Biodegradation of DDT using multi-species mixtures: From genome-mining prediction to practical assessment.

DDT (dichlorodiphenyltrichloroethane) is a commonly used insecticide that is recalcitrant and highly stable in the environment. Currently, DDT residue contamination, especially in agricultural soil, is still a concern in many countries, threatening human health and the environment. Among the approaches to resolve such an issue, novel biodegradation-based methods are now preferred to physicochemical methods, due to the sustainability and the effectiveness of the former. In this study, we explored the possibility of building mixed microbial cultures that can offer improved DDT-degrading efficiencies and be more environmentally transilient, based on genome annotation using the KEGG database and prediction of interactions between single strains using the obtained metabolic maps. We then proposed 10 potential DDT-degrading mixed cultures of different strain combinations and evaluated their DDT degradation performances in liquid, semi-solid and solid media. The results demonstrated the superiority of the mixtures over the single strains in terms of degrading DDT, particularly in a semi-solid medium, with up to 40-50% more efficiency. Not only did the mixed cultures degrade DDT more efficiently, but they also adapted to broader spectra of environmental conditions. The three best DDT-degrading and transilient mixtures were selected, and it turned out that their component strains seemed to have more metabolic interactions than those in the other mixtures. Thus, our study demonstrates the effectiveness of exploiting genome-mining techniques and the use of constructed mixed cultures in improving biodegradation.

RNA therapeutics for respiratory diseases.

It has become increasingly common to utilize RNA treatment to treat respiratory illnesses. Experimental research on both people and animals has advanced quickly since the turn of the twenty-first century in an effort to discover a treatment for respiratory ailments that could not be accomplished with earlier techniques, specifically in treating prevalent respiratory diseases such as lung cancer, chronic obstructive pulmonary disease (COPD), respiratory infections caused by viruses, and asthma. This chapter has provided a comprehensive overview of the scientific evidence in applying RNA therapy to treat respiratory diseases. The chapter describes the development of this therapy for respiratory diseases. At the same time, the types of RNA therapy for respiratory diseases have been highlighted. In addition, the mechanism of this therapy for respiratory diseases has also been covered. These insights are indispensable if this therapy is to be developed widely.

Effects of MAT1-2 Spore Ratios on Fruiting Body Formation and Degeneration in the Heterothallic Fungus Cordyceps militaris.

The medicinal mushroom Cordyceps militaris is widely exploited in traditional medicine and nutraceuticals in Asian countries. However, fruiting body production in C. militaris is facing degeneration through cultivation batches, and the molecular mechanism of this phenomenon remains unclear. This study showed that fruiting body formation in three different C. militaris strains, namely G12, B12, and HQ1, severely declined after three successive culturing generations using the spore isolation method. PCR analyses revealed that these strains exist as heterokaryons and possess both the mating-type loci, MAT1-1 and MAT1-2. Further, monokaryotic isolates carrying MAT1-1 or MAT1-2 were successfully separated from the fruiting bodies of all three heterokaryotic strains. A spore combination of the MAT1-1 monokaryotic isolate and the MAT1-2 monokaryotic isolate promoted fruiting body formation, while the single monokaryotic isolates could not do that themselves. Notably, we found that changes in ratios of the MAT1-2 spores strongly influenced fruiting body formation in these strains. When the ratios of the MAT1-2 spores increased to more than 15 times compared to the MAT1-1 spores, the fruiting body formation decreased sharply. In contrast, when MAT1-1 spores were increased proportionally, fruiting body formation was only slightly reduced. Our study also proposes a new solution to mitigate the degeneration in the heterokaryotic C. militaris strains caused by successive culturing generations.

Long-Term Epidemiology and Evolution of Swine Influenza Viruses, Vietnam.

Influenza A viruses are a One Health threat because they can spill over between host populations, including among humans, swine, and birds. Surveillance of swine influenza virus in Hanoi, Vietnam, during 2013-2019 revealed gene pool enrichment from imported swine from Asia and North America and showed long-term maintenance, persistence, and reassortment of virus lineages. Genome sequencing showed continuous enrichment of H1 and H3 diversity through repeat introduction of human virus variants and swine influenza viruses endemic in other countries. In particular, the North American H1-δ1a strain, which has a triple-reassortant backbone that potentially results in increased human adaptation, emerged as a virus that could pose a zoonotic threat. Co-circulation of H1-δ1a viruses with other swine influenza virus genotypes raises concerns for both human and animal health.

Echinochrome A Prevents Diabetic Nephropathy by Inhibiting the PKC-Iota Pathway and Enhancing Renal Mitochondrial Function in db/db Mice.

Echinochrome A (EchA) is a natural bioproduct extracted from sea urchins, and is an active component of the clinical drug, Histochrome®. EchA has antioxidant, anti-inflammatory, and antimicrobial effects. However, its effects on diabetic nephropathy (DN) remain poorly understood. In the present study, seven-week-old diabetic and obese db/db mice were injected with Histochrome (0.3 mL/kg/day; EchA equivalent of 3 mg/kg/day) intraperitoneally for 12 weeks, while db/db control mice and wild-type (WT) mice received an equal amount of sterile 0.9% saline. EchA improved glucose tolerance and reduced blood urea nitrogen (BUN) and serum creatinine levels but did not affect body weight. In addition, EchA decreased renal malondialdehyde (MDA) and lipid hydroperoxide levels, and increased ATP production. Histologically, EchA treatment ameliorated renal fibrosis. Mechanistically, EchA suppressed oxidative stress and fibrosis by inhibiting protein kinase C-iota (PKCι)/p38 mitogen-activated protein kinase (MAPK), downregulating p53 and c-Jun phosphorylation, attenuating NADPH oxidase 4 (NOX4), and transforming growth factor-beta 1 (TGFß1) signaling. Moreover, EchA enhanced AMPK phosphorylation and nuclear factor erythroid-2-related factor 2 (NRF2)/heme oxygenase 1 (HO-1) signaling, improving mitochondrial function and antioxidant activity. Collectively, these findings demonstrate that EchA prevents DN by inhibiting PKCι/p38 MAPK and upregulating the AMPKα/NRF2/HO-1 signaling pathways in db/db mice, and may provide a therapeutic option for DN.

A newly constructed Agrobacterium-mediated transformation system based on the hisB auxotrophic marker for genetic manipulation in Aspergillus niger.

The filamentous fungus Aspergillus niger is widely exploited as an industrial workhorse for producing enzymes and organic acids. So far, different genetic tools, including CRISPR/Cas9 genome editing strategies, have been developed for the engineering of A. niger. However, these tools usually require a suitable method for gene transfer into the fungal genome, like protoplast-mediated transformation (PMT) or Agrobacterium tumefaciens-mediated transformation (ATMT). Compared to PMT, ATMT is considered more advantageous because fungal spores can be used directly for genetic transformation instead of protoplasts. Although ATMT has been applied in many filamentous fungi, it remains less effective in A. niger. In the present study, we deleted the hisB gene and established an ATMT system for A. niger based on the histidine auxotrophic mechanism. Our results revealed that the ATMT system could achieve 300 transformants per 107 fungal spores under optimal transformation conditions. The ATMT efficiency in this work is 5 - 60 times higher than those of the previous ATMT studies in A. niger. The ATMT system was successfully applied to express the DsRed fluorescent protein-encoding gene from the Discosoma coral in A. niger. Furthermore, we showed that the ATMT system was efficient for gene targeting in A. niger. The deletion efficiency of the laeA regulatory gene using hisB as a selectable marker could reach 68 - 85% in A. niger strains. The ATMT system constructed in our work represents a promising genetic tool for heterologous expression and gene targeting in the industrially important fungus A. niger.

Penicillium digitatum as a Model Fungus for Detecting Antifungal Activity of Botanicals: An Evaluation on Vietnamese Medicinal Plant Extracts.

Medicinal plants play important roles in traditional medicine, and numerous compounds among them have been recognized for their antimicrobial activity. However, little is known about the potential of Vietnamese medicinal plants for antifungal activity. In this study, we examined the antagonistic activity of twelve medicinal plant species collected in Northern Vietnam against Penicillium digitatum, Aspergillus flavus, Aspergillus fumigatus, and Candida albicans. The results showed that the antifungal activities of the crude extracts from Mahonia bealei, Ficus semicordata, and Gnetum montanum were clearly detected with the citrus postharvest pathogen P. digitatum. These extracts could fully inhibit the growth of P. digitatum on the agar medium, and on the infected citrus fruits at concentrations of 300-1000 µg/mL. Meanwhile, the other tested fungi were less sensitive to the antagonistic activity of the plant extracts. In particular, we found that the ethanolic extract of M. bealei displayed a broad-spectrum antifungal activity against all four pathogenic fungi. Analysis of this crude extract by enrichment coupled with high-performance liquid chromatography revealed that berberine and palmatine are major metabolites. Additional inspections indicated berberine as the key compound responsible for the antifungal activity of the M. bealei ethanolic extract. Our study provides a better understanding of the potential of Vietnamese medicinal plant resources for combating fungal pathogens. This work also highlights that the citrus pathogen P. digitatum can be employed as a model fungus for screening the antifungal activity of botanicals.

A low-cost system for monitoring pH, dissolved oxygen and algal density in continuous culture of microalgae.

In a continuous and closed system of culturing microalgae, constantly monitoring and controlling pH, dissolved oxygen (DO) and microalgal density in the cultivation environment are paramount, which ultimately influence on the growth rate and quality of the microalgae products. Apart from the pH and DO parameters, the density of microalgae can be used to contemplate what light condition in the culture chamber is or when nutrients should be supplemented, which both also decide productivity of the cultivation. Moreover, the microalgal density is considered as an indicator indicating when the microalgae can be harvested. Therefore, this work proposes a low-cost monitoring equipment that can be employed to observe pH, DO and microalgal density over time in a culture environment. The measurements obtained by the proposed monitoring device can be utilized for not only real-time observations but also controlling other sub-systems in a continuous culture model including stirring, ventilating, nutrient supplying and harvesting, which leads to more efficiency in the microalgal production. More importantly, it is proposed to utilize the off-the-shelf materials to fabricate the equipment with a total cost of about 513 EUR, which makes it practical as well as widespread. The proposed monitoring apparatus was validated in a real-world closed system of cultivating a microalgae strain of Chlorella vulgaris. The obtained results indicate that the measurement accuracies are 0.3%, 3.8% and 8.6% for pH, DO and microalgae density quantities, respectively.

Notes on Culturable Endophytic Microorganisms Isolated from 14 Medicinal Plants in Vietnam: A Diversity Analysis to Predict the Host-Microbe Correlations.

Endophytes can generate a cornucopia of marvelous bioactive secondary metabolites useful for mankind but their biodiversity and associations with host plants are still elusive. In this study, we explored the culturable endophytic microorganisms associated with 14 medicinal plants that are of high socio-economic value and/or reportedly endemic to northern Vietnam. Specifically, we isolated the endophytic microorganisms by applying surface sterilization methods and identified them based on morphological and rDNA sequence analyses. Agglomerative Hierarchical Clustering (AHC) and Principal Component Analysis (PCA) were used to analyze the correlations between the taxonomic affiliations of the culturable endophytes and the characteristics of their hosts. Most of the culturable endophytes obtained were bacteria (80), and few of those were actinomycetes (15) and fungi (8). Many of them are reported to be endophytes of medicinal plants for the first time. A number of plants (5) are also reported for the first time to contain microbial endophytes, while some plants with powerful pharmaceutical potential harbor unique endophytes. Furthermore, our results reveal a strikingly close relation between the compositions of bacterial and fungal isolates from plants having anti-bacterial activity and those from plants having anti-inflammatory activity, or between the compositions of the microbial endophytic isolates from plants having anti-cancer activity and those from plants having antioxidant activity. Altogether, the results provide new findings which can be inspiring for further in-depth studies to explore and exploit the relationships between medicinal plants and their associated endophytes in northern Vietnam and world-wide.

Prevalence of left atrial appendage thrombus in patients with acute ischaemic stroke and sinus rhythm: a cross-sectional study.

OBJECTIVE: Thrombi originating in the left atrial appendage (LAA) mainly form because of atrial fibrillation (AF) and are a known cause of cardioembolic stroke. We aimed to investigate the prevalence of LAA thrombus in patients with acute ischaemic stroke (AIS) and sinus rhythm on 12-lead ECG. METHODS: From June 2019 to February 2021, we conducted a cross-sectional study wherein we performed transoesophageal echocardiography (TEE) in patients with AIS and sinus rhythm on 12-lead ECG who were referred for detection of LAA thrombus. After TEE, all patients underwent 24-hour ECG monitoring to screen for paroxysmal AF. Predictors of LAA thrombus were determined using logistic regression analysis. RESULTS: Overall, 223 patients (age: 66.2±11.3 years, men: 61.4%) were included in the study. LAA thrombus was detected in 15 patients (6.7%). Paroxysmal AF was detected in 14 of the 15 patients during 24-hour ECG monitoring. Compared with the non-thrombus group, the thrombus group had a statistically significant higher rate of spontaneous echo contrast (SEC), longer LAA, lower peak LAA emptying velocity and predominantly bilateral stroke. In the adjusted model, the presence of SEC increased the probability of LAA thrombus (OR 9.04; 95% CI 2.12 to 38.54; p=0.003). CONCLUSIONS: In patients with AIS and sinus rhythm on 12-lead ECG, our study revealed that the prevalence of LAA thrombus was 6.7% with the most prevalent aetiology being paroxysmal AF. The presence of SEC can be a predictor of LAA thrombus in these patients.

Growth and health of juvenile barramundi (Lates calcarifer) challenged with DO hypoxia after feeding various inclusions of germinated, fermented and untreated peanut meals.

Peanut (Arachis hypogaea) is mainly grown for oil extraction and the remaining oil-free seed referred as peanut meal (PM) leaves with high protein content which can be a possible substitute for fishmeal in aqua-diets. This study evaluates the suitability of three types of processed peanut seeds, namely untreated PM (UPM), fermented PM (FPM), and germinated PM (GPM) from peanut seeds to replace fishmeal in barramundi (Lates calcarifer) diets cultured under a commercial production environment. Nine formulated diets having 3 inclusion levels from the 3 different peanuts (15%, 30% and 60% fishmeal replacement) were evaluated against a control without PM. The performance of various types and levels of PMs was assessed by examining the growth, gut and liver condition and survival of fish after eight weeks of feeding the test diets. The immunological responses of juvenile barramundi were assessed by exposing the fish to the hypoxic conditions for 4 hours. The results showed that fermentation and germination significantly (P<0.05) reduced the tannins and alkaloid contents in the PMs. The fish fed 15% GPM diet grew faster and had higher survival than fish fed control diet, while fish fed diet including 60% GPM showed a significant reduction in growth and survival, and an increase in food conversion rate (FCR). FPM and UPM at any inclusion levels did not alter the growth, survival and FCR. Histology analysis revealed that fish fed 60% GPM and UPM showed higher amount of lipid droplets in liver, myodigeneration in fish muscle and a decrease number of acidic mucins in distal gut compare to all other test diets. Stress caused by reduced dissolved oxygen did not change the sodium, potassium, chlorides and alanine aminotransferase concentrations of plasma of fish fed any diet. However, the stress did increase plasma cortisol significantly (P<0.05) in fish fed 60% GPM, 30% and 60% UPM diets. These results suggest that the PMs can partly replace the fishmeal in juvenile barramundi diet and the processing further improves the PMs quality by reducing its antinutritional factors which in turn can increase either its inclusion level in the barramundi diets or improved growth and health status of the species.

A newly constructed Agrobacterium-mediated transformation system revealed the influence of nitrogen sources on the function of the LaeA regulator in Penicillium chrysogenum.

Penicillium chrysogenum is not only an industrially important filamentous fungus for penicillin production, but it also represents as a promising cell factory for production of natural products. Development of efficient transformation systems with suitable selection markers is essential for genetic manipulations in P. chrysogenum. In this study, we have constructed a new and efficient Agrobacterium tumefaciens-mediated transformation (ATMT) system with two different selection markers conferring the resistance to nourseothricin and phleomycin for P. chrysogenum. Under the optimized conditions for co-cultivation at 22 °C for 60 h with acetosyringone concentration of 200 µM, the transformation efficiency of the ATMT system could reach 5009 ± 96 transformants per 106 spores. The obtained transformants could be exploited as the T-DNA insertion mutants for screening genes involved in morphogenesis and secondary metabolism. Especially, the constructed ATMT system was applied successfully to generate a knockout mutant of the laeA regulatory gene and relevant complementation strains in a wild strain of P. chrysogenum. Our results indicated that the LaeA regulator controls growth, sporulation, osmotic stress response and antibiotic production in P. chrysogenum, but its function is reliant on nitrogen sources. Furthermore, we showed that the laeA orthologous genes from the citrus postharvest pathogen P. digitatum and from the industrial fungus Aspergillus niger could recover the phenotypic defects in the P. chrysogenum laeA deletion mutant. Conclusively, this work provides a new ATMT system, which can be employed for T-DNA insertional mutagenesis, heterologous gene expression or for molecular inspections of potential genes related to secondary metabolism in P. chrysogenum.

A Laboratory-Scale Study of the Applicability of a Halophilic Sediment Bioelectrochemical System for in situ Reclamation of Water and Sediment in Brackish Aquaculture Ponds: Effects of Operational Conditions on Performance.

Sediment bioelectrochemical systems (SBESs) can be integrated into brackish aquaculture ponds for in-situ bioremediation of the pond water and sediment. Such an in-situ system offers advantages including reduced treatment cost, reusability and simple handling. In order to realize such an application potential of the SBES, in this laboratory-scale study we investigated the effect of several controllable and uncontrollable operational factors on the in-situ bioremediation performance of a tank model of a brackish aquaculture pond, into which a SBES was integrated, in comparison with a natural degradation control model. The performance was evaluated in terms of electricity generation by the SBES, Chemical oxygen demand (COD) removal and nitrogen removal of both the tank water and the tank sediment. Real-life conditions of the operational parameters were also experimented to understand the most close-to-practice responses of the system to their changes. Predictable effects of controllable parameters including external resistance and electrode spacing, similar to those reported previously for the BESs, were shown by the results but exceptions were observed. Accordingly, while increasing the electrode spacing reduced the current densities but generally improved COD and nitrogen removal, increasing the external resistance could result in decreased COD removal but also increased nitrogen removal and decreased current densities. However, maximum electricity generation and COD removal efficiency difference of the SBES (versus the control) could be reached with an external resistance of 100 Ω, not with the lowest one of 10 Ω. The effects of uncontrollable parameters such as ambient temperature, salinity and pH of the pond (tank) water were rather unpredictable. Temperatures higher than 35°C seemed to have more accelaration effect on natural degradation than on bioelectrochemical processes. Changing salinity seriously changed the electricity generation but did not clearly affect the bioremediation performance of the SBES, although at 2.5% salinity the SBES displayed a significantly more efficient removal of nitrogen in the water, compared to the control. Variation of pH to practically extreme levels (5.5 and 8.8) led to increased electricity generations but poorer performances of the SBES (vs. the control) in removing COD and nitrogen. Altogether, the results suggest some distinct responses of the SBES under brackish conditions and imply that COD removal and nitrogen removal in the system are not completely linked to bioelectrochemical processes but electrochemically enriched bacteria can still perform nonbioelectrochemical COD and nitrogen removals more efficiently than natural ones. The results confirm the application potential of the SBES in brackish aquaculture bioremediation and help propose efficient practices to warrant the success of such application in real-life scenarios.

A Laboratory-Scale Study of the Applicability of a Halophilic Sediment Bioelectrochemical System for in situ Reclamation of Water and Sediment in Brackish Aquaculture Ponds: Establishment, Bacterial Community and Performance Evaluation.

In this study, we investigated the potential of using sediment bioelectrochemical systems (SBESs) for in situ treatment of the water and sediment in brackish aquaculture ponds polluted with uneaten feed. An SBES integrated into a laboratory-scale tank simulating a brackish aquaculture pond was established. This test tank and the control (not containing the SBES) were fed with shrimp feed in a scheme that mimics a situation where 50% of feed is uneaten. After the SBES was inoculated with microbial sources from actual shrimp pond sediments, electricity generation was well observed from the first experimental week, indicating successful enrichment of electrochemically active bacteria in the test tank sediment. The electricity generation became steady after 3 weeks of operation, with an average current density of 2.3 mA/m2 anode surface and an average power density of 0.05 mW/m2 anode surface. The SBES removed 20-30% more COD of the tank water, compared to the control. After 1 year, the SBES also reduced the amount of sediment in the tank by 40% and thus could remove approximately 40% more COD and approximately 52% more nitrogen from the sediment, compared to the control. Insignificant amounts of nitrite and nitrate were detected, suggesting complete removal of nitrogen by the system. PCR-DGGE-based analyses revealed the dominant presence of Methylophilus rhizosphaerae, Desulfatitalea tepidiphila and Thiothrix eikelboomii, which have not been found in bioelectrochemical systems before, in the bacterial community in the sediment of the SBES-containing tank. The results of this research demonstrate the potential application of SBESs in helping to reduce water pollution threats, fish and shrimp disease risks, and thus farmers' losses.

Draft Genome Sequence of Streptomyces cavourensis YBQ59, an Endophytic Producer of Antibiotics Bafilomycin D, Nonactic Acid, Prelactone B, and 5,11-Epoxy-10-Cadinanol.

This study reports the draft genome sequence of the endophytic Streptomyces cavourensis strain YBQ59, produces the antibiotics bafilomycin D, nonactic acid, prelactone B, and 5,11-epoxy-10-cadinanol. The draft genome sequence comprises ∼10.2 Mb, with a GC content of 64% and 8,958 predicted protein-coding genes, of which 14 gene clusters were found to associate with antibiotic biosynthetic pathways.

Antimicrobial and Cytotoxic Properties of Bioactive Metabolites Produced by Streptomyces cavourensis YBQ59 Isolated from Cinnamomum cassia Prels in Yen Bai Province of Vietnam.

The endophytic actinomycete strain YBQ59 was isolated from Cinnamomum cassia Prels in Yen Bai province (21°53'14″N; 104°35'9″E) of northern Vietnam. Based on analysis of morphological, physiological characteristics and 16S rRNA gene sequence (GenBank Acc. No. MF950891), the strain YBQ59 possessed high similarity to Streptomyces cavourensis subsp. cavourensis strain NRRL 2740, therefore assigned as S. cavourensis YBQ59. The ethyl acetate extract of the YBQ59 culture broth isolated eight pure secondary metabolites, identified as 1-monolinolein (1), bafilomycin D (2), nonactic acid (3), daidzein (4), 3'-hydroxydaidzein (5), 5,11-epoxy-10-cadinanol (6), prelactone B (7), and daucosterol (8). Compounds 1, 3-8 were reported for the first time from S. cavourensis. Compounds 1-5 exhibited antimicrobial activities against both methicillin-resistant Staphylococcus aureus ATCC 33591 (MRSA) and methicillin-resistant Staphylococcus epidermidis ATCC 35984 (MRSE) among which the compound 1 revealed the strongest effects with minimum inhibitory concentrations of 8.5 and 14.6 µg/mL, respectively. The compound 2 showed high potential effect against MRSA (MIC of 11.1 µg/mL) but less effect against MRSE (MIC of 30.3 µg/mL). The cytotoxicity of the compounds 1-7 was investigated against human lung adenocarcinoma EGFR-TKI-resistant cells, among which compounds 1, 2, and 5 exhibited the strong effect against A549 cells with IC50 values of 3.6, 6.7, and 7.8 µM, respectively. Taken together, the experimental findings in this study suggested that the compounds 1 and 2 could be reproducible metabolites applicable for inhibition of both drug-resistant bacteria and cancer cell lines.

A highly efficient Agrobacterium tumefaciens-mediated transformation system for the postharvest pathogen Penicillium digitatum using DsRed and GFP to visualize citrus host colonization.

Penicillium digitatum is a major postharvest pathogen of citrus crops. This fungus broadly spreads worldwide and causes green mold disease, which results in severe losses for citrus production. Understanding of the citrus infection by P. digitatum may help develop effective strategies for controlling this pathogen. In this study, we have characterized a virulent strain of P. digitatum isolated in Vietnam and established a highly efficient Agrobacterium tumefaciens-mediated transformation (ATMT) system for this fungal strain with two newly constructed binary vectors. These binary vectors harbor dominant selectable markers for hygromycin or nourseothricin resistance, and expression cassettes for the red fluorescent protein (DsRed) or the green fluorescent protein (GFP), respectively. Using the established ATMT system, the transformation efficiency of the Vietnamese strain could reach a very high yield of 1240±165 transformants per 106 spores. Interestingly, we found that GFP is much better than DsRed for in situ visualization of citrus fruit colonization by the fungus. Additionally, we showed that the transformation system can also be used to generate T-DNA insertion mutants for screening non-pathogenic or less virulent strains. Our work provides a new platform including a virulent tropical strain of P. digitatum, an optimized ATMT method and two newly constructed binary vectors for investigation of the postharvest pathogen. This platform will help develop strategies to dissect molecular mechanisms of host-pathogen interactions in more detail as well as to identify potential genes of pathogenicity by either insertional mutagenesis or gene disruption in this important pathogenic fungus.

Rhythmicity and plasticity of digestive physiology in a euryhaline teleost fish, permit (Trachinotus falcatus).

Digestive physiology is considered to be under circadian control, but there is little evidence in teleost fish. The present study explored the rhythmicity and plasticity to feeding schedules of enzymatic digestion in a candidate aquaculture fish, the permit (Trachinotus falcatus). The first experiment identified the rhythms of digestive factors throughout the light-dark (LD) cycle. Gastric luminal pH and pepsin activity showed significant daily variation albeit not rhythmic. These dynamic changes were likewise observed in several digestive enzymes, in which the activities of intestinal protease, chymotrypsin and lipase exhibited significant daily rhythms. In the second experiment, the existence of feed anticipatory activity in the digestive factors was investigated by subjecting the fish to either periodic or random feeding. Anticipatory gastric acidification prior to feeding was identified in periodically fed fish. However, pepsin activity did not exhibit such anticipation but a substantial postprandial increase was observed. Intestinal protease, leucine aminopeptidase and lipase anticipated periodic mealtime with elevated enzymatic activities. Plasma melatonin and cortisol demonstrated robust daily rhythms but feeding time manipulations revealed no significant impact. Plasma ghrelin level remained constant during the LD cycle and appeared to be unaffected by differing feeding regimes as well. Taken together, the digestive factors of permit were highly dynamic during the LD cycle. Periodic feeding entrained digestive physiology and mediated anticipatory gastric acidification and intestinal enzymatic activities. This knowledge will be essential in developing feeding protocols and husbandry-related welfare strategies that will further advance this candidate finfish as an aquaculture species.

A new and efficient approach for construction of uridine/uracil auxotrophic mutants in the filamentous fungus Aspergillus oryzae using Agrobacterium tumefaciens-mediated transformation.

Aspergillus oryzae is a filamentous fungus widely used in food industry and as a microbial cell factory for recombinant protein production. Due to the inherent resistance of A. oryzae to common antifungal compounds, genetic transformation of this mold usually requires auxotrophic mutants. In this study, we show that Agrobacterium tumefaciens-mediated transformation (ATMT) method is very efficient for deletion of the pyrG gene in different Aspergillus oryzae wild-type strains to generate uridine/uracil auxotrophic mutants. Our data indicated that all the obtained uridine/uracil auxotrophic transformants, which are 5- fluoroorotic acid (5-FOA) resistant, exist as the pyrG deletion mutants. Using these auxotrophic mutants and the pyrG selectable marker for genetic transformation via A. tumefaciens, we could get about 1060 transformants per 106 fungal spores. In addition, these A. oryzae mutants were also used successfully for expression of the DsRed fluorescent reporter gene under control of the A. oryzae amyB promoter by the ATMT method, which resulted in obvious red transformants on agar plates. Our work provides a new and effective approach for constructing the uridine/uracil auxotrophic mutants in the importantly industrial fungus A. oryzae. This strategy appears to be applicable to other filamentous fungi to develop similar genetic transformation systems based on auxotrophic/nutritional markers for food-grade recombinant applications.