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As a pathogen, Photobacterium damselae subsp. damselae (PDD) is distributed widely in the marine environment, and the host species of PDD are diverse. Although in the past decade the number of reports on the pathogenicity of PDD to marine animals has gradually increased, it is necessary to study this further in order to establish targeted prevention and technical control measures to reduce harm to marine organisms. In this study, a high-virulence PDD strain (PDD1608) was selected to explore the effect of the virulence gene dly on the biological characteristics and pathogenicity. The dly-deleted mutant strain Δdly PDD1608::Cm was successfully constructed using the λRed recombination technique. The biological characteristics of the wild-type and mutant strains were compared, including growth, swarming motility, drug susceptibility, physiological and biochemical characteristics, biofilm formation ability, and hemolytic and phospholipase activity of extracellular products (ECP). Meanwhile, Oryzias melastigma was used as the target host, and the pathogenicity of wild-type and mutant strains and their ECPs to O.melastigma was detected by the artificial infection test. There was a positive correlation between the results of the bacterial solution and ECP challenge, indicating that deletion of the dly gene affects the virulence of the PDD strain. Deletion of the virulence gene dly resulted in slower growth of the PDD mutant strain; reduced swarming and hemolytic and phospholipase activity; no change in the drug susceptibility or physiological and biochemical characteristics of the wild type and mutant strains; compared with the wild type strain, significant different (P < 0.05) biofilm formation ability of the mutant strain; the artificial infection test showed that the pathogenicity of the mutant strain and its ECP decreased. The results of this study revealed that the virulence gene dly affects many biological characteristics of PDD strains, such as growth, swarming motility, and hemolytic and phospholipase activity, and it was found to be closely related to the pathogenicity of the PDD strain and ECP.The results of this study contribute to further study of the influence of the virulence gene dly on the biological characteristics of the PDD strain in order to determine the appropriate prevention and control measures to curtail its spread and infection. The artificial infection test of the model organism O.melastigma helps provide an animal reference model for further exploration of the pathogenic mechanism and infection process of the PDD strain on the host. The results of the ECP experiments provide a theoretical reference for the research and development of PDD subunit vaccines.

Vibrio harveyi, Vibrio parahaemolyticus, Vibrio scophthalmi, Vibrio anguillarum, and Photobacterium damselae subsp. damselae are important pathogenic bacteria frequently reported in mariculture animal diseases in recent years. These pathogens exhibit strong pathogenicity, wide epidemic area, and high mortality rates, which usually cause serious economic losses in aquaculture. Strengthening the research on rapid detection technology of these pathogens can help to effectively prevent and control their transmission and infection, and reduce economic losses of the aquaculture industry. Therefore, rapid detection of multiple pathogens in this field can promote the disease control technology development of the aquaculture industry. In this study, the vhhA gene of V. harveyi, toxR gene of V. parahaemolyticus, luxR gene of V. scophthalmi, empA gene of V. anguillarum, and Mcp gene of P. damselae subsp. damselae were selected as the target genes. Specific primers were designed using Primer Premier 5.0 software. V. harveyi, V. parahaemolyticus, V. scophthalmi, V. anguillarum, and P. damselae subsp. damselae were used as the target bacteria, V. campbelii and 10 other bacterial species were used as the control group, and sterile water was used as the blank control. The specificity of the primers was verified by the laboratorial conventional real-time quantitative PCR. Thereafter, V. harveyi, V. parahaemolyticus, V. scophthalmi, V. anguillarum, and P. damselae subsp. damselae were amplified using the UF-150 microfluidic fluorescence quantitative PCR instrument to verify the feasibility and specificity of the designed primers on microfluidic fluorescence quantitative PCR. The PCR products were digested and recycled, then ligated to PMD-19T vector and transformed into DH-5α competent cells to construct the standard reference material. The extracted plasmid DNA was used as a template for fluorescence quantitative PCR amplification after 10-fold gradient dilution, and a standard curve was drawn. The DNA of V. harveyi, V. parahaemolyticus, V. scophthalmi, V. anguillarum, and P. damselae subsp. damselae were used to optimize the annealing temperatures for single microfluidics quantitative PCR. The annealing temperatures were set at 58 ℃, 59 ℃, 60 ℃, 61 ℃, 62 ℃, and 63 ℃, respectively, and the optimal common annealing temperatures of the five strains were finally screened. Meanwhile, the number of reaction cycles was set at 25, 30, 35 and 40, respectively, to verify the optimal number of reaction cycles. The designed specific primers of V. harveyi, V. parahaemolyticus, V. scophthalmi, V. anguillarum, and P. damselae subsp. damselae were integrated into the microfluidic chip; multiple microfluidic fluorescence quantitative PCR was performed on the integrated chip with the DNA template of the five strains to verify the cross-reaction between the primers. Based on the reaction systems and conditions of single detection methods, the reaction systems and conditions of multiple microfluidic fluorescence quantitative PCR were optimized, and the microfluidic chip was integrated to establish a multiple microfluidic fluorescence quantitative PCR detection technology that could simultaneously detect these five pathogens: V. harveyi, V. parahaemolyticus, V. scophthalmi, V. anguillarum and P. damselae subsp. damselae. Scophthalmus maximus, Litopenaeus vannamei, and Apostichopus japonicus were selected as the experimental orgainsms. The five pathogens were mixed with the tissues of S. maximus (muscle, gill and liver), L. vannamei (muscle and gill), and A. japonicus (respiratory tree and intestine). The established multiple microfluidic fluorescence quantitative PCR method was used to detect these tissues which contained the five pathogens, and the conventional real-time fluorescence quantitative PCR was performed for comparison. The results showed that the optimal reaction system of the established detection method was as follows: 2×Taq Pro Universal SYBR qPCR Master Mix 5 μL, primer F/R 1 μL, DNA template 2 μL, and ddH2O 1 μL. The reaction conditions were as follows: 95 ℃ for 30 s, 95 ℃ for 5 s, 61 ℃ for 30 s, and 30 cycles of amplification. The standard curves of V. harveyi, V. parahaemolyticus, V. scophthalmi, V. anguillarum, and P. damselae subsp. damselae showed good linearity in the range of 104–109 copies/μL. The linear equations were as follows: y= ‒2.972x+6.73, R2= 0.999; y= ‒3.287x+7.48, R2=0.998; y= ‒3.549x+8.14, R2=0.998; y= ‒3.912x+7.83, R2=0.999; y= ‒3.969x+7.07, R2=0.992, respectively. The method showed strong specificity and high sensitivity to V. harveyi, V. parahaemolyticus, V. scophthalmi, V. anguillarum, and P. damselae subsp. damselae, and the minimum detection limits for these bacteria were 40, 200, 200, 500 and 20 CFU/mL, respectively. The established multiplex microfluidic fluorescence quantitative PCR method was used to detect these pathogens in animal samples and compare the results to those of conventional fluorescence quantitative PCR. The results showed that the accuracy of the established multi-microfluidic fluorescence quantitative PCR method was higher than 96.2% of the conventional real-time fluorescence quantitative PCR method. The average detection time of the samples was only 26 min, which was significantly shorter than that of the conventional real-time fluorescence quantitative PCR reaction time of 1 h 40 min. The multiplex microfluidic fluorescence quantitative PCR method established in this study for the detection of five kinds of mariculture pathogens has strong specificity, high sensitivity, low environmental condition requirements, outstanding portability, and detection accuracy (same as that of conventional real-time fluorescence quantitative PCR), which is suitable for the development of rapid detection technology for aquatic pathogens.

Abstract Acoustic tweezers can control target movement through the momentum interaction between an acoustic wave and an object. This technology has advantages over optical tweezers for in-vivo cell manipulation due to its high tissue penetrability and strong acoustic radiation force. However, normal cells are difficult to acoustically manipulate because of their small size and the similarity between their acoustic impedance and that of the medium. In this study, we use the heterologous expression of gene clusters to generate genetically engineered bacteria that can produce numerous sub-micron gas vesicles in the bacterial cytoplasm. We show that the presence of the gas vesicles significantly enhances the acoustic sensitivity of the engineering bacteria, which can be manipulated by ultrasound. We find that by employing phased-array-based acoustic tweezers, the engineering bacteria can be trapped into clusters and manipulated in vitro and in vivo via electronically steered acoustic beams, enabling the counter flow or on-demand flow of these bacteria in the vasculature of live mice. Furthermore, we demonstrate that the aggregation efficiency of engineering bacteria in a tumour is improved by utilizing this technology. This study provides a platform for the in-vivo manipulation of live cells, which will promote the progress of cell-based biomedical applications.

Electroactive microorganisms (EAMs) play important roles in biogeochemical redox processes and have been of great interest in the fields of energy recovery, waste treatment, and environmental remediation. However, the currently identified EAMs are difficult to be widely used in complex and diverse environments, due to the existence of poor electron transfer capability, weak environmental adaptability, and difficulty with engineering modifications, etc. Therefore, rapid and efficient screening of high performance EAMs from environments is an effective strategy to facilitate applications of microbial fuel cells (MFCs). In this study, to achieve efficient degradation of methyl orange (MO) by MFC and electricity harvest, a more efficient exoelectrogen Shewanella carassii-D5 that belongs to Shewanella spp. was first isolated from activated sludge by WO3 nanocluster probe technique. Physiological properties experiments confirmed that S. carassii-D5 is a Gram-negative strain with rounded colonies and smooth, slightly reddish surface, which could survive in media containing lactate at 30 °C. Moreover, we found that S. carassii-D5 exhibited remarkable MO degradation ability, which could degrade 66% of MO within 72 h, 1.7 times higher than that of Shewanella oneidensis MR-1. Electrochemical measurements showed that MFCs inoculated with S. carassii-D5 could generate a maximum power density of 704.6 mW/m2, which was 5.6 times higher than that of S. oneidensis MR-1. Further investigation of the extracellular electron transfer (EET) mechanism found that S. carassii-D5 strain had high level of c-type cytochromes and strong biofilm formation ability compared with S. oneidensis MR-1, thus facilitating direct EET. Therefore, to enhance indirect electron transfer and MO degradation capacity, a synthetic gene cluster ribADEHC encoding riboflavin synthesis pathway from Bacillus subtilis was heterologously expressed in S. carassii-D5, increasing riboflavin yield from 1.9 to 9.0 mg/g DCW with 1286.3 mW/m2 power density output in lactate fed-MFCs. Furthermore, results showed that the high EET rate endowed a faster degradation efficient of MO from 66% to 86% with a maximum power density of 192.3 mW/m2, which was 1.3 and 1.6 times higher than that of S. carassii-D5, respectively. Our research suggests that screening and engineering high-efficient EAMs from sludge is a feasible strategy in treating organic pollutants.

A full understanding of bitumen fatigue cracking behavior is extremely important as this phenomenon has a considerable influence on bituminous pavement performance. The current framework for assessing this asphalt binder property is inconsistent in ranking bitumen fatigue performance in terms of the failure definition and damage characteristic curve (DCC) analysis. This study used four different types of asphalt binders: neat asphalt (NA), self-healing thermoplastic polyurethane (STP)-modified bitumen, self-healing poly (dimethyl siloxane) crosslinked with urea bond (IPA1w)-modified bitumen, and styrene–butadiene–styrene (SBS)-modified bitumen (SBSB). All the bitumens were subjected to short-term and long-term aging, and they were also tested by utilizing the linear amplitude sweep (LAS) test and the simplified viscoelastic continuum damage (S-VECD) model. LAS and S-VECD procedures were used to apply the newly proposed and current frameworks in order to analyze bitumen performance. The current framework showed that the bitumens that used a higher number of loading cycles (N) to reach their failure points (Nf) failed to exhibit greater fatigue performances in terms of DCC analysis. The developed framework (mainly based on the damage intensity [S] instead of N) was used to solve the inconsistency between the failure definition and DCC assessment in ranking bitumen performance. Additionally, the current framework (failure criterion) presented two R2 values below 0.1, but the developed framework (failure criterion) showed that all R2 values were greater than 0.9. The developed framework represents a turning point because, for the first time, this type of procedure is mainly being based on S instead of N. Although further tests are needed to confirm its efficiency, it eliminates the inconsistency between the failure definition and DCC assessment.

Trypanosoma brucei, the pathogen causing African sleeping sickness (trypanosomiasis) in humans, causes debilitating diseases in many regions of the world, but mainly in African countries with tropical and subtropical climates. Enormous efforts have been devoted to controlling trypanosomiasis, including expanding vector control programs, searching for novel anti-trypanosomial agents, and developing vaccines, but with limited success. In this study, we systematically investigated the effect of graphene quantum dots (GQDs) on trypanosomal parasites and their underlying mechanisms. Ultrasmall-sized GQDs can be efficiently endocytosed by T. brucei and with no toxicity to mammalian-derived cells, triggering a cascade of apoptotic reactions, including mitochondrial disorder, intracellular reactive oxygen species (ROS) elevation, Ca2+ accumulation, DNA fragmentation, adenosine triphosphate (ATP) synthesis impairment, and cell cycle arrest. All of these were caused by the direct interaction between GQDs and the proteins associated with cell apoptosis and anti-oxidation responses, such as trypanothione reductase (TryR), a key protein in anti-oxidation. GQDs specifically inhibited the enzymatic activity of TryR, leading to a reduction in the antioxidant capacity and, ultimately, parasite apoptotic death. These data, for the first time, provide a basis for the exploration of GQDs in the development of anti-trypanosomials.

[This corrects the article DOI: 10.1371/journal.pone.0228391.].

BACKGROUND:The respiratory syncytial virus (RSV) is the main cause of bronchiolitis in infants and interferon (IFN) α is a commercial antiviral drug. The nebulization of IFN α1b could be a viable treatment method. In this study, the therapeutic effects and safety of IFN α1b delivery via nebulization in infant bronchiolitis were investigated in this multi-center prospective study. METHODS AND FINDINGS:Bronchiolitis patients admitted to 22 hospitals who met the inclusion criteria were enrolled and randomly allocated to four groups: control, IFN Intramuscular Injection, IFN Nebulization 1 (1 μg/kg), and IFN Nebulization 2 (2 μg/kg) groups. All patients were observed for 7 days. The therapeutic effects and safety of different IFN delivery doses and delivery modes were evaluated. Coughing severity change, as scored by the researchers and parents, between days 1 and 3 was significantly different between the IFN Nebulization 2 and control groups. Lowell wheezing score change between days 3 and 5 was significantly different between IFN Nebulization 1 and control groups. There were no significant differences among the four groups regarding the number of consecutive days with fever, three-concave sign, fatigue and sleepiness, and loss of appetite. There were no cases of severe complications, no recurrence of fever, and no regression of mental status. CONCLUSIONS:IFN-α1b could more effectively alleviate coughing and wheezing in bronchiolitis. IFN-α1b nebulization had significant advantages in shortening the duration of wheezing and alleviating coughing.

Owing to their stability, doubly-fed induction generator (DFIG) integrated systems have gained considerable interest and are the most widely implemented type of wind turbines and due to the increasing escalation of the wind generation penetration rate in power systems. In this study, we investigate a DFIG integrated system comprising four modules: (1) a wind turbine that considers the maximum power point tracking and pitch-angle control, (2) induction generator, (3) rotor/grid-side converter with the corresponding control strategy, and (4) AC power grid. The detailed small-signal modeling of the entire system is performed by linearizing the dynamic characteristic equation at the steady-state value. Furthermore, a dichotomy method is proposed based on the maximum eigenvalue real part function to obtain the critical value of the parameters. Root-locus analysis is employed to analyze the impact of changes in the phase-locked loop, short-circuit ratio, and blade inertia on the system stability. Lastly, the accuracy of the small-signal model and the real and imaginary parts of the calculated dominant poles in the theoretical analysis are verified using PSCAD/EMTDC. [ABSTRACT FROM AUTHOR]