Duplex Real-Time Fluorescent Quantitative PCR Assays for the Detection of Toxigenic Microcystis Genotypes Based on SNP/InDel Variation in mcy Gene Cluster.

In this study, the toxigenic characteristics of 14 strains of Microcystis were analyzed, and single nucleotide polymorphism (SNP) and insertion/deletion (InDel) loci in microcystin synthetase (mcy) gene clusters were screened. Based on SNP and InDel loci associated with the toxigenic characteristics, primers and TaqMan or Cycling fluorescent probes were designed to develop duplex real-time fluorescent quantitative PCR (FQ-PCR) assays. After evaluating specificity and sensitivity, these assays were applied to detect the toxigenic Microcystis genotypes in a shrimp pond where Microcystis blooms occurred. The results showed a total of 2155 SNP loci and 66 InDel loci were obtained, of which 12 SNP loci and 5 InDel loci were associated with the toxigenic characteristics. Three duplex real-time FQ-PCR assays were developed, each of which could quantify two genotypes of toxigenic Microcystis. These FQ-PCR assays were highly specific, and two Cycling assays were more sensitive than TaqMan assay. In the shrimp pond, six genotypes of toxigenic Microcystis were detected using the developed FQ-PCR assays, indicating that above genotyping assays have the potential for quantitative analysis of the toxigenic Microcystis genotypes in natural water.

Polyamidoamine dendrimer-mediated hydrogel for solubility enhancement and anti-cancer drug delivery.

The application of hydrogels for anti-cancer drug delivery has garnered considerable interest in the medical field. Current cancer treatment approaches, such as chemotherapy and radiation therapy, often induce severe side effects, causing significant distress and substantial health complications to patients. Hydrogels present an appealing solution as they can be precisely injected into specific sites within the body, facilitating the sustainable release of encapsulated drugs. This localized treatment approach holds great potential for reducing toxicity levels and improving drug delivery efficacy. In this study we developed a hydrogel delivery system containing polyamidoamine (PAMAM) dendrimer and polyethylene glycol (PEG) for solubility enhancement and sustained delivery of hydrophobic anti-cancer drugs. The three selected model drugs, e.g. silibinin, camptothecin, and methotrexate, possess limited aqueous solubility and thus face restricted application. In the presence of vinyl sulfone functionalized PAMAM dendrimer at 45 mg/mL concentration, drug solubility is increased by 37-fold, 4-fold, and 10-fold for silibinin, camptothecin, and methotrexate, respectively. By further crosslinking of the functionalized PAMAM dendrimer and thiolated PEG, we successfully developed a fast-crosslinking hydrogel capable of encapsulating a significant payload of solubilized cancer drugs for sustained release. In water, the drug encapsulated hydrogels release 30%-80% of their loads in 1-4 days. MTT assays of J82 and MCF7 cells with various doses of drug encapsulated hydrogels reveal that cytotoxicity is observed for all three drugs on both J82 and MCF7 cell lines after 48 h. Notably, camptothecin exhibits higher cytotoxicity to both cell lines than silibinin and methotrexate, achieving up to 95% cell death at experimental conditions, despite its lower solubility. Our experiments provide evidence that the PAMAM dendrimer-mediated hydrogel system significantly improves the solubility of hydrophobic drugs and facilitates their sustained release. These findings position the system as a promising platform for controlled delivery of hydrophobic drugs for intratumoral cancer treatment.

Effects of microcystin on protein profile in hepatopancreas of Litopenaeus vannamei.

For intensive aquaculture in freshwater ponds, microcystin (MC-LR) generated from cyanobacterial blooms is one of the bottlenecks for the healthy and sustainable development of shrimp aquaculture industry. In this study, we measured the MC-LR content in the hepatopancreas and muscles of Litopenaeus vannamei stressed by MC-LR, and analyzed protein expression in the hepatopancreas using DIA high-throughput proteomics technology. The results showed that MC-LR content in the hepatopancreas and muscles reached the highest at 1 h after MC-LR injection, which was (6.12±0.45) µg·kg-1 and (5.00±0.19) µg·kg-1, respectively. Then, it decreased gra-dually, with that in the hepatopancreas being significantly higher than in muscles. We identified 820 differential expressed proteins, including 586 up-regulated and 234 down-regulated ones. Results of bioinformatics analysis showed that MC-LR stress significantly affected immune-related pathways such as lysosome, RIG-Ⅰ receptor signals and interleukin-2. It also altered energy metabolisms including citrate cycle, metabolism of starch and sucrose, and interconversion of pentose and glucoronate, which in turn led to the disorder of carbohydrate metabolism. In addition, MC-LR significantly upregulated 19 cytoskeleton-related blood shadow proteins and damaged the hepatopancreas cytoskeleton. It was concluded that MC-LR mainly affected the physiological processes associated with immunity, energy metabolism, and cytoskeleton in the hepatopancreas of L. vannamei.

The role of attached bacteria in the formation of Microcystis colony in Chentaizi River.

To further understand the role of attached bacteria in the formation of Microcystis colonies, we conducted a field investigation in Chentaizi River in Tianjin, China, which frequently suffers Microcystis blooms in summer. The results showed the average cell density of Microcystis was 2.31 × 107 cell/L from July 19 to July 27, 2021. Free-living and attached bacteria communities shared similar phylum diversity, but the abundance changed obviously. The colony size of Microcystis and attached bacterial number in the colony showed an increasing trend during the whole sampling period. There was a significant positive correlation between Microcystis colonial size and attached bacterial density (P < 0.01), indicating attached bacteria could contribute the colony formation of Microcystis. The genus composition in attached bacterial community varied in colonies of different sizes. The relative abundance of Acinetobacter, Cloacibacterium, Sphingobacterium, and Ralstonia in >90 µm colonies were significantly higher than those in 8-20 µm and 20-90 µm colonies (P < 0.05). These genera might have positive effects on the colony formation of Microcystis during sampling.

Significant Enhancement of Two-Photon Excited Fluorescence in Water-Soluble Triphenylamine-Based All-Organic Compounds.

Understanding water-soluble and environmentally friendly two-photon absorption (TPA) molecules benefits the design of superior organic complexes for biomedical, illumination, and display applications. In this work, we designed two triphenylamine-based all-organic compounds and explored the mechanism of enhanced TP fluorescence in water solutions for potential applications. Experimentally, we showed that adding protein into our TPA molecule solution can drastically boost the TP fluorescence. Numerical simulations reveal that the TPA molecules prefer to dock inside the protein complex. We hypothesize that the interaction between our triphenylamine-based all-organic compounds and water molecules lead to non-radiative decay processes, which prevent strong TP fluorescence in the water solution. Therefore, the protection by, for example, protein molecules from such interactions can be a universal strategy for superior functioning of organic TPA molecules. Further experiments and numerical simulations support our hypothesis. The present study may facilitate the design of superior water-soluble and environmentally friendly superior organic complexes.

Light limitation inducing overcompensatory growth of cyanobacteria and function of serine/threonine kinase (STK) genes involved.

The rapid overcompensatory growth that appears when cyanobacteria are supplied with adequate resources after a period of resource deprivation might contribute to the occurrence of cyanobacterial blooms. We investigated the changing characteristics of overcompensatory growth and serine/threonine kinase (STK) genes expression of cyanobacterium Microcystis aeruginosa in response to light limitation. The results showed M. aeruginosa exhibited overcompensatory growth for 2 days after light recovery, during which the increase in growth was inversely related to light intensity. Expression of STK genes, such as spkD, was upregulated significantly at 0.5-4 h after light recovery (P < 0.05). To investigate the function of STK genes in the overcompensatory growth, M. aeruginosa spkD was heterologously expressed in Synechocystis. Transgenic Synechocystis exhibited greater and longer overcompensatory growth than wild-type Synechocystis after light recovery. Relative expression levels of STK genes in transgenic Synechocystis were significantly higher than those in wild-type Synechocystis at 24 h of light recovery (P < 0.05). Heterologous expression of Microcystis spkD might stimulate overcompensatory growth of Synechocystis by affecting its STK gene expression.

Serine/threonine Kinases Play Important Roles in Regulating Polyunsaturated Fatty Acid Biosynthesis in Synechocystis sp. PCC6803.

Serine/threonine kinases (STKs) play important roles in prokaryotic cellular functions such as growth, differentiation, and secondary metabolism. When the external environment changes, prokaryotes rely on signal transduction systems, including STKs that quickly sense these changes and alter gene expression to induce the appropriate metabolic changes. In this study, we examined the roles of the STK genes spkD and spkG in fatty acid biosynthesis in the unicellular cyanobacterium Synechocystis sp. PCC6803, using targeted gene knockout. The linoleic acid (C18: 2), γ-linolenic acid (C18: 3n6), α-linolenic acid (C18: 3n3), and stearidonic acid (C18: 4) levels were significantly lower in spkD and spkG gene knockout mutants than in the wild type at a culture temperature of 30°C and a light intensity of 40 µmol⋅m-2⋅s-1. The expression levels of fatty acid desaturases and STK genes differed between the spkD and spkG gene knockout mutants. These observations suggest that spkD and spkG may directly or indirectly affect the fatty acid composition in Synechocystis sp. PCC6803 by regulating the expression of fatty acid desaturases genes. Therefore, the STK genes spkD and spkG play important roles in polyunsaturated fatty acid biosynthesis in Synechocystis sp. PCC6803. These findings could facilitate the development of cyanobacteria germplasm resources that yield high levels of fatty acids. In addition, they provide a theoretical basis for the genetic engineering of cyanobacteria with improved yields of secondary metabolites and increased economic benefits.

Enhanced Two-Photon Absorption in Two Triphenylamine-Based All-Organic Compounds.

Two-photon absorption (TPA) enables the excitation of molecules by comparatively lower energy photons with longer penetration depth and higher spatial precision control, which advances the uses of organic molecules in various applications. In this work, we report two simple all-organic molecules C42H33N (compound 3) and C138H168N4 (compound 14) with strong TPA and fluorescent emission activity. Density functional theory calculations show that the enhanced oscillator strengths could be responsible for improved TPA and emission activity for compound 14 compared to those for 3. The degradation of C138H168N4 under focused laser illumination without circulation is almost negligible within 5 h, making it a candidate for laser dyes. Solid-state measurements confirm the presence of a direct band gap for electron transition that determines the channel to release the absorbed energy and functionality of the studied molecules. This work emphasizes that a high TPA cross-section and selectable energy relaxation (fluorescent emission or heat dissipation) are equally important to the design of advanced functional TPA molecules.

Comparing effects of berberine on the growth and photosynthetic activities of Microcystis aeruginosa and Chlorella pyrenoidosa.

Berberine is a potent algicidal allelochemical of Microcystis aeruginosa. To optimize its application in the control of Microcystis blooms, the effects of berberine on the growth and photosynthetic activities of M. aeruginosa and a non-target green alga, Chlorella pyrenoidosa, were compared. The results showed that the algicidal activity of berberine on M. aeruginosa was light dependent. Berberine had no algicidal effects on C. pyrenoidosa with or without light exposure. Under light-dark conditions, berberine significantly decreased the chlorophyll fluorescence parameters in M. aeruginosa while no significant berberine-induced changes were observed under constant darkness. Significant reductions of photosystem II (PSII) and whole chain electron transport activities in M. aeruginosa exposed to berberine suggested that PSII was the important target site attacked by berberine. Contrary to M. aeruginosa, no berberine-induced inhibition in photosynthesis activities were observed in C. pyrenoidosa. The differences in photosynthetic apparatuses of these two algae might be responsible for their different sensitivities to berberine.

Microcystins distribution, bioaccumulation, and Microcystis genotype succession in a fish culture pond.

In freshwater aquaculture ponds, cyanobacterial blooms and microcystins (MCs) pollution have attracted considerable attention due to their toxic effects. To provide an insight into cyanobacterial problems in aquaculture ponds, MCs distribution, bioaccumulation, and Microcystis genotype succession in a fishpond were investigated from May 2017 to November 2017. The distribution of MCs in filtered water, seston, and sediment varied considerably among months. MCs concentrations in filtered water, seston, and sediment ranged from 1.16 to 3.66 µg/L, 0.64 to 13.98 µg/g DW, and 1.34 to 5.90 µg/g DW, respectively. In addition, chemical oxygen demand was positively correlated with sestonic MCs concentrations. MCs concentrations accumulated in different tissues of market-size fish were in the order of liver > kidney > intestine > muscle. MCs content in muscle was 4.3 times higher than the WHO recommended tolerable daily intake level. Twenty-four ITS genotypes of Microcystis were identified from a total of 653 sequences. During the survey period, considerable genotype variation and rapid genotype succession were observed and dominant genotype was absent. A redundancy analysis revealed that Microcystis genotypes could significantly influence the variations in the proportions of the potentially toxic Microcystis, which could in turn influence the MCs concentrations in seston.

A comparative study of the effects of microbial agents and anaerobic sludge on microalgal biotransformation into organic fertilizer.

The effects of Lactobacillus bulgaricus, Rhodopseudomonas palustris, Issatchenkia orientalis and anaerobic sludge on anaerobic digestion of microalgae to organic fertilizer were studied. High-throughput sequencing was used to analyze characteristics of microbial community structure during anaerobic digestion of microalgae using different inocula. Lactobacillales and Saccharomycetales were more likely to become dominant bacteria and eukaryotes. The relative abundance of Lactobacillales was 98.15%, 88.61% and 81.73% of total bacteria at the beginning, middle and end of the experiment, respectively. Meanwhile, the relative abundance of Saccharomycetales was 90.91%, 98.41% and 98.8% of eukaryotes at the beginning, middle and end of the experiment, respectively. At the end of digestion, the microcystin content in the reactor inoculated with Issatchenkia orientalis decreased to 0.71 µg/kg, which met drinking water standards. Rhodopseudomonas palustris did not become a dominant microorganism and had the most negative impact on the atmosphere. Volatile organic compounds were 11.92 mg/kg while the odor concentration reached 97,724 ou/m3. The organic matter content in reactors inoculated with specific groups of microbial agents, which was higher than the standard required for bio-organic fertilizer, occupying over 96% dry weight. In addition, the effective microorganism counts of Issatchenkia orientalis and Lactobacillus bulgaricus in fermentation products reached 1.8E+09 colony-forming units (cfu)/g and 1.6E+09 cfu/g, respectively, which are suitable values for microbial fertilizer.

Effects of Bacillus subtilis on the growth, colony maintenance, and attached bacterial community composition of colonial cyanobacteria.

In freshwater aquaculture ponds, application of algicidal Bacillus is a promising way in the control of cyanobacterial blooms. To best understand Bacillus algicidal characters and mechanisms in the field, different-sized colonial cyanobacteria were isolated from an aquaculture pond, and the effects of B. subtilis on their growth, colony maintenance, and colony-attached bacterial community composition were investigated. The results showed that B. subtilis could inhibit the growth of colonial cyanobacteria. Bigger-sized colonies isolated from the field could spontaneously disintegrate into smaller-sized colonies in the laboratory. Algicidal B. subtilis could accelerate the disintegration of colonies and decrease colony size. B. subtilis not only decreased the colony-attached bacterial community diversity but also changed its composition. B. subtilis increased the relative abundances of some attached bacterial genera, including Pseudomonas, Shewanella, Bacillus, Shinella, Rhizobium, and Ensifer. These bacteria with algicidal, microcystin-degrading, and flocculating activities might be an important contributor to algicidal effects of B. subtilis on colonial cyanobacteria.

Polyamidoamine dendrimer-PEG hydrogel and its mechanical property on differentiation of mesenchymal stem cells.

BACKGROUND: Biocompatible hydrogel systems with tunable mechanical properties have been reported to influence the behavior and differentiation of mesenchymal stem cells (MSCs). OBJECTIVE: To develop a functionalized hydrogel system with well-defined chemical structures and tunable mechanical property for regulation of stem cell differentiation. METHODS: An in situ-forming hydrogel system is developed by crosslinking vinyl sulfone functionalized polyamidoamine (PAMAM) dendrimer and multi-armed thiolated polyethylene glycol (PEG) through a thiol-ene Michael addition in aqueous conditions. The viability and differentiation of MSCs in hydrogels of different stiffness are conducted for 21 days under corresponding induction media. RESULTS: MSCs are viable in synthesized hydrogels after 48 hours of culture. By varying the concentrations of PAMAM dendrimer and PEG, hydrogels of different gelation time and stiffness are achieved. The MSC differentiation indicates that more osteogenic differentiation is observed in hard gel (5,663 Pa) and more adipogenic differentiation is observed in soft gel (77 Pa) in addition to the differentiation caused by each individual induction media during the process of culture. CONCLUSIONS: A biocompatible in situ-forming hydrogel system is successfully synthesized. This hydrogel system has shown influences on differentiation of MSCs and may potentially be important in developing therapeutic strategies in medical applications.

Effects of toxic Microcystis genotypes on natural colony formation and mechanism involved.

The cyanobacterium Microcystis occurs as colonies of different sizes with varying abundance of toxic genotypes versus non-toxic genotypes under natural conditions. To investigate the effects of toxic Microcystis genotypes on natural colony formation, samples collected from the mainstream of Haihe River from July to October 2015 were sieved into four colony classes with sizes of <8 µm, 8-20 µm, 20-90 µm and >90 µm. Each colony size class was analyzed for the proportion of toxic Microcystis genotypes, and microcystins (MCs) cellular production of toxic genotypes. The results showed the smallest size class of Microcystis colonies (<8 µm) showed the lowest proportion of toxic genotypes and the highest MC-RR and MC-YR cellular production. With the increasing colony sizes, the proportion of toxic Microcystis genotypes increased but the MC-RR and MC-YR cellular production decreased. A negative correlation between the MCs cellular production and the proportion of toxic genotypes was observed in all four colony size classes, suggesting that the less there were toxic Microcystis cells able to produce MCs, the more each toxic cell needed to produce that molecule. Toxic Microcystis played an important role in the colony formation in natural waters via producing MCs.

Influence of Cross-Linkers on the in Vitro Chondrogenesis of Mesenchymal Stem Cells in Hyaluronic Acid Hydrogels.

This study aims to investigate the effect of the structures of cross-linkers on the in vitro chondrogenic differentiation of bone mesenchymal stem cells (BMSCs) in hyaluronic acid (HA)-based hydrogels. The hydrogels were prepared by the covalent cross-linking of methacrylated HA with different types of thiol-tailored molecules, including dithiothreitol (DTT), 4-arm poly(ethylene glycol) (PEG), and multiarm polyamidoamine (PAMAM) dendrimer using thiol-ene "click" chemistry. The microstructure, mechanical properties, diffusivity, and degradation rates of the resultant hydrogels were controlled by the structural feature of different cross-linkers. BMSCs were then encapsulated in the resulting hydrogels and cultured in chondrogenic conditions. Overall, chondrogenic differentiation was highly enhanced in the PEG-cross-linked HA hydrogels, as measured by glycosaminoglycan (GAG) and collagen accumulation. The physical properties of hydrogels, especially the mechanical property and microarchitecture, were resulted from the structures of different cross-linkers, which subsequently modulated the fate of BMSC differentiation.

Sequestration and Distribution Characteristics of Cd(II) by Microcystis aeruginosa and Its Role in Colony Formation.

To investigate the sequestration and distribution characteristics of Cd(II) by Microcystis aeruginosa and its role in Microcystis colony formation, M. aeruginosa was exposed to six different Cd(II) concentrations for 10 days. Cd(II) exposure caused hormesis in the growth of M. aeruginosa. Low concentrations of Cd(II) significantly induced formation of small Microcystis colonies (P < 0.05) and increased the intracellular polysaccharide (IPS) and bound extracellular polysaccharide (bEPS) contents of M. aeruginosa significantly (P < 0.05). There was a linear relationship between the amount of Cd(II) sequestrated by algal cells and the amount added to cultures in the rapid adsorption process that occurred during the first 5 min of exposure. After 10 d, M. aeruginosa sequestrated nearly 80% of 0.2 mg L-1 added Cd(II), while >93% of Cd(II) was sequestrated in the groups with lower added concentrations of Cd(II). More than 80% of the sequestrated Cd(II) was bioadsorbed by bEPS. The Pearson correlation coefficients of exterior and interior factors related to colony formation of M. aeruginosa revealed that Cd(II) could stimulate the production of IPS and bEPS via increasing Cd(II) bioaccumulation and bioadsorption. Increased levels of cross-linking between Cd(II) and bEPS stimulated algal cell aggregation, which eventually promoted the formation of Microcystis colonies.

Thiol-ene crosslinking polyamidoamine dendrimer-hyaluronic acid hydrogel system for biomedical applications.

A series of alkene functionalized polyamidoamine (PAMAM) dendrimers were synthesized to prepare in situ forming hydrogels with varied gelation time and mechanical properties through crosslinking with thiolated hyaluronic acid (HS-HA). By varying the alkenyl groups on the dendrimers, the gelation time displayed a large range from 8 seconds to 18 hours, and the modulus of the hydrogels ranged from 36 to 183 Pa under experimental conditions. Investigation by (1)H-NMR spectroscopy revealed that the gelation time and the stiffness of the hydrogels were governed by the degree of electron deficiency of alkenyl groups on the dendrimers. This research provided a systematic study on the relationship between chemical structures versus gelation time and mechanical properties of hydrogels, which could guide the way to synthesize in situ forming hydrogels with designated gelation time and stiffness for biomedical applications. Further, a RGD peptide was attached to the PAMAM dendrimers to enhance cell attachment and proliferation. Viability assays of Human Umbilical Vein Endothelial Cells (HUVEC) in the synthesized hydrogels demonstrated the biocompatibility of the hydrogels after 48 hours of culturing, and the RGD peptide improved the viability of HUVEC cells in hydrogels. We believe the PAMAM/HA hydrogel system is a tuneable and biocompatible system for diverse biomedical applications.

Synthesis of PAMAM dendrimer-based fast cross-linking hydrogel for biofabrication.

Hydrogels possess great potential in biofabrication because they allow cell encapsulation and proliferation in a highly hydrated three-dimensional environment, and they provide biologically relevant chemical and physical signals. However, development of hydrogel systems that mimic the complexity of natural extracellular matrix remains a challenge. In this study, we report the development of a binary hydrogel system containing a synthetic poly(amido amine) (PAMAM) dendrimer and a natural polymer, i.e., hyaluronic acid (HA), to form a fast cross-linking hydrogel. Live cell staining experiment and cell viability assay of bone marrow stem cells demonstrated that cells were viable and proliferating in the in situ formed PAMAM/HA hydrogel system. Furthermore, introduction of a Arginylglycylaspartic acid (RGD) peptide into the hydrogel system significantly improved the cell viability, proliferation, and attachment. Therefore, this PAMAM/HA hydrogel system could be a promising platform for various applications in biofabrication.

Effect of environmental factors on allelopathic inhibition of Microcystis aeruginosa by berberine.

To understand how environmental conditions affect the allelopathic inhibition of toxic Microcystis aeruginosa by berberine, the independent effects of some environmental factors, including temperature, light, and aeration, on the growth and extracellular microcystin (MC) content of M. aeruginosa (FACHB 905) treated with 0.000 and 0.001% (w/v) berberine were investigated. The results showed that higher temperature and light density, and aeration in daytime were beneficial for the growth of M. aeruginosa under the measured environmental conditions. The allelopathic effects of berberine on M. aeruginosa were closely associated with the environmental conditions. Berberine had the best inhibitory effects when temperature, light and aeration were more optimal for growth. In darkness, no changes in the density of M. aeruginosa were observed with the prolongation of culture time and berberine could hardly exhibit algicidal effects. Disturbance in the photosynthesis process might be one of the main reasons responsible for algicidal function. Berberine could increase extracellular MC contents significantly via killing and lyzing algal cells. Other treatments coupled with berberine needed to be carried out to degrade or remove MC released from berberine-killed algal cells.