work underscores the immunologic potential of peptide-induced Abs and the importance of adjuvant selection in cancer vaccine design. Full article

Cyanobacteria are prolific producers of specialized metabolites of growing interest for blue biotechnology, transversal to various sectors such as cosmetics, foods and pharmaceuticals. In this work, the marine cyanobacterial strain Salileptolyngbya sp. LEGE 181209, from Cabo Verde, was systematically characterized to resolve its taxonomy, pigments profile, and cytotoxicity assessment. A polyphasic workflow combining 16S rRNA gene phylogenies, 16S–23S ITS secondary structures, p-distance, morphology, and scanning electron microscopy (SEM) was used to establish the taxonomic placement of the strain as a new species of the genus. PCR assays targeting the toxin biosynthetic genes mcyA and anaC, and cytotoxicity assays in HaCaT keratinocytes showed low-to-absent cytotoxicity, supporting a safety-forward profile for downstream use. A sequential extraction with solvents of different polarities yielded complementary pigment fractions profiled by HPLC-PDA and spectrophotometry. Total carotenoids reached 72.7 µg mg⁻¹ of dry extract (DE), the profile being dominated by β-carotene and zeaxanthin (≈42 and 8 µg mg⁻¹ of DE, respectively); chlorophyll-a was also very representative, reaching 85.6 µg mg⁻¹ of DE. Phycobiliproteins dominated the polar fraction, with phycocyanin reaching 150 µg mg⁻¹, followed by sugars (19.7 µg of glucose equivalents mg⁻¹) and phenols (8.8 µg of gallic acid equivalents mg⁻¹). Full article

In this paper, cultivable actinobacteria were isolated, cultured, and identified from the heavily algal-bloomed waters of Taihu Lake using 16S rRNA gene sequencing. Among the isolates, a single strain exhibiting vigorous cyanocidal activity against Microcystis aeruginosa FACHB-905 was selected for further investigation. The cyanocidal efficacy and underlying mechanisms of this strain, designated TH05, were assessed through using chlorophyll content, cyanobacterial inhibition rate, and cyanobacterial cell morphology measurements. In addition, oxidative stress responses, expression of key functional genes in FACHB-905, and variations in microcystin concentrations were comprehensively evaluated. Cyanobacterial blooms caused by Microcystis aeruginosa pose serious ecological and public health threats due to the release of microcystins (MCs). In this study, we evaluated the cyanocidal activity and mechanism of a novel actinomycete strain, Streptomyces sp. TH05. Optimization experiments revealed that a light–dark cycle of 12 h/12 h, temperature of 25 °C, and pH 7 significantly enhanced cyanocidal efficacy. Under these conditions, TH05 achieved an 84.31% inhibition rate after seven days of co-cultivation with M. aeruginosa. Scanning electron microscopy revealed two distinct cyanocidal modes: direct physical attachment of TH05 mycelia to cyanobacterial cells, causing cell wall disruption, and indirect membrane damage via extracellular bioactive compounds. Biochemical analyses showed increased levels of malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT) during the first five days, peaking at 2.47-, 2.12-, and 1.91-fold higher than control levels, respectively, indicating elevated oxidative stress. Gene expression analysis using elf-p as a reference showed that TH05 modulated key genes associated with photosynthesis (PsaB, PstD1, PstD2, RbcL), DNA repair and stress response (RecA, FtsH), and microcystin biosynthesis (McyA, McyD). All genes were upregulated except for RbcL, which was downregulated. In parallel, microcystin content peaked at 32.25 ng/L on day 1 and decreased to 16.16 ng/L by day 9, which was significantly lower than that of the control group on day 9 (29.03 ng/L). These findings suggest that strain TH05 exhibits potent and multifaceted cyanocidal activity, underscoring its potential for application in the biological control of cyanobacterial blooms. Full article

Microcystis, a potentially toxigenic cyanobacterium known to form extensive blooms in eutrophic lakes globally, was investigated in the cold oligotrophic Lake Baikal. We report the isolation of two Microcystis strains, Microcystis aeruginosa and M. novacekii, and document the presence of the latter species in Lake Baikal for the first time. In M. aeruginosa strain BN23, we detected the microcystin synthetase gene mcyE. Liquid chromatography-mass spectrometry revealed the presence of two microcystin variants in BN23, with microcystin-LR, a highly potent toxin, being the dominant form. The concentration of MC-LR reached 540 µg/g dry weight. In contrast, M. novacekii strain BT23 lacked both microcystin synthesis genes and detectable toxins. The habitat waters were characterized as oligotrophic with minor elements of mesotrophy, exhibiting low phytoplankton biomass dominated by the chrysophyte Dinobryon cylindricum (76–77% of biomass), with cyanobacteria contributing 8–10%. The contribution of Microcystis spp. to the total phytoplankton biomass could not be quantified as they were exclusively found in net samples. The water temperature at both sampling stations was ~19 °C, which is considerably lower than optimal for Microcystis spp. and potentially conducive to enhanced microcystin production in toxigenic genotypes. Full article

Chydorus sphaericus is often a dominant cladoceran zooplankton species in water bodies experiencing harmful cyanobacterial blooms. However, its relationship with toxin-producing algae remains largely unexplored. In this study, the feeding behavior of C. sphaericus on colonial cyanobacteria and potentially toxic Microcystis was investigated in a temperate, shallow, eutrophic lake. Liquid chromatographic analyses of phytoplankton marker pigments in C. sphaericus gut content revealed that pigments characteristic of cyanobacteria (identified a zeaxanthin, echinenone, and canthaxanthin) comprised the majority of its diet. Among them, colonial cyanobacteria (marked by the pigment canthaxanthin) were the highly preferred food source despite their minor contribution to phytoplankton biomass. qPCR targeting Microcystis genus-specific mcyE synthase genes, which are involved in microcystin biosynthesis, indicated that potentially toxic strains of Microcystis were present in C. sphaericus gut content throughout its temporal and spatial presence in the lake. The results suggest that the common small cladoceran in eutrophic waters, C. sphaericus, has a close trophic interaction with colonial cyanobacteria (including Microcystis) and may represent an important vector for transferring toxigenic Microcystis to the food web, even under conditions of low Microcystis biomass in the lake water. Full article

The proliferation of harmful cyanobacteria, particularly Microcystis, poses significant risks to drinking and recreational water resources, especially under the influence of climate change. Conventional monitoring methods based on microscopy for harmful cyanobacteria management systems are limited in detecting toxigenic genotypes, hindering accurate risk assessment. In this study, we developed a digital droplet PCR (ddPCR)-based method for the simultaneous quantification of total and toxigenic Microcystis in freshwater environments. We targeted the secA gene, specific to the Microcystis genus, and the mcyA gene, associated with microcystin biosynthesis. Custom-designed primers and probes showed high specificity and sensitivity, enabling accurate detection without cross-reactivity. The multiplex ddPCR assay allowed for concurrent quantification of both targets in a single reaction, reducing the analysis time and cost. Application to field samples demonstrated good agreement with microscopic counts and revealed seasonal shifts in toxigenic genotype abundance. Notably, ddPCR detected Microcystis at very low densities—down to 7 cells/mL in the mixed cyanobacterial communities of field samples—even when microscopy failed, highlighting its utility for early bloom detection. This approach provides a reliable and efficient tool for monitoring Microcystis dynamics and assessing toxin production potential, offering significant advantages for the early warning and proactive management of harmful cyanobacterial blooms. Full article

2,6-Dichloro-1,4-benzoquinone (2,6-DCBQ) is an emerging chlorinated disinfection byproduct (DBP) in bodies of water. However, this compound poses an unknown toxic effect on cyanobacteria. In this study, the toxicological mechanisms of 2,6-DCBQ in Microcystis aeruginosa (M. aeruginosa) were investigated through physiological and nontargeted metabolomic assessments. The results show that 2,6-DCBQ inhibited the growth of M. aeruginosa, reduced its photosynthetic pigment and protein contents, increased the levels of reactive oxygen species, damaged the antioxidant defense system, and aggravated the cytomembrane. Meanwhile, 2,6-DCBQ stimulated the production and release of microcystin-LR (MC-LR) and altered the transcripts of genes associated with its synthesis (mcyA, mcyD) and transport (mcyH). In addition, nontargeted metabolomics of M. aeruginosa cells exposed to 0.1 mg/L 2,6-DCBQ identified 208 differential metabolites belonging to 10 metabolic pathways and revealed the considerable interference caused by 2,6-DCBQ among ABC transporters, the two-component system, and folate biosynthesis. This study deepens the understanding of the physiological and nontargeted metabolomic responses of M. aeruginosa exposed to 2,6-DCBQ, offers insights into the toxic effect of 2,6-DCBQ on M. aeruginosa, and provides a theoretical basis for the ecological risk assessment of emerging DBPs in accordance with water quality criteria. Full article

Grazing by zooplankton can regulate bloom-forming cyanobacteria but can also transfer toxin-producing cells, as well as toxic metabolites, to the food web. While laboratory investigations have provided extensive knowledge on zooplankton and toxic cyanobacteria interactions, information on zooplankton feeding on toxin-producing cyanobacteria in natural water bodies remains scarce. In this study, we quantified Microcystis-specific mcyE synthase genes from the gut contents of various cladoceran and copepod taxa to assess the in situ crustacean community and taxon-specific ingestion of potentially toxic Microcystis in Lake Peipsi, a large eutrophic lake in Estonia, Northern Europe. Microcystis cells with mcyE genes were found in all crustaceans examined. However, some species, such as the cyclopoid copepod Mesocyclops leuckarti, were more efficient in ingesting potentially toxic Microcystis than other co-occurring cladocerans (Daphnia spp., Bosmina spp., Chydorus sphaericus) and copepods (Eudiaptomus gracilis). The amount of toxigenic Microcystis cells grazed by crustacean population changed temporarily, and copepods were the predominant consumers of toxigenic Microcystis during several months of the 5-month study period. Crustacean ingestion of toxigenic Microcystis was not related to Microcystis biomass or mcyE gene copy numbers in the environment but was instead related to the abundance of major crustacean grazers. Our findings emphasize the close interaction between crustacean zooplankton and toxigenic Microcystis, indicating that some species may play a more significant role in linking toxic cells within the food web than others. Full article

Citrullination, a post-translational modification (PTM), plays a critical role in rheumatoid arthritis (RA) by triggering immune responses to citrullinated self-antigens. Some HLA-DRB1 genes encode molecules with the shared epitope (QKRAA/QRRAA) sequence in the peptide-binding groove which preferentially presents citrulline-modified peptides, like vimentin, that intensifies the immune response in RA. In this study, we used computational approaches to evaluate intermolecular interactions between vimentin peptide-ligands (with/without PTM) and HLA-DRB1 alleles associated with a significantly increased risk for RA development. Crystal structures for HLA-DRB1*04:01, *04:04, and *04:05 bound to citrullinated peptides (PDB ID: 4MCY, 4MD5, 6BIR) were retrieved from the Protein Data Bank and non-citrullinated 3D structures were generated by mutating citrulline to arginine. The pHLA complexes were submitted to four rounds (50 ns each) of molecular dynamic simulations (MD) with Gromacs v.2022. Our results show that citrulline strengthens the interaction between vimentin and the HLA-DRB1 molecules, therefore impacting both the peptide affinity to the HLAs and pHLA stability; it also induces more intermolecular hydrogen bond formation during MD in the pHLA. Citrulline prevents repulsion between amino acid 71β and the P4-residue of native vimentin. Thus, vimentin citrullination seems to affect pHLA binding and dynamics, which may influence RA-related immune responses. Full article

Toxigenic cyanobacteria and microcystins in the oligotrophic pelagic zone and mesotrophic bay of Lake Onego—the second largest lake in Europe—were found for the first time. Microscopic analysis revealed that Dolichospermum lemmermannii, D. circinale and D. spiroides dominated in bloom spots in the oligotrophic zone of the lake and D. flos-aquae and Microcystis aeruginosa OKin the eutrophic bay. The abundance of cyanobacteria in bloom spots is potentially hazardous for humans and animals. PCR-analysis showed that mcyA gene involved in microcystin biosynthesis was found in cyanobacteria of the genera Dolichospermum and Microcystis. Five structural variants of intracellular microcystins were detected in a trace amount using high-performance liquid chromatography–mass-spectrometry of high resolution. The most hazardous hepatotoxin, MC-LR, was found only in the eutrophic bay. In the present study, the reasons for the low cyanotoxin content in the phytoplankton dominated by Dolichospermum are discussed. The findings of our study make a significant contribution to the accumulation of facts which state that toxigenic cyanobacterial blooms can occur in large oligotrophic lakes. Full article

Antibiotics can affect the photosynthetic system of Microcystis, potentially altering the balance of carbon and nitrogen, which may influence the synthesis of different microcystin (MC) congeners. However, the regulatory mechanisms by which antibiotics affect the synthesis of various MC congeners in Microcystis remain unknown. In this study, the effects of ciprofloxacin (CIP) on the growth, carbon and nitrogen balance, amino acid composition, mcyB gene expression, and production of different MC congeners were investigated in two toxin-producing strains of Microcystis aeruginosa. The results show that CIP exposure significantly inhibited the growth of both strains, achieving an inhibition rate of 71.75% in FACHB-315 and 41.13% in FACHB-915 at 8 μg/L CIP by the end of the cultivation. The intracellular C:N ratio in FACHB-315 increased by 51.47%, while no significant change was observed in FACHB-915. The levels of leucine, tyrosine, and arginine, as identified and quantified by UPLC-MS/MS, were significantly altered at higher CIP concentrations, leading to a reduction in leucine percentage and a notable increase in tyrosine in both strains, which contributed to a reduction in MC-LR proportion and an increase in MC-RR and MC-YR proportion. Additionally, the expression of the mcyB gene was upregulated by as much as 5.57 times, indicating that antibiotic stress could enhance MC synthesis at the genetic level, contributing to the increased toxicity of cyanobacteria. These findings emphasize the significant role of CIP in the biochemical processes of M. aeruginosa, particularly in MC synthesis and composition, providing valuable insights into the ecological risks posed by antibiotics and harmful cyanobacteria. Full article

Climate change threatens freshwater ecosystems, potentially intensifying cyanobacterial blooms and antibiotic resistance. We investigated these risks in Cosseys Reservoir, New Zealand, using short-term warming simulations (22 °C, 24 °C, and 27 °C) with additional oxidative stress treatments. A metagenomic analysis revealed significant community shifts under warming. The cyanobacterial abundance increased from 6.11% to 20.53% at 24 °C, with Microcystaceae and Nostocaceae proliferating considerably. The microcystin synthesis gene (mcy) cluster showed a strong association with cyanobacterial abundance. Cyanobacteria exhibited enhanced nutrient acquisition (pstS gene) and an upregulated nitrogen metabolism under warming. Concurrently, antibiotic resistance genes (ARGs) increased, particularly multidrug resistance genes (50.82% of total ARGs). A co-association network analysis identified the key antibiotic-resistant bacteria (e.g., Streptococcus pneumoniae and Acinetobacter baylyi) and ARGs (e.g., acrB, MexK, rpoB2, and bacA) central to resistance dissemination under warming conditions. Oxidative stress exacerbated both cyanobacterial growth and ARGs’ proliferation, especially efflux pump genes (e.g., acrB, adeJ, ceoB, emrB, MexK, and muxB). This study demonstrated that even modest warming (2–5 °C) could promote both toxic cyanobacteria and antibiotic resistance. These findings underscore the synergistic effects of temperature and oxidative stress posed by climate change on water quality and public health, emphasizing the need for targeted management strategies in freshwater ecosystems. Future research should focus on long-term impacts and potential mitigation measures. Full article

Cyanobacteria are cosmopolitan organisms; nonetheless, climate change and eutrophication are increasing the occurrence of cyanobacteria blooms (cyanoblooms), thereby raising the risk of cyanotoxins in water sources used for drinking, agriculture, and livestock. This study aimed to determine the presence of cyanobacteria, including toxigenic cyanobacteria and the occurrence of cyanotoxins in the El Pañe reservoir located in the high-Andean region, Arequipa, Peru, to support water quality management. The study included morphological observation of cyanobacteria, molecular determination of cyanobacteria (16S rRNA analysis), and analysis of cyanotoxins encoding genes (mcyA for microcystins, cyrJ for cylindrospermopsins, sxtl for saxitoxins, and AnaC for anatoxins). In parallel, chemical analysis using Liquid Chromatography coupled with Mass Spectrometry (LC-MS/MS) was performed to detect the presence of cyanotoxins (microcystins, cylindrospermopsin, saxitoxin, and anatoxin, among others) and quantification of Microcystin-LR. Morphological data show the presence of Dolichospermum sp., which was confirmed by molecular analysis. Microcystis sp. was also detected through 16S rRNA analysis and the presence of mcyA gene related to microcystin production was found in both cyanobacteria. Furthermore, microcystin-LR and demethylated microcystin-LR were identified by chemical analysis. The highest concentrations of microcystin-LR were 40.60 and 25.18 µg/L, in May and November 2022, respectively. Microcystins were detected in cyanobacteria biomass. In contrast, toxins in water (dissolved) were not detected. Microcystin concentrations exceeded many times the values established in Peruvian regulation and the World Health Organization (WHO) in water intended for human consumption (1 µg/L). This first comprehensive report integrates morphological, molecular, and chemical data and confirms the presence of two toxigenic cyanobacteria and the presence of microcystins in El Pañe reservoir. This work points out the need to implement continuous monitoring of cyanobacteria and cyanotoxins in the reservoir and effective water management measures to protect the human population from exposure to these contaminants. Full article

Cyanobacteria are adaptable and dominant organisms that exist in many harsh and extreme environments due to their great ecological tolerance. They produce various secondary metabolites, including cyanotoxins. While cyanobacteria are well studied in surface waters and some aerial habitats, numerous other habitats and niches remain underexplored. We collected 61 samples of: (i) biofilms from springs, (ii) aerial microbial mats from buildings and subaerial mats from caves, and (iii) water from borehole wells, caves, alkaline, saline, sulphidic, thermal, and iron springs, rivers, seas, and melted cave ice from five countries (Croatia, Georgia, Italy, Serbia, and Slovenia). We used (q)PCR to detect cyanobacteria (phycocyanin intergenic spacer—PC-IGS and cyanobacteria-specific 16S rRNA gene) and cyanotoxin genes (microcystins—mcyE, saxitoxins—sxtA, cylindrospermopsins—cyrJ), as well as amplicon sequencing and morphological observations for taxonomic identification. Cyanobacteria were detected in samples from caves, a saline spring, and an alkaline spring. While mcyE or sxtA genes were not observed in any sample, cyrJ results showed the presence of a potential cylindrospermopsin producer in a biofilm from a sulphidic spring in Slovenia. This study contributes to our understanding of cyanobacteria occurrence in diverse habitats, including rare and extreme ones, and provides relevant methodological considerations for future research in such environments. Full article

A new microcystin-producing mangrove cyanobacterium, Aliinostoc bakau sp. nov., was isolated from a tropical mangrove in Penang, Malaysia, and characterized using combined morphological and phylogenetic approaches. Cultures were established in liquid media of different salinities (0, 7, 14, 21, 28, and 35 ppt). Optimal growth observed at both 7 and 14 ppt was consistent with the origin of the strain from an estuarine mangrove environment. Phylogenetic analysis based on the 16S rRNA gene strongly indicated that the strain is a member of the genus Aliinostoc and is distinct from other currently sequenced species in the genus. The sequences and secondary structure of the 16S–23S ITS region D1–D1’ and Box–B helices provided further confirmation that the new species is clearly distinct from previously described Aliinostoc species. Amplification of the mcyE gene fragment associated with the production of microcystin in A. bakau revealed that it is identical to that in other known microcystin-producing cyanobacteria. Analysis of the extracts obtained from this strain by HPLC-MS/MS confirmed the presence of microcystin variants (MC-LR and -YR) at concentrations of 0.60 μg/L and MC-RR at a concentration of 0.30 μg/L. This is the first record of microcystin production from Aliinostoc species in tropical mangrove habitats. Full article