Biosynthetic potential of uncultured anammox community bacteria revealed through multi-omics analysis.

Microbial secondary metabolites (SMs) and their derivatives have been widely used in medicine, agriculture, and energy. Growing needs for renewable energy and the challenges posed by antibiotic resistance, cancer, and pesticides emphasize the crucial hunt for new SMs. Anaerobic ammonium-oxidation (anammox) systems harbor many uncultured or underexplored bacteria, representing potential resources for discovering novel SMs. Leveraging HiFi long-read metagenomic sequencing, 1,040 biosynthetic gene clusters (BGCs) were unearthed from the anammox microbiome with 58% being complete and showcasing rich diversity. Most of them showed distant relations to known BGCs, implying novelty. Members of the underexplored lineages (Chloroflexota and Planctomycetota) and Proteobacteria contained lots of BGCs, showcasing substantial biosynthetic potential. Metaproteomic results indicated that Planctomycetota members harbored the most active BGCs, particularly those involved in producing potential biofuel-ladderane. Overall, these findings underscore that anammox microbiomes could serve as valuable resources for mining novel BGCs and discovering new SMs for practical application.

Hyperbaric oxygen therapy promotes the browning of white fat and contributes to the healing of diabetic wounds.

Non-healing wounds are one of the chronic complications of diabetes and have remained a worldwide challenge as one of the major health problems. Hyperbaric oxygen (HBO) therapy is proven to be very successful for diabetic wound treatment, for which the molecular basis is not understood. Adipocytes regulate multiple aspects of repair and may be therapeutic for inflammatory diseases and defective wound healing associated with aging and diabetes. Endothelial cell-derived extracellular vesicles could promote wound healing in diabetes. To study the mechanism by which HBO promotes wound healing in diabetes, we investigated the effect of HBO on fat cells in diabetic mice. A diabetic wound mouse model was established and treated with HBO. Haematoxylin and eosin (H&E) staining and immunofluorescence were used for the analysis of wound healing. To further explore the mechanism, we performed whole-genome sequencing on extracellular vesicles (EVs). Furthermore, we conducted in vitro experiments. Specifically, exosomes were collected from human umbilical vein endothelial cell (HUVEC) cells after HBO treatment, and then these exosomes were co-incubated with adipose tissue. The wound healing rate in diabetic mice treated with HBO was significantly higher. HBO therapy promotes the proliferation of adipose precursor cells. HUVEC-derived exosomes treated with HBO significantly promoted fat cell browning. These data clarify that HBO therapy may promote vascular endothelial cell proliferation and migration, and promote browning of fat cells through vascular endothelial cells derived exosomes, thereby promoting diabetic wound healing. This provides new ideas for the application of HBO therapy in the treatment of diabetic trauma.

New insights into functional divergence and adaptive evolution of uncultured bacteria in anammox community by complete genome-centric analysis.

Anaerobic ammonium-oxidation (anammox) bacteria play a crucial role in global nitrogen cycling and wastewater nitrogen removal, but they share symbiotic relationships with various other microorganisms. Functional divergence and adaptive evolution of uncultured bacteria in anammox community remain underexplored. Although shotgun metagenomics based on short reads has been widely used in anammox research, metagenome-assembled genomes (MAGs) are often discontinuous and highly contaminated, which limits in-depth analyses of anammox communities. Here, for the first time, we performed Pacific Biosciences high-fidelity (HiFi) long-read sequencing on the anammox granule sludge sample from a lab-scale bioreactor, and obtained 30 accurate and complete metagenome-assembled genomes (cMAGs). These cMAGs were obtained by selecting high-quality circular contigs from initial assemblies of long reads generated by HiFi sequencing, eliminating the need for Illumina short reads, binning, and reassembly. One new anammox species affiliated with Candidatus Jettenia and three species affiliated with novel families were found in this anammox community. cMAG-centric analysis revealed functional divergence in general and nitrogen metabolism among the anammox community members, and they might adopt a cross-feeding strategy in organic matter, cofactors, and vitamins. Furthermore, we identified 63 mobile genetic elements (MGEs) and 50 putative horizontal gene transfer (HGT) events within these cMAGs. The results suggest that HGT events and MGEs related to phage and integration or excision, particularly transposons containing tnpA in anammox bacteria, might play important roles in the adaptive evolution of this anammox community. The cMAGs generated in the present study could be used to establish of a comprehensive database for anammox bacteria and associated microorganisms. These findings highlight the advantages of HiFi sequencing for the studies of complex mixed cultures and advance the understanding of anammox communities.

Motility behavior and physiological response mechanisms of aerobic denitrifier, Enterobacter cloacae strain HNR under high salt stress: Insights from individual cells to populations.

The motility behaviors at the individual-cell level and the collective physiological responsive behaviors of aerobic denitrifier, Enterobacter cloacae strain HNR under high salt stress were investigated. The results revealed that as salinity increased, electron transport activity and adenosine triphosphate content decreased from 15.75 µg O2/g/min and 593.51 mM/L to 3.27 µg O2/g/min and 5.34 mM/L, respectively, at 40 g/L, leading to a reduction in the rotation velocity and vibration amplitude of strain HNR. High salinity stress (40 g/L) down-regulated genes involved in ABC transporters (amino acids, sugars, metal ions, and inorganic ions) and activated the biofilm-related motility regulation mechanism in strain HNR, resulting in a further decrease in flagellar motility capacity and an increase in extracellular polymeric substances secretion (4.08 mg/g cell of PS and 40.03 mg/g cell of PN at 40 g/L). These responses facilitated biofilm formation and proved effective in countering elevated salt stress in strain HNR. Moreover, the genetic diversity associated with biofilm-related motility regulation in strain HNR enhanced the adaptability and stability of the strain HNR populations to salinity stress. This study enables a deeper understanding of the response mechanism of aerobic denitrifiers to high salt stress.

Fate of iron nanoparticles in anammox system: Dissolution, migration and transformation.

Iron nanoparticles (FeNPs) are commonly used in various industrial processes, leading to their release into the environment and eventual entrance into wastewater treatment plants (WWTPs). FeNPs undergo dissolution, migration, and transformation in WWTPs, which can potentially affect the stable operation of anaerobic ammonia oxidation (anammox) systems and may be discharged with wastewater or biomass. To better understand the fate of FeNPs in anammox systems, exposure experiments were conducted using anammox granular sludges (AnGS) and FeNPs. Results demonstrated that FeNPs released Fe2+ upon contact with water, with a portion being bound to functional groups in extracellular polymeric substances (EPS) and the rest entering the bacteria to form highly absorbable substances. A significant amount of FeNPs was observed to cover the surface of AnGS or aggregate and deposit at the bottom of the reactor, eventually converting into Fe3O4 and stably existing within the anammox system. The findings of this study clarify the fate of FeNPs in anammox systems and provide important insights into the stable operation of anammox systems under FeNPs exposure.

Unraveling the structure and function of bacterioferritin in Candidatus Kuenenia stuttgartiensis: Iron storage sites maintain cellular iron homeostasis.

Anammox bacteria rely heavily on iron and have many iron storage sites. However, the biological significance of these iron storage sites has not been clearly defined. In this study, we explored the properties and location of iron storage sites to better understand their cellular function. To do this, the Candidatus Kuenenia stuttgartiensis iron storage protein, bacterioferritin (K.S Bfr), was successfully expressed and purified. In vitro, correctly assembled globulins were observed by transmission electron microscopy. The self-assembled K.S Bfr has active redox and can bind Fe2+ and mineralize it in the protein cavity. In vivo, engineered bacteria with K.S Bfr showed good adaptability to Fe2+, with a survival rate of 78.9% when exposed to 5 mM Fe2+, compared with only 66.0% for wild-type bacteria lacking K.S Bfr. A potential iron regulatory strategy similar to that of Anammox was identified in transcriptomic analysis of engineered bacteria. This system may be controlled by the iron uptake regulator Furto transport Fe2+ via FeoB and store excess Fe2+ in K.S Bfr to maintain cellular homeostasis. K.S Bfr has superior iron storage capacity both intracellularly and in vitro. The discovery of K.S Bfr reveals the storage location of iron-rich nanoparticles, increases our understanding of the adaptability of iron-dependent bacteria to Fe2+, and suggests possible iron regulation strategies in Anammox bacteria.

Biofilm formation during wastewater treatment: Motility and physiological response of aerobic denitrifying bacteria under ammonia stress based on surface plasmon resonance imaging.

A bacterial image analysis system based on surface plasmon resonance imaging was established to investigate the effect of bacterial motility on biofilm formation under high ammonia nitrogen at the single-cell level. The results showed that the bacterial mean rotation speed and vertical motility distance decreased with the increasing concentration of ammonia nitrogen. Ammonia nitrogen inhibited the metabolic activity of the bacteria, decreasing bacterial motility. Bacterial motility was negatively correlated with the biofilm-formation ability. The biofilm formation ability of Enterobacter cloacae strain HNR exposed to ammonia nitrogen was enhanced by reducing its movement and promoting EPS secretion. Genes related to the tricarboxylic acid cycle and oxidative phosphorylation were down-regulated, indicating inhibition of microbial energy metabolism. Genes related to bacterial secretion and lipopolysaccharide synthesis were up-regulated, facilitating the formation of biofilms and enabling the bacteria to resist ammonia nitrogen stress. This study provides new insights into the biofilm formation under ammonia stress.

Acceleration of start-up of moving bed biofilm reactor at low temperature by adding specialized quorum sensing bacteria.

This study aims to accelerate biofilm formation and operational performance of moving bed biofilm reactor (MBBR) at 5 ℃ by adding specialized Quorum Sensing bacteria (sphingomonas rubra BH3T). Results showed that bio augmented MBBR (RS) achieved a higher chemical oxygen demand and NH4+-N removal rate (93% and 75%), which in accordance with its increased biofilm thickness, higher biofilm activity, and nitrifying bacteria abundance (Nitrospira). The increased biofilm thickness (60.23 %) during the whole operating time, accompanied by more potent adhesion force (61.59 %), was related to increased polysaccharides and proteins in the biofilm. Pyrosequencing analysis indicated that BH3T contributed to higher species richness and triggered the rapid growth of precursor microorganisms (Nakamurella, Micropruina, and Zoogloea) and the enrichment of multifunctional microorganisms (Pseudomonas, Aeromonas, Arcobacter, Dechloromonas, and Flavobacterium) at low temperatures. This study provides an economical and practical new insight into accelerating start-up of MBBR system at low temperature.

Potential role of nanobubbles in dynamically modulating the structure and stability of anammox granular sludge within biological nitrogen removal process.

The generation of visible macrobubbles considerably affects the structure and function of anammox granules in the anammox granular sludge (AnGS) system. However, the existence of nanobubbles (NBs) and their role in maintaining the AnGS structure and stability are unclear because of the complexity of the system and lack of effective analytical methods. In this study, methods for NB analysis and assessment of their effects were developed to investigate the formation and characteristics of NBs in an AnGS system and the effects of NBs on the properties and function of AnGS. The results indicated that dissolved gas supersaturation caused by AnGS generated NBs of 2.75 × 108 bubbles/mL inside an AnGS reactor after running for 300 min at 30 °C. The increasing absolute value of the zeta potential of NBs with time indicated that the NBs in the AnGS system were gradually stable. The size of the stable NBs ranged from 150 nm to 400 nm. NB formation also increased the space and pressure between cells, leading to the breakage of the cell cluster and causing structural changes in granules. Changes in the local granular microstructure caused by NBs were favorable for the porous structure of granules to avoid granular disintegration and flotation caused by the excessive secretion of extracellular polymeric substances blocking gas channels. The formation and stability of NBs penetrating the cell clusters played a crucial role in the formation and stability of nanopores around or inside the cell clusters, further providing a basis for the formation of high-porosity structures and efficient mass transfer of AnGS.

Effects of lung-protective ventilation strategy on lung aeration loss and postoperative pulmonary complications in moderate-risk patients undergoing abdominal surgery.

BACKGROUND: There is a controversy about whether the use of a lung-protective ventilation strategy(LPVS) can reduce the incidence of postoperative pulmonary complications (PPCs) and improve the clinical outcomes in moderate-risk patients were assessed by the Assess Respiratory Risk in Surgical Patients in Catalonia(ARISCAT). METHODS: One hundred moderate-risk patients predicted by the ARISCAT, scheduled to undergo abdominal surgery were randomized into two groups: conventional ventilation strategy group (G0) and lung-protective ventilation strategy group (G1). Lung ultrasonography (LUS) and the LUS score were performed before induction of anesthesia (T0), 30min after extubation (T1), and 24h (T2), 72h (T3) after surgery. The incidence and severity of PPCs within the postoperative 7 days, the duration of postoperative oxygen supplementation, and postoperative hospital stay (PHS) were recorded. RESULTS: The LUS score of both groups at T1-3 was higher than those at T0 (P<0.05), moreover, the LUS score of G1 was lower than that of G0 at T1-3. The incidence of PPCs of G1 (10.9%) was lower than that of G0 (29.8%) (relative risk, 0.37; 95% confidence interval [CI], 0.14 to 0.93; P=0.02) and the severity of PPCs of G1 were lower than those of G0 (P<0.05). The PHS of G1 was less than that of G0 (8[7-10] vs. 9[8-11], P<0.05). CONCLUSIONS: The LPVS can decrease lung aeration loss assessed by LUS and reduce the incidence of PPCs in moderate-risk patients.

[Effect of Recycled Water Irrieation on Heavy Metal Pollution in Irrigation Soil].

With acceleration of urbanization, water shortages will become a serious problem. Usage of reclaimed water for flushing and watering of the green areas will be common in the future. To study the heavy metal contamination of soils after green area irrigation using recycled wastewater from special industries, we selected sewage and laboratory wastewater as water source for integrated oxidation ditch treatment, and the effluent was used as irrigation water of the green area. The irrigation units included broad-leaved forest, bush and lawn. Six samples sites were selected, and 0-20 cm soil of them were collected. Analysis of the heavy metals including Cr, Mn, Ni, Cu, Zn, As, Cd and Pb in the soil showed no significant differences with heavy metals concentration in soil irrigated with tap water. The heavy metals in the soil irrigated with recycled water were mainly enriched in the surface layer, among which the contents of Cr, Ni, Cu, Zn and Pb were below the soil background values of Beijing. A slight pollution of As and Cd was found in the soil irrigated by recycled water, which needs to be noticed.

PPARγ ligands decrease hydrostatic pressure-induced platelet aggregation and proinflammatory activity.

Hypertension is known to be associated with platelet overactivity, but the direct effects of hydrostatic pressure on platelet function remain unclear. The present study sought to investigate whether elevated hydrostatic pressure is responsible for platelet activation and to address the potential role of peroxisome proliferator-activated receptor-γ (PPARγ). We observed that hypertensive patients had significantly higher platelet volume and rate of ADP-induced platelets aggregation compared to the controls. In vitro, Primary human platelets were cultured under standard (0 mmHg) or increased (120, 180, 240 mmHg) hydrostatic pressure for 18 h. Exposure to elevated pressure was associated with morphological changes in platelets. Platelet aggregation and PAC-1 (the active confirmation of GPIIb/IIIa) binding were increased, CD40L was translocated from cytoplasm to the surface of platelet and soluble CD40L (sCD40L) was released into the medium in response to elevated hydrostatic pressure (180 and 240 mmHg). The PPARγ activity was up-regulated as the pressure was increased from 120 mmHg to 180 mmHg. Pressure-induced platelet aggregation, PAC-1 binding, and translocation and release of CD40L were all attenuated by the PPARγ agonist Thiazolidinediones (TZDs). These results demonstrate that platelet activation and aggregation are increased by exposure to elevated pressure and that PPARγ may modulate platelet activation induced by high hydrostatic pressure.

Formulation optimization of prostaglandin E1-loaded lipid emulsion: enhanced stability and reduced biodegradation.

The purpose of this study is to develop a new formulation for prostaglandin E1 (PGE1)-loaded lipid emulsion (Lipo-PGE1) with improved stability and reduced biodegradation. High-pressure homogenization was used to prepare the Lipo-PGE1, and high-performance liquid chromatography and accelerated test were used to evaluate its physicochemical stability. A tissue homogenate incubation test was firstly established and validated to assess its biodegradation. The factors influencing the stability of Lipo-PGE1, including oil phase, emulsifier, pH value, and drug concentration were systematically investigated. The optimized formulation consisting of poloxamer188 1.5% (w/v), egg lecithin 0.5% (w/v), soybean oil 10.0% (w/v), oleic acid 0.24% (w/v), and glycerol 2.2% (w/v), with the pH value at 4.0, was defined and characterized. When compared with the currently available commercial product of Lipo-PGE1, the degradation percentage of this optimized Lipo-PGE1 reduced by 47.1% after sterilization, the drug remaining percentage increased by 13.9% after storage at 4°C over 6 months. Also, a significant reduction in biodegradation of the optimized Lipo-PGE1 in comparison with the commercial Lipo-PGE1 was observed by a tissue homogenate incubation test. Overall, we provided a novel formulation for Lipo-PGE1 with a better physicochemical stability and a less biodegradation than the currently available commercial product of Lipo-PGE1, indicating its potential superiority in clinical application.

Growth-inhibitory and apoptosis-inducing effects of tanshinones on hematological malignancy cells and their structure-activity relationship.

This study has investigated the growth-inhibitory and apoptosis-inducing effects of dihydrotanshinone, tanshinone I, tanshinone IIA, and cryptotanshinone on hematological malignancy cell lines, aiming to explore their structure-activity relationship. The growth-inhibitory effects of the tanshinones on K562 and Raji cells were assessed using a modified MTT assay; the apoptosis-inducing effects were assessed by fluorescence microscopy and flow cytometry analysis. The changes in cellular morphology were observed using an inverted phase-contrast microscope. MTT results revealed that these tanshinones inhibited cell proliferation in a concentration-dependent and time-dependent manner. The IC50 values of dihydrotanshinone, tanshinone I, tanshinone IIA, and cryptotanshinone for K562 cells were 3.50, 13.52, 19.32, and 47.52 µmol/l at 24 h; 1.36, 4.70, 5.67, and 22.72 µmol/l at 48 h; and 1.15, 1.59, 2.82, and 19.53 µmol/l at 72 h, and the values for Raji cells were 3.30, 4.37, 12.92, and 52.36 µmol/l at 24 h; 1.55, 1.71, 6.54, and 25.45 µmol/l at 48 h; and 1.07, 1.38, 1.89, and 18.47 µmol/l at 72 h. The flow cytometry analysis demonstrated that these tanshinones induced apoptosis of K562 cells in a concentration-dependent manner, and dihydrotanshinone as well as tanshinone I were more potent than tanshinone IIA and cryptotanshinone. Some noticeable apoptotic morphologies could be observed by fluorescence microscopy on tanshinones-treated Raji cells. Collectively, these tanshinones caused growth inhibition and apoptosis in hematological malignancy cell lines, with dihydrotanshinone being the most potent, followed by tanshinone I, tanshinone IIA, and cryptotanshinone. These results suggested that the structure of aromatic ring A enhanced the cytotoxicity and the structure of ring C may have contributed to the cytotoxicity, but the mechanisms need to be further investigated.