A ratiometric fluorescence assay for the detection of DNA methylation based on an alkaline phosphatase triggered in situ fluorogenic reaction.

The accurate and sensitive quantification of DNA methylation is significant for the early diagnosis of cancer. In this work, an alkaline phosphatase (ALP) triggered in situ fluorogenic reaction between ascorbic acid (AA) and 2,3-DAN was employed as a ratiometric fluorescent probe for the accurate and sensitive detection of DNA methylation with the assistance of ALP encapsulated liposomes. The quinoxaline derivative with a yellow fluorescence emission (I525) was generated from the reaction between AA and 2,3-DAN. Meanwhile, the consumption of 2,3-DAN declined its fluorescence intensity (I386). A ratiometric fluorescent probe (I525/I386) constructed by the above in situ fluorogenic reaction was applied for the accurate detection of DNA methylation. The methylated DNA was first captured by its complementary DNA in 96-well plates. Then, 5mC antibody (Ab) linked liposomes that were encapsulated with ALP recognized and combined with the methylation sites of the target DNA. After the liposomes were lysed by Triton X-100, the released ALP triggered the hydrolysis of ascorbic acid diphosphate (AAP) to form AA, participating in the fluorogenic reaction with 2,3-DAN to produce a quinoxaline derivative. Thus, the ratiometric fluorescence detection of DNA methylation was achieved using I525/I386 values. Using the ALP-enzyme catalyzed reaction and liposomes as signal amplifiers, a low detection limit of 82 fM was obtained for DNA methylation detection. Moreover, the accuracy of the assay could be improved using ratiometric fluorescent probes. We hope that the proposed assay will pave a new way for the accurate determination of low-abundance biomarkers.

Compensatory Guaiacyl Lignin Biosynthesis at the Expense of Syringyl Lignin in 4CL1-Knockout Poplar.

The lignin biosynthetic pathway is highly conserved in angiosperms, yet pathway manipulations give rise to a variety of taxon-specific outcomes. Knockout of lignin-associated 4-coumarate:CoA ligases (4CLs) in herbaceous species mainly reduces guaiacyl (G) lignin and enhances cell wall saccharification. Here we show that CRISPR-knockout of 4CL1 in poplar (Populus tremula × alba) preferentially reduced syringyl (S) lignin, with negligible effects on biomass recalcitrance. Concordant with reduced S-lignin was downregulation of ferulate 5-hydroxylases (F5Hs). Lignification was largely sustained by 4CL5, a low-affinity paralog of 4CL1 typically with only minor xylem expression or activity. Levels of caffeate, the preferred substrate of 4CL5, increased in line with significant upregulation of caffeoyl shikimate esterase1 Upregulation of caffeoyl-CoA O-methyltransferase1 and downregulation of F5Hs are consistent with preferential funneling of 4CL5 products toward G-lignin biosynthesis at the expense of S-lignin. Thus, transcriptional and metabolic adaptations to 4CL1-knockout appear to have enabled 4CL5 catalysis at a level sufficient to sustain lignification. Finally, genes involved in sulfur assimilation, the glutathione-ascorbate cycle, and various antioxidant systems were upregulated in the mutants, suggesting cascading responses to perturbed thioesterification in lignin biosynthesis.

Effects of microplastics on the structure and function of bacterial communities in sediments of a freshwater lake.

As an emerging pollutant, microplastics (MPs) cause widespread concern around the world owing to the serious threat they pose to ecosystems. In particular, sediments are thought to be the long-term sink for the continual accumulation of MPs in freshwater ecosystems. Polyethylene (PE) and polyethylene terephthalate (PET) have been frequently detected with large concentration variations in freshwater sediments from the lower reaches of the Yangtze River, one of the most economically developed regions in China, characterized by accelerated urbanization and industrialization, high population density and high plastics consumption. However, the impact of PE and PET on the sedimental bacterial community composition and its function has not been well reported for this specific region. Herein, PE and PET particles were added to freshwater sediments to assess the effects of different MP types on the bacterial community and its function, using three concentrations (500, 1500 and 2500 items/kg) per MP and incubations of 35, 105 and 175 days, respectively. This study identified a total of 68 phyla, 211 classes, 518 orders, 853 families and 1745 genera. Specifically, Proteobacteria, Chloroflexi, Acidobacteriota, Actinobacteriota and Firmicutes were the top five phyla. A higher bacterial diversity was obtained in control sediments than in the MP-treated sediments. The presence of MPs, whether PET or PE, had significant impact on the bacterial diversity, community structure and community composition. PICRUSt2 and FAPOTAX predictions demonstrated that MPs could potentially affect the metabolic pathways and ecologically functional groups of bacteria in the sediment. Besides the MP-related factors, such as the type, concentration and incubation time, the physicochemical parameters had an effect on the structure and function of the bacterial community in the freshwater sediment. Taken together, this study provides useful information for further understanding how MPs affect bacterial communities in the freshwater sediment of the lower reaches of the Yangtze River, China.

Temporal dynamics of bacterial colonization on five types of microplastics in a freshwater lake.

Microplastics (MPs), as a new substrate, provide a unique niche for microbial colonization in the freshwater ecosystems; however, the impacts of long-term MP exposure on colonized bacteria are still unclear. In this study, five MP types were exposed in a freshwater lake for approximately one year, and the MP particles, together with the surrounding water, were collected on days 60, 150, 250 and 330 during the in situ field experiment. Bacteria on the MP surface, as well as free-living bacteria in the surrounding water, were analyzed to evaluate the temporal dynamics of these bacterial communities. Results show that all five MP types exhibited signs of degradation during the exposure process. Additionally, the alpha diversity, community structure and composition of MP-attached bacteria significantly differed from that of the free-living bacteria in the surrounding water, indicating that the five MP types could provide a preferable niche for bacterial colonization in a freshwater environment. Proteobacteria, Chloroflexi, Verrucomicrobiota, Actinobacteriota and Firmicutes were the top five dominant phyla. Some plastic-degrading bacteria included in these phyla were detected, verifying that MP-attached biofilms had a certain degree of MP degradation potential. Some potentially pathogenic bacteria were also detected, suggesting an ecological threat for spreading disease in the aquatic ecosystem. Furthermore, the bacterial community and some metabolic pathways were significantly affected by the MP type (P < 0.01) and exposure time (P < 0.01), indicating that the presence of MPs not only alters the bacterial community structure and composition, but also influences their potential functional properties in freshwater ecosystems. Multiple factors, including the physicochemical properties related to MPs and the environmental parameters of the surrounding water, affect the community composition and the function of MP-attached bacteria to different degrees. Our findings indicate that the presence of MPs has a potential ecological impact on freshwater ecosystems.

Nanoplastic toxicity induces metabolic shifts in Populus × euramericana cv. '74/76' revealed by multi-omics analysis.

There is increasing global concern regarding the pervasive issue of plastic pollution. We investigated the response of Populus × euramericana cv. '74/76' to nanoplastic toxicity via phenotypic, microanatomical, physiological, transcriptomic, and metabolomic approaches. Polystyrene nanoplastics (PS-NPs) were distributed throughout the test plants after the application of PS-NPs. Nanoplastics principally accumulated in the roots; minimal fractions were translocated to the leaves. In leaves, however, PS-NPs easily penetrated membranes and became concentrated in chloroplasts, causing thylakoid disintegration and chlorophyll degradation. Finally, oxidant damage from the influx of PS-NPs led to diminished photosynthesis, stunted growth, and etiolation and/or wilting. By integrating dual-omics data, we found that plants could counteract mild PS-NP-induced oxidative stress through the antioxidant enzyme system without initiating secondary metabolic defense mechanisms. In contrast, severe PS-NP treatments promoted a shift in metabolic pattern from primary metabolism to secondary metabolic defense mechanisms, an effect that was particularly pronounced during the upregulation of flavonoid biosynthesis. Our findings provide a useful framework from which to further clarify the roles of key biochemical pathways in plant responses to nanoplastic toxicity. Our work also supports the development of effective strategies to mitigate the environmental risks of nanoplastics by biologically immobilizing them in contaminated lands.

DPA-Zinc around Polyplexes Acts Like PEG to Reduce Protein Binding While Targeting Cancer Cells.

Gene therapy holds great promise as an effective treatment for many diseases of genetic origin. Gene therapy works by employing cationic polymers, liposomes, and nanoparticles to condense DNA into polyplexes via electronic interactions. Then, a therapeutic gene is introduced into target cells, thereby restoring or changing cellular function. However, gene transfection efficiency remains low in vivo due to high protein binding, poor targeting ability, and substantial endosomal entrapment. Artificial sheaths containing PEG, anions, or zwitterions can be introduced onto the surface of gene carriers to prevent interaction with proteins; however, they reduce the cellular uptake efficacy, endosomal escape, targeting ability, thereby, lowering gene transfection. Here, it is reported that linking dipicolylamine-zinc (DPA-Zn) ions onto polyplex nanoparticles can produce a strong hydration water layer around the polyplex, mimicking the function of PEGylation to reduce protein binding while targeting cancer cells, augmenting cellular uptake and endosomal escape. The polyplexes with a strong hydration water layer on the surface can achieve a high gene transfection even in a 50% serum environment. This strategy provides a new solution for preventing protein adsorption while improving cellular uptake and endosomal escape.

Effect evaluation of microplastics on activated sludge nitrification and denitrification.

A large amount of microplastics have entered conventional wastewater treatment plants, and their effects on activated sludge nitrification and denitrification are rarely reported. This study investigated the effects of microplastics on activated sludge nitrification and denitrification using five typical microplastics, namely, polyvinyl chloride (PVC), polypropylene, polyethylene, polystyrene, and polyester (PES) with concentrations of 0, 1000, 5000, and 10,000 particles/L. Results indicated that microplastics had negative effects on ammonia oxidation rate and low effect on nitrite oxidation rate during nitrification. The total inorganic nitrogen did not have much difference during 3 h nitrification under all the tested conditions. The addition of microplastics showed positive effects on denitrification, especially for PVC and PES at microplastic concentration of 5000 particles/L. Nitrification and denitrification did not evidently stop under all the tested conditions, indicating that the selected microplastic types and concentrations were not toxic to nitrification and denitrification within 3 h. The high abundance of PVC microplastics remarkably increased the nitrous oxide (N2O) emission during denitrification. The N2O emission in the test with 10,000 particle/L of PVC was 4.6times higher than the blank control. This study indicated that microplastics with <10,000 particle/L concentration in wastewater had low effects on nitrification and denitrification, whereas they had high effects on the N2O emission during denitrification.

A chloroplast-inspired nanoplatform for targeting cancer and synergistic photodynamic/photothermal therapy.

Specific targeting capabilities and effective phototherapeutic functions are the key demands for precise cancer phototherapeutic agents. Herein, a bioinspired nanoplatform composed of Cu(ii)-chlorophyll-hyaluronic acid nanoparticles (Cu(ii)Chl-HA NPs) was developed for targeting cancer and synergistic photodynamic/photothermal therapy. Inspired by the photonic biosystem of the chloroplast, Cu(ii) chlorophyll was used as a photosensitive substituent to covalently connect with a hydrophilic HA tail rather than a natural phytol tail, and this conjugate further assembled into a nanoparticle-like morphology under non-covalent interaction. Time-dependent density functional theory calculations reveal that the Cu(ii) chlorophyll has a much smaller energy gap between an excited singlet and excited triplet, and theoretically leads to rapid electron intersystem crossing that would benefit the PDT effect. In addition, a series of experiments have proven that, under 650 nm laser irradiation, the nanoplatform of Cu(ii)Chl-HA can produce a high amount of singlet oxygen and exhibit an outstanding photothermal conversion capability. More interestingly, owing to the specific interactions between the HA component and the CD44 receptor on the cell membrane, the HA tails impart Cu(ii)Chl-HA NPs an excellent receptor-mediated targeting performance toward CD44-overexpressing cancer cells. Based on these features, the nanoplatform of Cu(ii)Chl-HA NPs presents active targeting and outstanding dual modality synergistic PDT/PTT performance of cancer both in vitro and in vivo. Thus, this work opens up a new strategy to fabricate a bioinspired multifunctional cancer phototherapy nanoplatform.

Function of self-forming dynamic membrane and biokinetic parameters' determination by microelectrode.

The self-forming dynamic membrane coupled bioreactor (SFDMBR), which uses coarse pore-sized material to separate solids and liquids in a bioreactor, has some advantages compared with membrane bioreactor (MBR) using micro-/ultra-filtration membranes such as low module cost and high flux. In this study, we investigate the microbial activity change of a self-forming dynamic membrane (DM) during its bio-fouling process by a microelectrode for O2. At a high flux of 40 L/m2h, the dissolved oxygen was determined to be depleted at the depth of 1.5-2.0 mm in the self-forming DM. Based on the dissolved oxygen concentration profiles in the self-forming DM, a reliable and simple model and computational procedure were developed to estimate the biokinetic parameters in the self-forming DM. Sensitivity analysis of the model revealed that the dissolved oxygen profiles are sufficiently sensitive to the maximum specific rates of oxygen uptake (q down curvemax,20), which were computed to be within the range of 3.8-11.1 mg O2/gSS h. q down curvemax,20 decreased sharply in the first 5 days with the development of the bio-fouling process in the surface cake layer of the self-forming DM and then reached a relatively steady state afterwards. The Monod half-saturation coefficient for oxygen (Ko) was computed to be in the range of 0.16-0.75 mgO2/L. In summary, the results gave new experimental evidence on the change of microbial activity in the self-forming DM during its bio-fouling process.

Adipose-derived stem cells transfected with pEGFP-OSX enhance bone formation during distraction osteogenesis.

This study was designed to investigate the effects of local delivery of adipose-derived stem cells (ADSCs) transfected with transcription factor osterix (OSX) on bone formation during distraction osteogenesis. New Zealand white rabbits (n=54) were randomly divided into three groups (18 rabbits per group). A directed cloning technique was used for the construction of recombinant plasmid pEGFP-OSX, where EGFP is the enhanced green fluorescence protein. After osteodistraction of the right mandible of all experimental rabbits, rabbits in group A were treated with ADSCs transfected with pEGFP-OSX, group B with ADSCs transfected with pEGFP-N1, and group C with physiological saline. Radiographic and histological examinations were processed after half of the animals within each group were humanely killed by injection of sodium pentothal at Week 2 or 6 after surgery. The distraction bone density was measured as its projectional bone mineral density (BMD). Three parameters were measured, namely, the thickness of new trabeculae (TNT), and the volumes of the newly generated cortical bone (NBV1) and the cancellous bone (NBV2) of the distracted regions. Good bone generation in the distraction areas was found in group A, which had the highest BMD, TNT, and NBV in the distraction zones among the groups. There was no significant difference in bone generation in the distraction areas between groups B and C. The results indicate that the transplantation of ADSCs transfected with pEGFP-OSX can effectively promote bone generation during distraction in vivo.

[Application of midline partial glossectomy for OSAHS].

OBJECTIVE: To evaluate the prospective effect of applying midline partial glossectomy with Uvulopalatopharyngoplasty in the treatment of OSAHS. METHOD: The data of 40 patients with OSAHS operated with midline partial glossectomy with UPPP were analyzed. RESULT: The follow-up polysomnography (6-month follow-up) revealed that the mean apnea and hypopnea index decreased from 62.39+/-21.68 to 18.60+/-5.01 (P<0.01), and the mean minimal oxygen saturation (SaO2) increased from 64.40%+/-14.96% to 88.39%+/-9.21% (P<0.01). In general, 29 patients were with excellent curative effect, 8 with good effect, and 3 with bad effect. Total effective rate was 92.5%. CONCLUSION: The study confirms that the same-stage midline partial glossectomy with UPPP for OSAHS is a safe and effective procedure.

[Study on denitrification characteristics of dynamic membrane based on nitrate liquid-membrane microelectrodes].

Nitrate microelectrodes and ORP microelectrodes were fabricated to study the denitrification characteristics of dynamic membrane at different COD loadings. The denitrification process was found at 0.6-1 mm depth beneath the interface of biofilm/bulk. The results of ORP microelectrode also demonstrated that the ORP value in the range of denitrification area was between 88.6 approximately -128.4 mV which was appropriate to denitrification. When the COD loading was 0.45 kg/(m3 x d), the denitrification rate (NO3- -N) was the maximum of 0.6347 x 10(-6 mol/(L x s). With the increase of COD loading, the denitrification area was increasing and two layers with different denitrification rates emerged in the dynamic membrane. The phenomenon implied the effect of organic concentration, oxygen concentration and bacterial competition on the denitrification rate.

[Characteristics of dynamic membrane based on oxygen microelectrode].

Self-forming dynamic membrane coupled bioreactor is a technology using coarse pore-sized material modules to separate solid and liquid in the activated sludge. The cake layer formed on the coarse pore-sized material, i.e. dynamic membrane, can remove turbidity efficiently and some organic pollutants. Its biological activity can be studied by microelectrode. For measurement in-situ, a small bioreactor is designed to simulate the real filtration process. A microelectrode with micromanipulator is used to measure the dissolved oxygen profile in the dynamic membrane. The results indicate that bubble emerged in the dynamic membrane during operation of bioreactor. The dissolved oxygen concentration decreased with the increasing depth, and fell to 0 at 2.0 - 2.5mm depth. The specific oxygen uptake rates of the surface part of dynamic membrane were calculated to be 34.3, 10.6 and 12.4 mg/(g x h) respectively at the 1st, 5th and 8th day of the filtration.