An efficient Agrobacterium-mediated system based on the pyrG auxotrophic marker for recombinant expression in the filamentous fungus Penicillium rubens.

OBJECTIVES: This work aimed to construct a versatile, effective, and food-grade Agrobacterium tumefaciens-mediated transformation (ATMT) system for recombinant expression in the filamentous fungus Penicillium rubens (also known as Pencillium chrysogenum). RESULTS: In this study, the wild-type P. chrysogenum VTCC 31172 strain was re-classified as P. rubens by a multilocus sequencing analysis. Further, the pyrG gene required for uridine/uracil biosynthesis was successfully deleted in the VTCC 31172 strain by homologous recombination to generate a stable uridine/uracil auxotrophic mutant (ΔpyrG). The growth of the P. rubens ΔpyrG strain could be restored by uridine/uracil supplementation, and a new ATMT system based on the uridine/uracil auxotrophic mechanism was established for this strain. The optimal ATMT efficiency could reach 1750 transformants for 106 spores (equivalent to 0.18%). In addition, supplementation of uridine/uracil at the concentrations of 0.005-0.02% during the co-cultivation process significantly promoted transformation efficiency. Especially, we demonstrated that the pyrG marker and the amyB promoter from the koji mold Aspergillus oryzae were fully functional in P. rubens ΔpyrG. Expression of the DsRed reporter gene under the regulation of the A. oryzae amyB promoter lighted up the mycelium of P. rubens with a robust red signal under fluorescence microscopy. Furthermore, genomic integration of multiple copies of the Aspergillus fumigatus phyA gene under the control of the amyB promoter significantly enhanced phytase activity in P. rubens. CONCLUSIONS: The ATMT system developed in our work provides a safe genetic platform for producing recombinant products in P. rubens without using drug resistance markers.

Evaluation of the fermentation potential of lactic acid bacteria isolated from herbs, fruits and vegetables as starter cultures in nut-based milk alternatives.

Fermentation of plant-based milk alternatives (PBMAs), including nut-based products, has the potential to generate new foods with improved sensorial properties. In this study, we screened 593 lactic acid bacteria (LAB) isolates from herbs, fruits and vegetables for their ability to acidify an almond-based milk alternative. The majority of the strongest acidifying plant-based isolates were identified as Lactococcus lactis, which were found to lower the pH of almond milk faster than dairy yoghurt cultures. Whole genome sequencing (WGS) of 18 plant-based Lc. lactis isolates revealed the presence of sucrose utilisation genes (sacR, sacA, sacB and sacK) in the strongly acidifying strains (n = 17), which were absent in one non-acidifying strain. To confirm the importance of Lc. lactis sucrose metabolism in efficient acidification of nut-based milk alternatives, we obtained spontaneous mutants defective in sucrose utilisation and confirmed their mutations by WGS. One mutant containing a sucrose-6-phosphate hydrolase gene (sacA) frameshift mutation was unable to efficiently acidify almond, cashew and macadamia nut milk alternatives. Plant-based Lc. lactis isolates were heterogeneous in their possession of the nisin gene operon near the sucrose gene cluster. The results of this work show that sucrose-utilising plant-based Lc. lactis have potential as starter cultures for nut-based milk alternatives.

Bicistronic reporter mice for monitoring of Fgf21 expression.

Fibroblast growth factor 21 (FGF21) is a metabolic hormone that is synthesized and secreted by cellular and metabolic stresses. Serum FGF21 levels are associated with clinical parameters in patients with various diseases, including metabolic disorders. Animal models that allow FGF21 levels to be monitored in vivo are important for research and clinical applications of FGF21. Here, a novel Fgf21-reporter mouse strain (Fgf21+/Luc2-tdT) expressing luciferase and tandem dimer tomato (tdT) fluorescence proteins under the control of the endogenous Fgf21 promoter was generated, which provided an in vitro and in vivo monitoring tool for the Fgf21 expression. Luciferase activity, in vivo bioluminescence, and tdT fluorescence were analyzed in adult mice fed or fasted for 24 h. Luciferase activities were significantly increased in the liver but slightly decreased in the pancreas of fasted mice compared with those of fed mice. In vivo bioluminescence signal was increased in the liver of fasted mice. Obvious tdT fluorescence was detected in the pancreas. These results suggest that Fgf21-reporter mice have great potential for research and clinical applications of FGF21.

Findings from the first colorectal cancer screening among 103 542 individuals in Vietnam with systematic review of colorectal cancer screening programs in Asia-Pacific region.

BACKGROUND: Colorectal cancer is a leading cancer incidence and cause of death worldwide and in Vietnam. Although screening is considered an effective measure to prevent and control colorectal cancer, there is no such effort in Vietnam. METHODS: Between 01 January 2018 and 31 October 2019, a population-based colorectal cancer screening program was conducted in Hanoi, Vietnam. A health advocacy campaign and follow-up phone calls were used to enroll residents aged ≥40 years old to complete an immunochemical-fecal occult blood testing. Positive immunochemical-fecal occult blood testing was followed by a colonoscopy. We also conducted a systematic review of the colorectal cancer screening programs in the Asia-Pacific region that used similar approach by searching Ovid Medline and PubMed databases. RESULTS: During study period, 103 542 individuals among 672 742 eligible residents attended the screening of whom 81.5% participants finished immunochemical-fecal occult blood testing test and the positive rate was 6.1%. The coverage rate for immunochemical-fecal occult blood testing test was 11.9%. Among 2278 individuals who underwent colonoscopy, 3.5% were histologically diagnosed with cancer, 17.8% with advanced adenomas, and 23.1% with non-advanced adenomas. Males had significantly higher detection rate of advanced adenomas, cancer or ≥ two polyps/tumor than females (P < 0.0001). The systematic review showed that in two-step modality (i.e. immunochemical-fecal occult blood testing/fecal immunochemical test and colonoscopy), the test positive was from 4.1 to 10.6%. Once colonoscopy was performed subsequently, the rate of cancer among positive participants was from 1.7 to 16.4% and that of advanced adenomas was from 7.1 to 23.1%. CONCLUSION: We showed that the two-step modality is a promising strategy for colorectal cancer screening in Vietnam that might apply to similar settings with limited resources.

Association between body mass index and colorectal adenomas: Findings from a case-control study in Vietnam.

Colorectal cancer is a leading cancer worldwide and in Vietnam. Adenomas (adenomatous polyps) is an important precursor of colorectal cancer. There is currently no study to determine the modifiable risk factors for colorectal adenomas, including body mass index (BMI) in Vietnam. We conducted an individually matched case-control study of 1149 colorectal adenomas and 1145 controls in a large-scale colorectal screening program involving 103 542 individuals aged 40-75 years old in Hanoi, Vietnam. Conditional logistic regression was used to evaluate the association between BMI and colorectal adenomas prevalence, after controlling for potential confounders. Overall, comparing to normal weight (ie, 18.5-22.9 kg/m2 ), underweight (ie, BMI < 18.5) was associated with a non-statistically significant increased prevalence of colorectal adenomas (odd ratio [OR] = 1.29 and 95% confident interval [CI]: 0.88-1.87). This association became significant among male (OR = 1.98, 95% CI: 1.20-3.27), male who were ever smokers (OR = 2.59, 95% CI: 1.33-5.03), nonregular exercise (OR = 2.44, 95% CI: 1.26-4.73) and individuals with cardiometabolic disorders (OR = 3.46, 95% CI: 1.19-10.00). The association between underweight and colorectal adenomas did not vary by smoking status, drinking status, family history of cancer, adenomas types or cardiometabolic disorders. No association was observed among obese individuals (BMI ≥ 25). In the population with low prevalence of obesity, we found that the association between BMI and colorectal adenomas followed a reversed J-shape that underweight was associated with increased prevalence. Further studies are, therefore, warranted to replicate our results and to investigate the biologic mechanism the effect of underweight on colorectal adenomas prevalence.

Enhanced uptake of potassium or glycine betaine or export of cyclic-di-AMP restores osmoresistance in a high cyclic-di-AMP Lactococcus lactis mutant.

The broadly conserved bacterial signalling molecule cyclic-di-adenosine monophosphate (c-di-AMP) controls osmoresistance via its regulation of potassium (K+) and compatible solute uptake. High levels of c-di-AMP resulting from inactivation of c-di-AMP phosphodiesterase activity leads to poor growth of bacteria under high osmotic conditions. To better understand how bacteria can adjust in response to excessive c-di-AMP levels and to identify signals that feed into the c-di-AMP network, we characterised genes identified in a screen for osmoresistant suppressor mutants of the high c-di-AMP Lactococcus ΔgdpP strain. Mutations were identified which increased the uptake of osmoprotectants, including gain-of-function mutations in a Kup family K+ importer (KupB) and inactivation of the glycine betaine transporter transcriptional repressor BusR. The KupB mutations increased the intracellular K+ level while BusR inactivation increased the glycine betaine level. In addition, BusR was found to directly bind c-di-AMP and repress expression of the glycine betaine transporter in response to elevated c-di-AMP. Interestingly, overactive KupB activity or loss of BusR triggered c-di-AMP accumulation, suggesting turgor pressure changes act as a signal for this second messenger. In another group of suppressors, overexpression of an operon encoding an EmrB family multidrug resistance protein allowed cells to lower their intracellular level of c-di-AMP through active export. Lastly evidence is provided that c-di-AMP levels in several bacteria are rapidly responsive to environmental osmolarity changes. Taken together, this work provides evidence for a model in which high c-di-AMP containing cells are dehydrated due to lower K+ and compatible solute levels and that this osmoregulation system is able to sense and respond to cellular water stress.

Structural and functional studies of pyruvate carboxylase regulation by cyclic di-AMP in lactic acid bacteria.

Cyclic di-3',5'-adenosine monophosphate (c-di-AMP) is a broadly conserved bacterial second messenger that has been implicated in a wide range of cellular processes. Our earlier studies showed that c-di-AMP regulates central metabolism in Listeria monocytogenes by inhibiting its pyruvate carboxylase (LmPC), a biotin-dependent enzyme with biotin carboxylase (BC) and carboxyltransferase (CT) activities. We report here structural, biochemical, and functional studies on the inhibition of Lactococcus lactis PC (LlPC) by c-di-AMP. The compound is bound at the dimer interface of the CT domain, at a site equivalent to that in LmPC, although it has a distinct binding mode in the LlPC complex. This binding site is not well conserved among PCs, and only a subset of these bacterial enzymes are sensitive to c-di-AMP. Conformational changes in the CT dimer induced by c-di-AMP binding may be the molecular mechanism for its inhibitory activity. Mutations of residues in the binding site can abolish c-di-AMP inhibition. In L. lactis, LlPC is required for efficient milk acidification through its essential role in aspartate biosynthesis. The aspartate pool in L. lactis is negatively regulated by c-di-AMP, and high aspartate levels can be restored by expression of a c-di-AMP-insensitive LlPC. LlPC has high intrinsic catalytic activity and is not sensitive to acetyl-CoA activation, in contrast to other PC enzymes.

Onward and [K+]Upward: a New Potassium Importer under the Spell of Cyclic di-AMP.

Cyclic di-AMP (c-di-AMP) is a second messenger which plays a major role in osmotic homeostasis in bacteria. In work by Quintana et al. (I. M. Quintana, J. Gibhardt, A. Turdiev, E. Hammer, et al., J Bacteriol 201:e00028-19, 2019, https://doi.org/10.1128/jb.00028-19), two Kup homologs from Lactococcus lactis were identified as high-affinity K+ importers whose activities are inhibited by direct binding of c-di-AMP. The results broaden the scope of K+ level regulation by c-di-AMP, with Kup homologs found in a number of pathogenic, commensal, and industrial bacteria.

Survey data on energy-saving policies, energy price, crisis and household energy-saving behavior.

The state policies and energy prices are evaluated to play a crucial role in the context of crises occurring in each country. The authors collected data on state policies and energy prices concerning energy-saving behavior during crises, with a particular focus on the Covid-19 crisis. The data was gathered from 1216 respondents, who are households. The data's reliability was assessed using Smart-PLS software. The data will provide research ideas related to state policies, energy prices, and energy-saving behavior associated with crises similar to Covid-19.

Cyclic-di-AMP signalling in lactic acid bacteria.

Cyclic dimeric adenosine monophosphate (cyclic-di-AMP) is a nucleotide second messenger present in Gram-positive bacteria, Gram-negative bacteria and some Archaea. The intracellular concentration of cyclic-di-AMP is adjusted in response to environmental and cellular cues, primarily through the activities of synthesis and degradation enzymes. It performs its role by binding to protein and riboswitch receptors, many of which contribute to osmoregulation. Imbalances in cyclic-di-AMP can lead to pleiotropic phenotypes, affecting aspects such as growth, biofilm formation, virulence, and resistance to osmotic, acid, and antibiotic stressors. This review focuses on cyclic-di-AMP signalling in lactic acid bacteria (LAB) incorporating recent experimental discoveries and presenting a genomic analysis of signalling components from a variety of LAB, including those found in food, and commensal, probiotic, and pathogenic species. All LAB possess enzymes for the synthesis and degradation of cyclic-di-AMP, but are highly variable with regards to the receptors they possess. Studies in Lactococcus and Streptococcus have revealed a conserved function for cyclic-di-AMP in inhibiting the transport of potassium and glycine betaine, either through direct binding to transporters or to a transcriptional regulator. Structural analysis of several cyclic-di-AMP receptors from LAB has also provided insights into how this nucleotide exerts its influence.

Survey dataset on student perceptions and experiences of quality assurance in Vietnamese universities.

This paper presents a dataset from a survey of student perceptions and experiences of quality assurance in Vietnamese higher education institutions. Data were collected from July to September 2020 using the online survey via Google Forms. The survey was sent to students via their email and social media, and there were 1323 valid responses. The data collection instrument was developed based on an international survey administered by UNESCO. The survey was designed to elicit data with respect to students' views on institutional quality policy and model, quality assurance procedures and tools, and student survey. The dataset serves as an insightful reference for institutional practitioners and policymakers in quality assurance to revise internal quality assurance policies and instruments to enhance the quality of teaching and learning. Moreover, the dataset could be of interest to other educational researchers who can use it to investigate students' understanding and viewpoints on quality assurance.

Short communication: Antibacterial effects of essential oils from Cinnamomum cassia bark and Eucalyptus globulus leaves-The involvements of major constituents.

Essential oils from Cinnamomum cassia bark and Eucalyptus globulus leaves have been traditionally applied for bacterial infections, through both of aromatherapy and oral application. (E)-cinnamaldehyde and 1,8 cineole have been identified as their major secondary metabolites, and are also generally considered as the main active ingredients responsible for their medicinal applications. However, ethnobotanical doctors still prefer to use whole essentials oils over purified compounds in bacterial infections. We therefore hypothesized that multi-compound extracts might exert better effects than isolated ingredients. In order to verify the hypothesis about advantages of whole materials, we examined antibacterial properties of the 2 plant essential oils in the comparison with their isolated major compounds, such as (E)-cinnamaldehyde and 1,8 cineole. Effects of liquid- and vapor-phase were examined on a set of 6 gram-positive and -negative bacteria, applying broth dilution, agar well diffusion and disc volatilization methods. In all 3 investigations, we observed that whole cinnamon and eucalyptus oils, with the lower concentrations of (E)-cinnamaldehyde (89.1%) and 1,8 cineole (61.2%), were able to induce better effects than the purified active compounds (≥ 99%). These results partly explain the advantages of using whole essential oils over isolated ingredients, and therefore support the application of traditional dosage forms for bacterial infections in ethnomedicine.

Associations between Ileal Juice Bile Acids and Colorectal Advanced Adenoma.

BACKGROUND: There is an urgent need to identify biomarkers for advanced adenoma, an important precursor of colorectal cancer (CRC). We aimed to determine alterations in ileal juice bile acids associated with colorectal advanced adenoma. METHODS: We quantified a comprehensive panel of primary and secondary bile acids and their conjugates using an ultraperformance liquid chromatography triple-quadrupole mass spectrometric assay in ileal juice collected at colonoscopy from 46 study subjects (i.e., 14 biopsy-confirmed advanced adenomas and 32 controls free of adenoma or cancer). Using analysis of covariance (ANCOVA), we examined the differences in bile acid concentrations by disease status, adjusting for age, sex, body mass index, smoking status and type 2 diabetes. RESULTS: The concentrations of hyodeoxycholic acid (HCA) species in ileal juice of the advanced adenoma patients (geometric mean = 4501.9 nM) were significantly higher than those of controls (geometric mean = 1292.3 nM, p = 0.001). The relative abundance of ursodeoxycholic acid (UDCA) in total bile acids was significantly reduced in cases than controls (0.73% in cases vs. 1.33% in controls; p = 0.046). No significant difference between cases and controls was observed for concentrations of total or specific primary bile acids (i.e., cholic acid (CA), chenodeoxycholic acid (CDCA) and their glycine- and taurine-conjugates) and total and specific major secondary bile acids (i.e., deoxycholic acid and lithocholic acid). CONCLUSIONS: Colorectal advanced adenoma was associated with altered bile acids in ileal juice. The HCA species may promote the development of colorectal advanced adenoma, whereas gut microbiota responsible for the conversion of CDCA to UDCA may protect against it. Our findings have important implications for the use of bile acids as biomarkers in early detection of colorectal cancer.

Association between Sleep Duration and Colorectal Adenomas: Findings from a Case-Control Study in Vietnam.

BACKGROUND: Colorectal cancer is one of the leading cancers worldwide and in Vietnam. Adenomas are important precursors of colorectal cancer. Study on the association between sleep duration and development of colorectal adenoma (CRA) is limited, particularly among Vietnamese population. METHODS: We conducted an individually matched case-control study of 870 CRA cases and 870 controls in a large-scale colorectal screening program involving 103,542 individuals ages ≥40 years old in Hanoi, Vietnam. Sleep duration was categorized in three groups: short: ≤6 hours/day, normal: 7 to 8 hours/day, and long: >8 hours/day. Conditional logistic regression was used to evaluate the association between sleep duration and adenomas risk after controlling for potential confounders. RESULTS: Overall, short-sleep duration was associated with increased risk of having CRA compared with normal duration [OR, 1.48; 95% confidence interval (CI), 1.12-1.97]. This pattern was present in both females (OR, 1.58; 95% CI, 1.14-2.18) and males (OR, 1.45; 95% CI, 1.08-1.93), with advanced adenomas (OR, 1.61; 95% CI, 1.09-2.38) and non-advanced adenomas (OR, 1.66; 95% CI, 1.19-2.32). Furthermore, the association between CRA development and short-sleep duration was more apparent among females who were nondrinker, nonobese, physically active, with proximal or both sided adenomas and with cardiometabolic disorder. Among males, the short-sleep duration was associated with CRA risk among never-smoking, cardiometabolic disorders, and obese. CONCLUSIONS: Short-sleep duration was associated with increased prevalence of both advanced and non-advanced CRAs among Vietnamese population. IMPACT: Findings from this study showed that maintaining an adequate sleep duration may have an important implication for colorectal adenoma prevention and control.

The symptoms of postpartum depression observed by family members: A pilot study.

Postpartum Depression (PPD) is a burden on women's mental health after delivery, predominantly occurring in the 1st year. PPD poses a threat to the mother's life and affects the quality of childcare. Early detection by family members of depressive symptoms is critical. This study aimed to examine the role of family members in reporting depressive symptoms of PPD among new mothers. A cross-sectional study was conducted, where 56 family members were asked to report depressive symptoms observed in new mothers. At the same time, the new mothers were also screened for PPD using the Edinburgh Postpartum Depression Scale (EPDS). Binary logistic regression was performed. Depressive symptoms of new mothers reported by family members, including emotional and behavioral disturbance, being under stress, high anxiety, isolation, changing lifestyle, and inability to take care of their children, were found as predictors of PPD.

Effectiveness of a standard clinical training program in new graduate nurses' competencies in Vietnam: A quasi-experimental longitudinal study with a difference-in-differences design.

OBJECTIVE: This study aimed to estimate the effectiveness of a standard clinical training program for new graduate nurses in Vietnam. METHODS: A quasi-experimental longitudinal study with a difference-in-differences design was conducted. A total of 280 new graduate nurses completed a self-administered questionnaire. The intervention group consisted of 206 respondents (those having participated in standard clinical training) and the control group (those that did not receive training) of 74. Differences in mean increases in competency scores between the intervention and control groups were estimated. The effect size of the intervention was estimated by calculating Cohen's d. A generalized linear model was employed to identify the factors associated with mean increases. RESULTS: The mean increase in total competency scores (range: 0-6 points) in the intervention group was 0.73 points greater than in the control group with an intermediate effect size (Cohen's d = 0.53; 95% CI 0.26 to 0.80). A greater reduction in standard deviation of total competency scores in the intervention group was confirmed. Participation in standard clinical training produced a positive association with a mean increase in total competency score without significance (ß = 0.04, P = 0.321). Provincial hospitals as clinical training venues had a significantly positive association (ß = 0.11, P = 0.007) with the mean increase in total competency scores. Competency at pre-clinical training was negatively (ß = -0.75, P < 0.001) associated with the mean increase. CONCLUSION: Findings implied that the standard clinical training program could contribute to both increasing and standardizing new graduate nurses' competencies in Vietnam. Further studies are needed to more precisely examine the attribution of standard clinical training to better increase new graduate nurses' competencies.

Nurses' perception of individual and organizational changes caused by a novel clinical training system for new graduate nurses: A qualitative research using photovoice.

BACKGROUND: Clinical training for new graduate nurses has a positive impact on their clinical competence; however, there is limited evidence on the impact of this training at the organizational level and on the processes that mediate these impacts. OBJECTIVES: To identify the individual and organizational impacts of a clinical training system and the mechanism that produces them by exploring nurses' perceptions of the changes in health facilities after the introduction of a standardized clinical training system in Vietnam. DESIGN: Qualitative research using an ethnographic approach; photovoice was used to obtain insights into nurses' perceptions. SETTING: Health facilities of four provinces and one city in Vietnam participated in the Project for Strengthening of the Clinical Training System for New Graduate Nurses. PARTICIPANTS: New graduate nurses, preceptors, and managers who engaged in the clinical training program. METHODS: Qualitative data, including photographs, were collected through photovoice sessions and inductively analyzed using thematic analysis. RESULTS: Sixteen photovoice sessions were conducted with 94 nurses from 22 hospitals. Three themes emerged: "acquiring competency" as an individual change, "fostering mutual learning culture" as an organizational change, and "improved quality of care and nurse empowerment" as the impact. The clinical training system was found to enhance diverse competencies of new graduate nurses, preceptors, and nurse managers, while facilitating collaboration among different professions, departments, and organizations and cultivating a better learning environment. Organizational changes were initiated with the hospital director's support. These changes were interactively related and produced the impact. CONCLUSIONS: The novel clinical training system for new graduate nurses could strengthen nurses' competencies and facilitate organizational changes to actualize a positive impact on nursing care and nurses' status. These findings could facilitate the design of an effective training program to stimulate organizational learning to produce better health outcome.

Cyclic di-AMP Oversight of Counter-Ion Osmolyte Pools Impacts Intrinsic Cefuroxime Resistance in Lactococcus lactis.

The broadly conserved cyclic di-AMP (c-di-AMP) is a conditionally essential bacterial second messenger. The pool of c-di-AMP is fine-tuned through diadenylate cyclase and phosphodiesterase activities, and direct binding of c-di-AMP to proteins and riboswitches allows the regulation of a broad spectrum of cellular processes. c-di-AMP has a significant impact on intrinsic ß-lactam antibiotic resistance in Gram-positive bacteria; however, the reason for this is currently unclear. In this work, genetic studies revealed that suppressor mutations that decrease the activity of the potassium (K+) importer KupB or the glutamine importer GlnPQ restore cefuroxime (CEF) resistance in diadenylate cyclase (cdaA) mutants of Lactococcus lactis Metabolite analyses showed that glutamine is imported by GlnPQ and then rapidly converted to glutamate, and GlnPQ mutations or c-di-AMP negatively affects the pools of the most abundant free amino acids (glutamate and aspartate) during growth. In a high-c-di-AMP mutant, GlnPQ activity could be increased by raising the internal K+ level through the overexpression of a c-di-AMP-insensitive KupB variant. These results demonstrate that c-di-AMP reduces GlnPQ activity and, therefore, the level of the major free anions in L. lactis through its inhibition of K+ import. Excessive ion accumulation in cdaA mutants results in greater spontaneous cell lysis under hypotonic conditions, while CEF-resistant suppressors exhibit reduced cell lysis and lower osmoresistance. This work demonstrates that the overaccumulation of major counter-ion osmolyte pools in c-di-AMP-defective mutants of L. lactis causes cefuroxime sensitivity.IMPORTANCE The bacterial second messenger cyclic di-AMP (c-di-AMP) is a global regulator of potassium homeostasis and compatible solute uptake in many Gram-positive bacteria, making it essential for osmoregulation. The role that c-di-AMP plays in ß-lactam resistance, however, is unclear despite being first identified a decade ago. Here, we demonstrate that the overaccumulation of potassium or free amino acids leads to cefuroxime sensitivity in Lactococcus lactis mutants partially defective in c-di-AMP synthesis. It was shown that c-di-AMP negatively affects the levels of the most abundant free amino acids (glutamate and aspartate) in L. lactis Regulation of these major free anions was found to occur via the glutamine transporter GlnPQ, whose activity increased in response to intracellular potassium levels, which are under c-di-AMP control. Evidence is also presented showing that they are major osmolytes that enhance osmoresistance and cell lysis. The regulatory reach of c-di-AMP can be extended to include the main free anions in bacteria.

Fate of carotenoid-producing Bacillus aquimaris SH6 colour spores in shrimp gut and their dose-dependent probiotic activities.

Bacillus aquimaris SH6 spores produce carotenoids that are beneficial to white-leg shrimp (Litopenaeus vannamei) health. However, the optimal dose and mechanisms behind these effects are not well understood. We investigated the fate of SH6 spores in the gut of L. vannamei. Shrimp were divided into six groups administrated with either feed only (negative control) or SH6 spores at 5 × 106 CFU/g pellet (high dose, SH6 spore-H group), 1 × 106 CFU/g pellet (medium dose, SH6 spore-M group), 2 × 105 CFU/g pellet (low dose, SH6 spore-L group), astaxanthin at 0.5 mg/g pellet (Carophyll group), or carotenoids from SH6 vegetative cells at 5 µg/g pellet (SH6 carotenoid group). The growth rate was highest in SH6 spore-H (3.38%/day), followed by SH6 spore-M (2.84%/day) and SH6 spore-L (2.25%/day), which was significantly higher than the control (1.45%/day), Carophyll (1.53%/day) or SH6 carotenoid (1.57%/day) groups. The astaxanthin levels (1.9-2.0 µg/g shrimp) and red-colour scores (21-22) in SH6 spore-H/M were higher than the control (astaxanthin: 1.2 µg/g shrimp; red score: 20) or SH6 spore-L, but lower than the Carophyll and SH6 carotenoids. Feeding with medium and high doses of SH6 spores after 28 days resulted in respective 1.3-2-fold increases in phenol oxidase activity and 8-9 fold increases in Rho mRNA expression compared to the control and low dose group. The live-counts of SH6 in the gut gradually increased during the 28-day feeding period with SH6 spores at different concentrations, starting from 4.1, 8.2, and 5.4 × 104 CFU/g gut at day 1 and reaching 5.3, 5.1, and 4.4 × 105 CFU/g gut in the SH6-H/M/L groups, respectively, at day 28. Gut microbiota became more diversified, resulting in a 2-8-fold increase in total bacterial live-counts compared to the controls. SH6 spore germination was detected by measuring the mRNA expression of a specific sequence coding for SH6 amylase at 4 h, reaching saturation at 24 h. Our results confirm that SH6 spores colonize and germinate in the gut to improve the microbial diversity and boost the immune system of shrimp, exhibiting beneficial effects at >1 × 106 CFU/g pellet.