Impact of Organic Carbons Addition on the Enrichment Culture of Nitrifying Biofloc from Aquaculture Water: Process, Efficiency, and Microbial Community.

In this study, we developed a rapid and effective method for enriching the culture of nitrifying bioflocs (NBF) from aquacultural brackish water. The self-designed mixotrophic mediums with a single or mixed addition of sodium acetate, sodium citrate, and sucrose were used to investigate the enrichment process and nitrification efficiency of NBF in small-scale reactors. The results showed that NBF with an MLVSSs from 1170.4 mg L-1 to 2588.0 mg L-1 were successfully enriched in a period of less than 16 days. The citrate group performed the fastest enrichment time of 10 days, while the sucrose group had the highest biomass of 2588.0 ± 384.7 mg L-1. In situ testing showed that the highest nitrification efficiency was achieved in the citrate group, with an ammonia oxidation rate of 1.45 ± 0.34 mg N L-1 h-1, a net nitrification rate of 2.02 ± 0.20 mg N L-1 h-1, and a specific nitrification rate of 0.72 ± 0.14 mg N g-1 h-1. Metagenomic sequencing revealed that Nitrosomonas (0.0~1.0%) and Nitrobacter (10.1~26.5%) were dominant genera for AOB and NOB, respectively, both of which had the highest relative abundances in the citrate group. Linear regression analysis further demonstrated significantly positive linear relations between nitrification efficiencies and nitrifying bacterial genera and gene abundance in NBF. The results of this study provide an efficient enrichment culture method of NBF for the operation of biofloc technology aquaculture systems, which will further promote its wide application in modern intensive aquaculture.

Nanoreactor Confined and Enriched Intermediates for Electroreduction of CO2 to C2+ Products.

In the process of electroreduction of carbon dioxide (eCO2RR) to multi-carbon (C2+) products, it is imperative to enhance the concentration of key intermediate species on the catalyst surface. The utilization of micro-nano reactors to achieve confinement effects has been widely observed in various catalytic reactions, yet it has seldom been employed in eCO2RR. Here, we present a novel nanoreactor composed of stacked CuS nanosheets for eCO2RR to C2+ products. In comparison to catalyst comprising of nanosheet with open space, the C-C coupling within this confined nanospace is significantly enhanced, resulting in the increase of Faraday efficiency (FE) of C2+ products to 53 %. In situ infrared (IR) spectroscopy reveals the confinement and enrichment of key intermediate by the nanoreactor. Our research findings demonstrate that a meticulously designed nanoreactor can elevate the selectivity of C2+ products, thereby aiding in the design of eCO2RR catalysts.

Concentrations, distribution, and key influencing factors of antibiotic resistance genes and bacterial community in water and reared fish tissues in a typical tilapia farm in South China.

Although previous studies have investigated the occurrence of antibiotic resistance genes (ARGs) in aquaculture, few have monitored the concentrations and propagation of ARGs in biological tissues or investigated the key factors influencing their spread in aquaculture. This study investigated the concentration, propagation, and distribution of ARGs and bacterial communities in water sources, pond water, and tilapia tissues, and their key influencing factors, in a typical tilapia farm. ErmF, sul1, and sul2 were the dominant ARGs with high concentrations. The total concentrations of ARGs (TCAs) in tilapia tissues decreased in the following order: stomach > scales > intestine > gills (P < 0.05). Redundancy analysis and multiple linear regression revealed that suspended solids (SS) and chemical oxygen demand (COD) were positively correlated with the dominant ARGs ermF sul2, and the TCAs (P < 0.05); additionally, Chloroflexi and Bacteroidetes in tilapia aquaculture water were positively correlated with the dominant ARGs ermF and sul2, as well as the TCAs (P < 0.05). This study suggests that SS and COD were the key factors driving the distribution and spread of ARGs in tilapia aquaculture water. Additionally, Chloroflexi and Bacteroidetes were the key bacterial flora affecting the propagation of ARGs in tilapia aquaculture systems.

Effect of bacterial community succession on environmental factors during litter decomposition of the seaweed Gracilaria lemaneiformis.

In large-scale seaweed farming, an understanding of the decomposition process plays a pivotal role in optimizing cultivation practices by considering the influence of the bacterial community. Therefore, we assessed the bacterial community structure and its influence on environmental factors during Gracilaria lemaneiformis decomposition, utilizing both microcosms and in-situ simulations. The decomposition rates in the microcosms and in situ simulations reached 79 % within 180 days and 81 % within 50 days, respectively In the microcosms, the dissolved oxygen content decreased from 5.3 to 0.4 mg/L, while the concentrations of total organic carbon, nitrogen, and phosphorus in the water increased by 165 %, 1636 %, and 2360 %, respectively. The common dominant bacteria included Proteobacteria, Planctomycetes, Firmicutes, Bacteroidetes, and Spirochaetae. Planctomycetes and Firmicutes were positively correlated with the total organic carbon, nitrogen, and phosphorus concentrations. Planctomycetes species played significant roles during the decomposition process. The overall findings of this study could inform more sustainable seaweed cultivation practices.

Responses of sediment nitrogen forms and bacterial communities to different aquatic nitrogen conditions in three submerged macrophyte-type ecological treatment systems.

Ecological treatment system (ETS) has been recognized as a promising technology for mitigating agricultural non-point pollution, whereas it remains to be seen how nitrogen (N) forms and bacterial communities in ETS sediments respond to different aquatic N conditions. Therefore, a four-month microcosm experiment was conducted to investigate the effects of three aquatic N conditions (2 mg/L NH4+-N, 2 mg/L NO3--N and 1 mg/L NH4+-N + 1 mg/L NO3--N) on sediment N forms and bacterial communities in three ETSs vegetated by Potamogeton malaianus, Vallisneria natans and artificial aquatic plant, respectively. Through analysis of four transferable N fractions, the valence states of N in ion-exchange and weak acid extractable fractions were found to be mainly determined by aquatic N conditions, while significant N accumulation was observed only in strong oxidant extractable and strong alkali extractable fractions. Sediment N profiles were primarily influenced by time and plant types, with N condition having secondary effect, while sediment bacterial community structures experienced a significant shift over time and were slightly influenced by plant types. Sediment functional genes related to N fixation, nitrification, assimilable nitrate reduction, dissimilatory nitrite reduction (DNRA) and denitrification were substantially enriched in month 4, and the bacterial co-occurrence network exhibited less complexity but more stability under NO3- condition compared to others. Furthermore, certain sediment N fractions were found to have strong relationships with specific sediment bacteria, such as nitrifiers, denitrifiers and DNRA bacteria. Our findings highlight the significant influence of aquatic N condition in submerged macrophyte-type ETSs on sediment N forms and bacterial communities.

Single Atom Bi Decorated Copper Alloy Enables C-C Coupling for Electrocatalytic Reduction of CO2 into C2+ Products.

Single atom alloy (SAA) catalysts have been recently explored for promotion of various heterogeneous catalysis, but it remains unexplored for selective electrocatalytic reduction of carbon dioxide (CO2 ) into multi-carbon (C2+ ) products involving C-C coupling. Herein we report a single-atomic Bi decorated Cu alloy (denoted as BiCu-SAA) electrocatalyst that could effectively modulate selectivity of CO2 reduction into C2+ products instead of previous C1 ones. The BiCu-SAA catalyst exhibits remarkably superior selectivity of C2+ products with optimal Faradaic efficiency (FE) of 73.4 % compared to the pure copper nanoparticle or Bi nanoparticles-decorated Cu nanocomposites, and its structure and performance can be well maintained at current density of 400 mA cm-2 under the flow cell system. Based on our in situ characterizations and density functional theory calculations, the BiCu-SAA is found to favor the activation of CO2 and subsequent C-C coupling during the electrocatalytic reaction, as should be responsible for its extraordinary C2+ selectivity.

Temporal variations, distribution, and dissemination of antibiotic resistance genes and changes of bacterial communities in a biofloc-based zero-water-exchange mariculture system.

Antibiotic resistance genes (ARGs) have obtained an increasing number of global concerns for the severe risks they pose to food safety and public health. Studies have investigated the concentrations and distribution of ARGs in the environment. However, the distribution and dissemination of ARGs, the bacterial communities, and the key influencing factors during the entire rearing period in the biofloc-based zero-water-exchange mariculture system (BBZWEMS) remain unclear. The current study investigated the concentrations, temporal variations, distribution, and dissemination of ARGs, the changes in the bacterial communities, as well as the key influencing factors during the rearing period in the BBZWEMS. Sul1 and sul2 were dominant ARGs. Total concentrations of ARGs followed a trend of decrease in pond water, while they followed a trend of increase in source water, biofloc, and shrimp gut. Total concentrations of targeted ARGs in the water source were higher than those in the pond water and biofloc samples for each corresponding rearing stage by 2.25-122.97-fold (p < 0.05). The bacterial communities in biofloc and pond water did not change much, while they changed considerably in the shrimp gut samples during the rearing period. Pearson correlation, redundancy analysis, and multivariable linear regression analysis showed that suspended substances and Planctomycetes were positively correlated with the concentrations of ARGs (p < 0.05). The current study indicates that the water source may be a critical source of ARGs, and that suspended substances is a key factor influencing the distribution and dissemination of ARGs in the BBZWEMS. Early intervention measures on ARGs in water sources should be implemented to aid in the prevention and control of resistance genes in aquaculture industry, and reduce the potential risks of ARGs to public health and food safety.

Rational design of heterogenized molecular phthalocyanine hybrid single-atom electrocatalyst towards two-electron oxygen reduction.

Single-atom catalysts supported on solid substrates have inspired extensive interest, but the rational design of high-efficiency single-atom catalysts is still plagued by ambiguous structure determination of active sites and its local support effect. Here, we report hybrid single-atom catalysts by an axial coordination linkage of molecular cobalt phthalocyanine with carbon nanotubes for selective oxygen reduction reaction by screening from a series of metal phthalocyanines via preferential density-functional theory calculations. Different from conventional heterogeneous single-atom catalysts, the hybrid single-atom catalysts are proven to facilitate rational screening of target catalysts as well as understanding of its underlying oxygen reduction reaction mechanism due to its well-defined active site structure and clear coordination linkage in the hybrid single-atom catalysts. Consequently, the optimized Co hybrid single-atom catalysts exhibit improved 2e- oxygen reduction reaction performance compared to the corresponding homogeneous molecular catalyst in terms of activity and selectivity. When prepared as an air cathode in an air-breathing flow cell device, the optimized hybrid catalysts enable the oxygen reduction reaction at 300 mA cm-2 exhibiting a stable Faradaic efficiency exceeding 90% for 25 h.

Cultivation-dependent and cultivation-independent investigation of O-methylated pollutant-producing bacteria in three drinking water treatment plants.

O-methylated pollutants (OMPs) are emerging contaminants in drinking water and mainly produced through bacterial O-methylation. However, the information of OMP-producing bacteria (OMPPB) in drinking water treatment plant (DWTP) is largely unknown so far. In this study, the OMPPB in water samples from three DWTPs (XL, JX and NX) were investigated by using cultivation-dependent and cultivation-independent technologies. Four OMPs were detected and their odor and toxicity risks were assessed. Formation potentials (FPs) of 2,4,6-trichloanisole, 2,3,6-trichloanisole, 2,4,6-tribromoanisole, pentachloroanisole and diclofenac methyl ester were determined in water samples and their values shifted significantly among DWTPs. A most probable number (MPN) method was established to quantify OMPPB numbers and the relationships between total haloanisole FPs (HAFPs) (y) and OMPPB numbers (x) in three DWTPs could be described by the following functions: y = 0.496×0.373 (XL), y = 0.041×0.465 (JX) and y = 0.218×0.237 (NX). Several genera like Bacillus, Ralstonia, Brevundimonas, etc. were newly found OMPPB among the cultivable bacteria, and their OMP products were evaluated in terms of quantity and environment risks (odor, toxicity and bioaccumulation). High-throughput sequencing revealed treatment process was the main driving factor to shape the OMPPB community structures and Mantel test showed HAFP profile was significantly influenced by Mycobacterium and Pelomonas. PICURSt2 analysis discovered four phenolic O-methyltransferases (OMTs) and four carboxylic OMTs which might be responsible for OMP formation. Several strategies were recommended to assess risk and control contamination brought by OMPPB in DWTPs.

Toxicity of the microcystin-producing cyanobacteria Microcystis aeruginosa to shrimp Litopenaeus vannamei.

Microcystis aeruginosa is reported to cause cyanobacterial blooms in shrimp breeding ponds, which can result in significant shrimp mortality. However, the toxic effects of M. aeruginosa on Litopenaeus vannamei are still not completely understood. In this paper, the toxicity of M. aeruginosa cells to L. vannamei was examined, and the toxic components in the cells were analyzed through high-pressure liquid chromatography (HLPC). In addition, the immune response of shrimp to the M. aeruginosa cell extract was assessed by measuring the activity of immune-related enzymes, as well as the transcription of the relevant genes. The results showed that M. aeruginosa cells, extract and cell-free cultured medium resulted in a 100%, 98.3%, and 1.7% mortality rate in shrimp, respectively. HPLC analysis results revealed the presence of microcystin-LR (MC-LR) at a concentration of 190.40 mg/kg of cells. In addition, the activity and gene transcription of two immune related enzymes, SOD and LZM, were both significantly reduced in shrimp hepatopancreas (p < 0.05) after injection with extract. However, reduced glutathione (GSH) content was slightly increased, but the ratio of GSH to GSSG decreased. The transcription of gst gene function as detoxification, was significantly downregulated (p < 0.05). The results demonstrated that M. aeruginosa cell extract was highly toxic to L. vannamei, and exerted a negative effect on shrimp immunity including reduction of antioxidant capacity, antibacterial activity and detoxification activity, due to toxins including microcystin-LR.

Metagenomic analysis of the abundances, diversity, and distribution of antibiotic resistance genes and their potential bacterial hosts in two types of shrimp-rearing farms in South China.

Antibiotic resistance genes (ARGs) are attracting increasing concern worldwide. Many previous studies have investigated the occurrence and concentrations of ARGs in aquaculture. However, the sources of ARGs and the links with their potential bacterial hosts have not yet been explored. This study investigated the abundances and diversity of ARGs in two types of shrimp farms in South China through metagenomic sequencing. In total, 14 ARG types were detected. Tetracycline was the dominant ARG type. The abundances of ARGs in samples decreased in the order of duck feces > water source > sediment > shrimp gut > pond water. The samples from the duck-shrimp integrated farm contained 1.29-3.81-fold more abundant ARGs than those from the shrimp monoculture farm (p < 0.05). Several ARGs, that were most predominant in the duck feces samples, were also the most predominant in the shrimp gut samples from the duck-shrimp integrated farm. Redundancy analysis indicated that the abundances and distribution of ARGs formed three clusters: duck feces, water samples, and sediment and shrimp gut samples. The dominant genera in duck feces known as human pathogenic bacteria were potential hosts of ARGs, and were also dominant in the shrimp gut samples in the duck-shrimp integrated farm. Additionally, the abundances of dominant genera in the shrimp gut samples of the duck-shrimp integrated farm were 1.74-35.07-fold higher than those in the shrimp monoculture farm (p < 0.01). The duck-shrimp integrated farm had 3.36-fold and 4.94-fold higher abundances of ARGs and mobile genetic elements in the shrimp gut samples than those from the shrimp monoculture farm, respectively (p < 0.05). The results indicate that duck feces may be a crucial source of diverse and abundant ARGs spreading to reared shrimps in duck-shrimp integrated farms, posing a severe risk to public health.

Effect of Input C/N Ratio on Bacterial Community of Water Biofloc and Shrimp Gut in a Commercial Zero-Exchange System with Intensive Production of Penaeus vannamei.

Although increasing attention has been attracted to the study and application of biofloc technology (BFT) in aquaculture, few details have been reported about the bacterial community of biofloc and its manipulation strategy for commercial shrimp production. An 8-week trial was conducted to investigate the effects of three input C/N ratios (8:1, 12:1 and 16:1) on the bacterial community of water biofloc and shrimp gut in a commercial BFT tank system with intensive aquaculture of P. vannamei. Each C/N ratio group had three randomly assigned replicate tanks (culture water volume of 30 m3), and each tank was stocked with juvenile shrimp at a density of 300 shrimp m-3. The tank systems were operated with zero-water exchange, pH maintenance and biofloc control. During the trial, the microbial biomass and bacterial density of water biofloc showed similar variation trends, with no significant difference under respective biofloc control measures for the three C/N ratio groups. Significant changes were found in the alpha diversity, composition and relative abundance of bacterial communities across the stages of the trial, and they showed differences in water biofloc and shrimp gut among the three C/N ratio groups. Meanwhile, high similarity could be found in the composition of the bacterial community between water biofloc and shrimp gut. Additionally, nitrogen dynamics in culture water showed some differences while shrimp performance showed no significant difference among the three C/N ratio groups. Together, these results confirm that the manipulation of input C/N ratio could affect the bacterial community of both water biofloc and shrimp gut in the environment of a commercial BFT system with intensive production of P. vannamei. Moreover, there should be different operations for the nitrogen dynamics and biofloc management during shrimp production process under different C/N ratios.

Systematic Pan-Cancer Analysis of KLRB1 with Prognostic Value and Immunological Activity across Human Tumors.

INTRODUCTION: KLRB1 is a gene encoding CD161 expressed in NK cells and some T cell subsets. At present, KLRB1 is believed to affect tumorigenesis and development by regulating the cytotoxicity of NK cells in several cancers. However, there is a lack of systematic reviews of KLRB1 in a variety of malignancies. OBJECTIVES: Hence, our research is aimed at providing a relatively comprehensive understanding of the role of KLRB1 in different types of cancer, paving the way for further research on the molecular mechanism and immunotherapy potential of KLRB1. METHODS: In this study, we used relevant public databases, including TCGA (The Cancer Genome Atlas), GEO (Gene Expression Omnibus), CCLE (Cancer Cell Line Encyclopedia), GTEx (Genotype Tissue-Expression), and HPA (Human Protein Atlas), to perform a pan-cancer analysis of KLRB1 across 33 types of cancer. We explored the potential molecular mechanism of KLRB1 in clinical prognosis and tumor immunity from the aspects of gene expression, survival status, clinical phenotype, immune infiltration, immunotherapy response, and chemotherapeutic drug sensitivity. RESULTS: KLRB1 was downregulated in 13 cancers while upregulated in kidney cancer. Patients with high expression of KLRB1 have a better prognosis in most types of cancer. Moreover, the KLRB1 expression level is related to TMB and MSI and related to various immune signatures of tumor. The expression of KLRB1 can affect tumor immune cell infiltration. KLRB1 expression level can also affect the sensitivity of chemotherapy drugs. CONCLUSIONS: KLRB1 may be a prognostic and immunological biomarker across tumors. At the same time, KLRB1 expression can reflect the sensitivity of cancer patients to chemotherapy drugs. KLRB1 may become a new target for immunotherapy.

Diversity, abundances and distribution of antibiotic resistance genes and virulence factors in the South China Sea revealed by metagenomic sequencing.

Antibiotic resistance genes (ARGs) and virulence factors (VFs) pose considerable health risks to humans. The occurrence and abundance of several typical ARGs in the sea have been widely investigated. However, the full profiles and abundances of the antibiotic resistome and VFs in the South China Sea remain unexplored. Therefore, in this study, we investigated the full profiles of the ARGs and VFs, as well as their abundances and distribution, in the South China Sea using metagenomic approaches. In total, 140 ARG subtypes and 155 VFs were detected. The most abundant ARG was multidrug resistance gene, followed by bacitracin resistance gene. Flagella was the most abundant VF. Pearson correlation analysis revealed a strong and positive correlation between the abundances of ARGs and VFs. Redundancy analysis and co-occurrence network analysis showed that the predominant VFs were positively correlated with the predominant ARGs in the South China Sea. Nonmetric multidimensional scaling and Procrustes analyses demonstrated that the sampling sites were clustered into three compartments according to the geographical location, i.e., offshore, open sea, and reef zones. The abundances of ARGs and VFs in the offshore zone were much higher than those in the open sea and reef zones (p < 0.05). Several physico-chemical factors most closely associated with anthropogenic activities, i.e., nitrate, lead, copper, and zinc, were positively correlated with the predominant ARGs and VFs in the South China Sea. Our results suggest that the ocean is a large reservoir of diverse and abundant ARGs and VFs, which may threaten human health and seafood safety. These findings improve the understanding of the relationship between ARG dissemination and intensive anthropogenic activities and can aid in improving ocean management and seafood product safety.

Prediction for pathological and immunohistochemical characteristics of triple-negative invasive breast carcinomas: the performance comparison between quantitative and qualitative sonographic feature analysis.

OBJECTIVE: Sonographic features are associated with pathological and immunohistochemical characteristics of triple-negative breast cancer (TNBC). To predict the biological property of TNBC, the performance using quantitative high-throughput sonographic feature analysis was compared with that using qualitative feature assessment. METHODS: We retrospectively reviewed ultrasound images, clinical, pathological, and immunohistochemical (IHC) data of 252 female TNBC patients. All patients were subgrouped according to the histological grade, Ki67 expression level, and human epidermal growth factor receptor 2 (HER2) score. Qualitative sonographic feature assessment included shape, margin, posterior acoustic pattern, and calcification referring to the Breast Imaging Reporting and Data System (BI-RADS). Quantitative sonographic features were acquired based on the computer-aided radiomics analysis. Breast cancer masses were manually segmented from the surrounding breast tissues. For each ultrasound image, 1688 radiomics features of 7 feature classes were extracted. The principal component analysis (PCA), least absolute shrinkage and selection operator (LASSO), and support vector machine (SVM) were used to determine the high-throughput radiomics features that were highly correlated to biological properties. The performance using both quantitative and qualitative sonographic features to predict biological properties of TNBC was represented by the area under the receiver operating characteristic curve (AUC). RESULTS: In the qualitative assessment, regular tumor shape, no angular or spiculated margin, posterior acoustic enhancement, and no calcification were used as the independent sonographic features for TNBC. Using the combination of these four features to predict the histological grade, Ki67, HER2, axillary lymph node metastasis (ALNM), and lymphovascular invasion (LVI), the AUC was 0.673, 0.680, 0.651, 0.587, and 0.566, respectively. The number of high-throughput features that closely correlated with biological properties was 34 for histological grade (AUC 0.942), 27 for Ki67 (AUC 0.732), 25 for HER2 (AUC 0.730), 34 for ALNM (AUC 0.804), and 34 for LVI (AUC 0.795). CONCLUSION: High-throughput quantitative sonographic features are superior to traditional qualitative ultrasound features in predicting the biological behavior of TNBC. KEY POINTS: • Sonographic appearances of TNBCs showed a great variety in accordance with its biological and clinical characteristics. • Both qualitative and quantitative sonographic features of TNBCs are associated with tumor biological characteristics. • The quantitative high-throughput feature analysis is superior to two-dimensional sonographic feature assessment in predicting tumor biological property.

Design, Synthesis, and Antibacterial Evaluation of Novel Ocotillol Derivatives and Their Synergistic Effects with Conventional Antibiotics.

The improper use of antibiotics has led to the development of bacterial resistance, resulting in fewer antibiotics for many bacterial infections. Especially, the drug resistance of hospital-acquired methicillin-resistant Staphylococcus aureus (HA-MRSA) is distinctly serious. This research designed and synthesized two series of 3-substituted ocotillol derivatives in order to improve their anti-HA-MRSA potency and synergistic antibacterial activity. Among the synthesized compounds, 20-31 showed minimum inhibitory concentration (MIC) values of 1-64 µg/mL in vitro against HA-MRSA 18-19, 18-20, and S. aureus ATCC29213. Compound 21 showed the best antibacterial activity, with an MIC of 1 µg/mL and had synergistic inhibitory effects. The fractional inhibitory concentration index (FICI) value was 0.375, when combined with chloramphenicol (CHL) or kanamycin (KAN). The structure-activity relationships (SARs) of ocotillol-type derivatives were also summarized. Compound 21 has the potential to be developed as a novel antibacterial agent or potentiator against HA-MRSA.

Dynamic changes in Beclin-1, LC3B and p62 at various time points in mice with temporary middle cerebral artery occlusion and reperfusion (tMCAO).

Ischaemic stroke is attributable to cerebrovascular disease and is associated with high morbidity, disability, mortality and recurrence. Autophagy is a critical mediator and plays dual roles in ischaemic stroke. Autophagy can protect against ischaemic brain injury during the early stage of ischaemic stroke, while excessive autophagy can induce apoptosis and exacerbate brain injury. However, the time-dependent variations in autophagy in ischaemic stroke are unknown. C57BL/6 mice were used to establish a model of temporary middle cerebral artery occlusion and reperfusion (tMCAO). The neurological functional scores and infarct volumes were determined at 1 d, 3 d, 5 d, and 7 d after modelling. The levels of Beclin-1, LC3B, p62, GFAP, TNF-α, IL-6, IL-10, ROS, 4-HNE and 8-OHDG were measured by ELISA, RT-PCR, immunofluorescence analysis and western blotting. The morphology of autophagosomes of ischaemic penumbra was observed by transmission electron microscopy (TEM). Beclin-1, LC3B, ROS, 4-HNE, 8-OHDG, GFAP, TNF-α and IL-6 levels increased (P < 0.01), while p62 and IL-10 levels decreased (P < 0.01) after tMCAO compared to those in the sham group. Beclin-1, LC3B, ROS, 4-HNE, 8-OHDG, GFAP, TNF-α and IL-6 levels were reduced in tMCAO mice at 3 d, 5 d and 7 d (P<0.05), and p62 and IL-10 levels were enhanced (P < 0.05) compared to those at 1 d. In addition, Beclin-1 positively correlated with LC3B, GFAP, TNF-α, IL-6, ROS, 4-HNE and 8-OHDG (P < 0.05), and Beclin-1 negatively correlated with p62 and IL-10 (P < 0.05). The number of autophagosomes was consistent with the expression of autophagy marker proteins, both showing a steady decrease. In summary, autophagy was activated within 7 d of tMCAO induction, and it strengthened at 1 d and then weakened steadily from 3 to 7 d. In addition, this study verified that autophagy positively correlated with the inflammatory response and oxidative stress at 7 d after tMCAO.

Removal of antibiotic resistance genes and inactivation of antibiotic-resistant bacteria by oxidative treatments.

The persistence of antibiotics in the environment because of human activities, such as seafood cultivation, has attracted great attention as they can give rise to antibiotic resistance genes (ARGs) and antibiotic-resistant bacteria (ARB). In this study, we explored the inactivation and removal efficiencies of Escherichia coli SR1 and sul1 (plasmid-encoded ARGs), respectively, in their extracellular and intracellular forms (eARGs and iARGs) by three commonly used fishery oxidants, namely chlorine, bromine, and potassium permanganate (KMnO4), at the practical effective concentration range (0.5, 5, and 15 mg/L). Kinetics data were obtained using laboratory phosphate-buffered saline (PBS). Following the same fishery oxidation methods, the determined kinetics models were tested by studying the SR1 and sul1 disinfection efficiencies in (sterilized) pond water matrix. At concentrations of 5 and 15 mg/L, all three oxidants achieved sufficient cumulative integrated exposure (CT values) to completely inactivate SR1 and efficiently remove sul1 (up to 4.0-log). The oxidation methods were then applied to an unsterilized pond water matrix in order to study and evaluate the indigenous ARB and ARGs disinfection efficiencies in aquaculture, which reached 1.4-log and 1.0-log during treatment with fishery oxidants used in pond preparation at high concentrations before stocking (5-15 mg/L), respectively. A high chlorine concentration (15 mg/L) could efficiently remove ARGs (or iARGs) from pond water, and the iARG removal efficiency was higher than that of eARGs in pond water. The method and results of this study could aid in guiding future research and practical disinfection to control the spread of ARGs and ARB in aquaculture.

Reconstruction of the glutamate decarboxylase system in Lactococcus lactis for biosynthesis of food-grade γ-aminobutyric acid.

Gamma-aminobutyric acid (GABA), an important bioactive compound, is synthesized through the decarboxylation of L-glutamate (L-Glu) by glutamate decarboxylase (GAD). The use of lactic acid bacteria (LAB) as catalysts opens interesting avenues for the biosynthesis of food-grade GABA. However, a key obstacle involved in the improvement of GABA production is how to resolve the discrepancy of optimal pH between the intracellular GAD activity and cell growth. In this work, a potential GAD candidate (LpGadB) from Lactobacillus plantarum was heterologously expressed in Escherichia coli. Recombinant LpGadB existed as a homodimer under the native conditions with a molecular mass of 109.6 kDa and exhibited maximal activity at 40°C and pH 5.0. The Km value and catalytic efficiency (kcat/Km) of LpGadB for L-Glu was 21.33 mM and 1.19 mM-1s-1, respectively, with the specific activity of 26.67 µM/min/mg protein. Subsequently, four C-terminally truncated LpGadB mutants (GadBΔC10, GadBΔC11, GadBΔC12, GadBΔC13) were constructed based on homology modeling. Among them, the mutant GadBΔC11 with highest catalytic activity at near-neutral pH values was selected. In further, the GadBΔC11 and Glu/GABA antiporter (GadC) of Lactococcus lactis were co-overexpressed in the host L. lactis NZ3900. Finally, after 48 h of batch fermentation, the engineered strain L. lactis NZ3900/pNZ8149-gadBΔC11C yielded GABA concentration up to 33.52 g/L by applying a two-stage pH control strategy. Remarkably, this is the highest yield obtained to date for GABA from fermentation with L. lactis as a microbial cell factory.Key points• The GadB from L. plantarum was heterologously expressed in E. coli and biochemically characterized.• Deletion of the C-plug in GadB shifted its pH-dependent activity toward a higher pH.• Reconstructing the GAD system of L. lactis is an effective approach for improving its GABA production.