Storage of plasma-derived exosomes: evaluation of anticoagulant use and preserving temperatures.

Exosomes carry large cargo of proteins, lipids, and nucleic acids, serving as versatile biomarkers for disease diagnosis and vehicles for drug delivery. However, up to date, no well recognized standard procedures for exosome storage were available for clinical application. This study aimed to determine the optimal storage conditions and the anticoagulants for plasma-derived exosome isolation. Fresh whole blood samples were collected from healthy participants and preserved in four different anticoagulants including sodium citrate (SC1/4), sodium citrate (SC1/9), lithium heparin (LH), or Ethylenediamine tetraacetic acid (EDTA), respectively. Exosomes were extracted from the plasma by differential ultracentrifugation and stored at three different temperatures, 4°C, -20°C or - 80°C for a duration ranging from one week to six months. All plasma samples for storage conditions comparison were pretreated with LH anticoagulant. Exosome features including morphological characteristics, pariticles size diameter, and surface protein profiles (TSG101, CD63, CD81, CD9, CALNEXIN) were assessed by transmission electron microscopy, Nanoparticle Tracking Analysis, and Western Blotting, respectively. Exosomes preserved in LH and SC1/4 group tended to remain intact microstructure with highly abundant protein biomarkers. Exosomes stored at 4°C for short time were prone to be more stable compared to thos at -80°C. Exosomes stored in plasma were superior in terms of ultrastructure, size diameter and surface protein expression to those stored in PBS. In conclusion, plasma-dervied exosome characteristics strictly depend on the anticoagulants and storage temperature and duration.

What is the context? Effective isolation of exosomes is a prerequisite for subsequent investigation into its involvemnt in disease development as well as potentialtherapeutic applications.Anticoagulants, storage temperature and durations might change the microscopical structure, integrity and also the stability of plasma-derived exosomes. However, no internationally recognized standard of exosome storage procedure was available for clinical use.What is new? Our finding evaluated the effect of anticoagulants and storage on plasma exosome characteristics.Exosomes isolated from plasma preserved with Li-heparin and sodium citrate (1/4) showed better physical properties and surface marker protein expression.Isolated exosomes appeared more stable in a short time for 4°C compared to −80°C. Storage of exosomes in plasma showed better physical properties and surface marker protein expression than in PBS.What is the impact? Our findings inform the significance of standardizing procedure of exosome isolation and preservation.

Sodium citrate buffer improves pazopanib solubility and absorption in gastric acid-suppressed rat model.

Pazopanib exhibits pH-dependent solubility and its absorption depends primarily on the stomach pH. Significant decrease of pazopanib absorption by coadministration with proton pump inhibitors in clinical situation need to be overcome. Thus, the purpose of this study is firstly to investigate the effect of acidic beverages and sodium citrate buffer on the solubility of pazopanib and secondly to examine the effect of sodium citrate buffer on pazopanib absorption in a rat model with esomeprazole-mediated gastric acid suppression. Pazopanib solubility decreased with increasing pH of sodium citrate buffer in vitro. Interestingly, its solubility in some acidic beverages was significantly lower than that in sodium citrate buffer of the same pH. The AUC0-24h of pazopanib administered in tap water to rats treated with esomeprazole (ESP rats) was 66 % lower than that in the control rats treated with saline. However, AUC0-24h was 4.8 times higher in ESP rats that received pazopanib with sodium citrate buffer (pH 2.3) compared to ESP rats that received pazopanib with tap water. Our results indicate that the drug-drug interactions between pazopanib and proton pump inhibitors can be overcome, at least in part, by suspending pazopanib in sodium citrate buffer.

Multianticoagulant Pseudothrombocytopenia in a Patient with Primary Carcinoma of the Liver with Hypersplenism.

BACKGROUND: Pseudothrombocytopenia (PTCP) is a relatively rare phenomenon in vitro, the mechanism is not completely clear, and there is no unified solution for it. How to identify and solve PTCP accurately is a challenge for laboratory personnel. METHODS: According to the patient's clinical manifestations, thrombocytopenia caused by hypersplenism was excluded. PTCP was confirmed by platelet volume histograms, scattergrams and platelet clumps on the blood smears. Commonly used alternative anticoagulants such as sodium citrate or heparin were used for platelet counting. The corrective effect of the platelet count was not good, so non-anticoagulant blood was collected and tested immediately, and blood smears were used to count platelets manually. RESULTS: The PTCP of the patient could not be solved using sodium citrate and heparin anticoagulation. By collecting non-anticoagulant blood and testing immediately, the platelet count returned to normal (180 x 109/L), which is consistent with the results of manual counting on the patient's blood smears (175 x 109/L). CONCLUSIONS: When PTCP is confirmed, commonly used alternative anticoagulants can be used. If these do not work, non-anticoagulant blood can be collected and tested immediately, and blood smears can be used to count platelets manually.

Sodium citrate versus sodium bicarbonate for metabolic acidosis in patients with chronic kidney disease: A randomized controlled trial.

BACKGROUND: Metabolic acidosis (MA) is frequently associated with chronic kidney disease (CKD) progression. Our aim was to compare the effect of oral sodium citrate (SC) with that of oral sodium bicarbonate (SB) on renal function and serum bicarbonate correction, as well as to evaluate their safety profile in patients with MA of CKD. METHODS: We conducted a prospective, single-center, randomized 1:1, parallel, controlled, unblinded clinical trial of 124 patients with MA and CKD stages 3b and 4. The primary outcome was the mean change in estimated glomerular filtration rate (eGFR). The secondary outcomes were mean change in serum bicarbonate level, eGFR decrease by 30%, eGFR decrease by 50%, dialysis, death or prolonged hospitalization, and a combined endpoint. RESULTS: No significant difference was found between the groups in terms of mean eGFR change [adjusted mean difference = -0.99 mL/min/1.73 m2 (95% CI: -2.51 to 0.93, P = .20)]. We observed a mean serum bicarbonate change of 6.15 mmol/L [(95% CI: 5.55-6.74), P < .001] in the SC group and of 6.19 mmol/L [(95% CI: 5.54-6.83), P < .001] in the SB group, but no significant difference between the 2 groups [adjusted mean difference = 0.31 mmol/L (-0.22 to 0.85), P = .25]. Cox proportional hazard analysis showed similar risks regarding eGFR decrease by 30% (P = .77), eGFR decrease by 50% (P = .50), dialysis (P = .85), death or prolonged hospitalization (P = .29), and combined endpoint (P = .57). Study drug discontinuation due to adverse events was significantly more common in the SB group (17.7% vs 4.8%, P = .02). CONCLUSIONS: SC and SB have a similar effect on kidney function decline, both improve serum bicarbonate level, but SB is associated with higher rates of medication discontinuation due to adverse events.

Extracellular vesicles separated from goat milk by differential centrifugation coupled with sodium citrate pretreatments.

Satisfactory separation of milk-derived extracellular vesicles (MEVs) is important for the downstream analysis of the functions and properties of MEVs. However, the presence of abundant proteins in milk hindered the separation of MEVs. In this study, three pretreatment methods, including sodium citrate (SC), acetic acid (AA), and high-speed centrifugation, were adopted to separate MEVs from goat milk while minimizing the impact of protein. The MEVs were then characterized by nanoparticle tracking, transmission electron microscopy and western blotting experiments. The results indicated that pretreatments with AA and SC greatly decreased the impact of casein, but AA pretreatment damaged the surface structure of MEVs. Additionally, the differential centrifugation process resulted in a slight loss of MEVs. Overall, MEVs with small size and high purity can be obtained under 125 k × g centrifugation combined with SC pretreatment, which suggests a promising method for separation of MEVs from goat milk.

Inverse vulcanization induced oxygen modified porous polysulfides for efficient sorption of heavy metals.

The applications of polysulfides derived from natural plant oil and sulfur via the inverse vulcanization in the removal of heavy metals from aqueous solutions suffered from their low porosity and scarce surface functionality because of their hydrophobic surfaces and bulk characteristics. In this study, polysulfides from sulfur and palm oil (PSPs) with significantly enhanced porosity (13.7-24.1 m2/g) and surface oxygen-containing functional groups (6.9-8.6 wt.%) were synthesized with the optimization of process conditions including reaction time, temperature, and mass ratios of sulfur/palm oil/NaCl/sodium citrate. PSPs were applied as sorbents to remove heavy metals present in aqueous solutions. The integration of porosity and oxygen modification allowed a fast kinetic (4.0 h) and enhanced maximum sorption capacities for Pb(II) (218.5 mg/g), Cu(II) (74.8 mg/g), and Cr(III) (68.4 mg/g) at pH 5.0 and T 298 K comparing with polysulfides made without NaCl/sodium citrate. The sorption behaviors of Pb(II), Cu(II), and Cr(III) on PSPs were highly dependent on the solution pH values and ionic strength. The sorption presented excellent anti-interference capability for the coexisting cations and anions. The sorption processes were endothermic and spontaneous. This work would guide the preparation of porous polysulfides with surface modification as efficient sorbents to remediate heavy metals from aqueous solutions.

Effect of Pre-exercise Sodium Citrate Ingestion on Repeated Sprint Performance in Soccer Players.

ABSTRACT: Kuru, D, Aktitiz, S, Atakan, MM, Köse, MG, Turnagöl, HH, and Kosar, SN. Effect of pre-exercise sodium citrate ingestion on repeated sprint performance in soccer players. J Strength Cond Res 38(3): 556-562, 2024-This study aimed to test the hypothesis that sodium citrate (CIT) administered 180 minutes before exercise improves repeated sprint performance in athletes within a field-based setting. Twenty male soccer players (mean ± SD : age = 20.9 ± 2.3 years; body mass [BM] = 73.8 ± 5.9 kg) performed a running-based anaerobic sprint test (RAST) with 0.5 g·kg -1 BM of CIT or with placebo (PLC; NaCl) ingestion 180 minutes before exercise in a randomized, crossover, and double-blind design, with at least 6 days between the trials. Blood samples were collected before exercise and at first, third, fifth, and seventh minutes after exercise to analyze blood pH, bicarbonate, and lactate levels. Gastrointestinal symptoms were also monitored at 30-minute intervals for 180 minutes after CIT and PLC ingestion. Pre-exercise blood pH (CIT = 7.49 ± 0.03 vs. PLC = 7.41 ± 0.02) and bicarbonate (CIT = 30.57 ± 1.33 vs. PLC = 25.25 ± 1.52) increased with CIT compared with PLC ( p < 0.001). Blood pH, bicarbonate, and lactate at the first, third, fifth, and seventh minutes after RAST with CIT were higher than PLC ( p < 0.05), except for lactate at first minute ( p > 0.05). Compared with PLC, CIT ingestion significantly improved minimum power output ( p = 0.024) and percentage decrement score ( p = 0.023). Gastrointestinal symptoms were significantly higher after CIT ingestion vs. PLC at 30th ( p = 0.003) and 60th minutes ( p = 0.010). However, there were no significant differences at 90th, 120th, 150th, or 180th minutes ( p > 0.05). The ingestion of 0.5 g·kg -1 BM of CIT 180 minutes before exercise is an effective ergogenic aid for improving repeated sprint ability as evidenced by improvements in minimum power output and percentage decrement score.

The effect of photolysis of sodium citrate treated with gold chloride using coloured light on the generation of gold nanoparticles and the repression of WiDr colon cancer cells.

Gold nanoparticles (GNPs) are usually formed via a wet chemical method using gold (III) chloride trihydrate (GC), which is treated with stable reducing agents such as sodium citrate (SC). This study determines the effect of coloured light on the formation of GNPs by irradiation of SC after the addition of GC (SCGC) and the effect of the SCGC photolytic procedure on the suppression of WiDr colon cancer cells by forming reactive oxygen species. The absorbance of surface plasmon resonance peaks at 523 nm are 0.069 and 0.219 for SCGC when treated with blue light illumination (BLI) and violet light irradiation (VLI), respectively, whereas green and red light treatments have little or no effect. Most GNPs have diameters ranging from 3 to 15 nm, with a mean of 6 nm, when SCGC is exposed to VLI for 1.5 h. Anionic superoxide radicals (O2•-) are formed in a charge-transfer process after SCGC under VLI treatment; however, BLI treatment produces no significant reaction. Moreover, SCGC under VLI treatment proves to be considerably more effective at inhibiting WiDr cells than BLI treatment, as firstly reported in this study. The reduction rates for WiDr cells treated with SCGC under BLI and VLI at an intensity of 2.0 mW/cm2 for 1.5 h (energy dose, 10.8 J/cm2) are 4.1% and 57.7%, respectively. The suppression rates for WiDr cells treated with SCGC are inhibited in an irradiance-dependent manner, the inhibition percentages being 57.7%, 63.3%, and 80.2% achieved at VLI intensities of 2.0, 4.0, and 6.0 mW/cm2 for 1.5 h, respectively. Propidium iodide is a fluorescent dye that detects DNA changes after cell death. The number of propidium iodide-positive nuclei significantly increases in WiDr cells treated with SCGC under VLI, suggesting that SCGC photolysis under VLI is a potential treatment option for the photodynamic therapy process.

Potassium sodium hydrogen citrate intervention on gut microbiota and clinical features in uric acid stone patients.

The high recurrence rate of renal uric acid stone (UAS) poses a significant challenge for urologists, and potassium sodium hydrogen citrate (PSHC) has been proven to be an effective oral dissolution drug. However, no studies have investigated the impact of PSHC on gut microbiota and its metabolites during stone dissolution therapy. We prospectively recruited 37 UAS patients and 40 healthy subjects, of which 12 patients completed a 3-month pharmacological intervention. Fasting vein blood was extracted and mid-stream urine was retained for biochemical testing. Fecal samples were collected for 16S ribosomal RNA (rRNA) gene sequencing and short chain fatty acids (SCFAs) content determination. UAS patients exhibited comorbidities such as obesity, hypertension, gout, and dyslipidemia. The richness and diversity of the gut microbiota were significantly decreased in UAS patients, Bacteroides and Fusobacterium were dominant genera while Subdoligranulum and Bifidobacterium were poorly enriched. After PSHC intervention, there was a significant reduction in stone size accompanied by decreased serum uric acid and increased urinary pH levels. The abundance of pathogenic bacterium Fusobacterium was significantly downregulated following the intervention, whereas there was an upregulation observed in SCFA-producing bacteria Lachnoclostridium and Parasutterella, leading to a significant elevation in butyric acid content. Functions related to fatty acid synthesis and amino acid metabolism within the microbiota showed upregulation following PSHC intervention. The correlation analysis revealed a positive association between stone pathogenic bacteria abundance and clinical factors for stone formation, while a negative correlation with SCFAs contents. Our preliminary study revealed that alterations in gut microbiota and metabolites were the crucial physiological adaptation to PSHC intervention. Targeted regulation of microbiota and SCFA holds promise for enhancing drug therapy efficacy and preventing stone recurrence. KEY POINTS: • Bacteroides and Fusobacterium were identified as dominant genera for UAS patients • After PSHC intervention, Fusobacterium decreased and butyric acid content increased • The microbiota increased capacity for fatty acid synthesis after PSHC intervention.

Decision-Making Error in Platelet Transfusion Caused by EDTA-Dependent Pseudothrombocytopenia: a Case Report and Literature Review.

BACKGROUND: Ethylenediaminetetraacetic acid-dependent pseudothrombocytopenia (EDTA-PTCP) is a rare phenomenon characterized by pseudo low platelet counts when using EDTA as anticoagulant and can result in false decision making of platelet transfusion. METHODS: An application for platelet transfusion from a patient who planned to undergo spinal surgery was received by the Department of Transfusion service. The preoperative laboratory test results showed thrombocytopenia (platelet counts: 27 x 109/L). The surgeon planned to transfuse platelets before the operation to avoid bleeding in operation due to thrombocytopenia. However, the lab technologist found that there was aggregation of platelets under the microscope. Samples used with sodium citrate and heparin as anticoagulants were rechecked. RESULTS: The platelet count of the patient was normal in sodium citrate and heparin anticoagulant tubes. The patient had no history and clinical symptoms of thrombocytopenia. Therefore, the doctor canceled the platelet order. We also reviewed the relevant literature of EDTA-PTCP. CONCLUSIONS: EDTA-PTCP is rare and may result of a wrong decision of platelet transfusion. Correct understanding and treatment of this situation can avoid unnecessary platelet transfusion.

Precise glucose measurement in sodium fluoride-citrate plasma affects estimates of prevalence in diabetes and prediabetes.

OBJECTIVES: Estimates of glucose concentrations vary among types of blood samples, which impact on the assessment of diabetes prevalence. Guidelines recommend a conversion factor to calculate plasma glucose from measurements of glucose in whole blood. The American Diabetes Association recommends the use of blood drawing tubes containing sodium fluoride (NaF) and citrate, which have not yet been evaluated regarding possible differences in glucose concentration and conversion factors. Thus, we compared glucose measurements in NaF-citrate plasma and venous whole blood and estimated the impact of differences on diabetes and prediabetes prevalence. METHODS: Glucose differences were calculated by Bland-Altman analysis with pairwise comparison of glucose measurements from whole blood and NaF-citrate plasma (n=578) in clinical studies of the German Diabetes Center. Subsequently, we computed the impact of the glucose difference on diabetes and prediabetes prevalence in the population-based National Health and Nutrition Examination Survey (NHANES). RESULTS: Even upon conversion of whole blood to plasma glucose concentrations using the recommended conversion factor, mean glucose concentration difference remained 4.72 % higher in NaF-citrate plasma. Applying the higher glucose estimates, increases the population-based diabetes and prediabetes prevalence by 13.67 and 33.97 % or more than 7.2 and 13 million people in NHANES, respectively. Additional economic burden could be about 20 $ billion per year due to undiagnosed diabetes. CONCLUSIONS: The recommended conversion factor is not valid for NaF-citrate plasma. Systematic bias of glucose measurements due to sampling type leads to clinically relevant higher estimates of diabetes and prediabetes prevalence.

Quality indicators in prolonged hemodialysis with regional citrate anticoagulation with the genius system: retrospective cohort of critical patients with acute kidney injury.

BACKGROUND: Prolonged hemodialysis (HD) is performed from 6 to 12 h and can last up to 24 h. To prevent system clotting some studies suggest that Regional Citrate Anticoagulation (RCA) use reduces bleeding rates relative to systemic heparin. However, there may be difficulties in the patient's clinical management and completing the prescribed HD with Genius system using RCA. OBJECTIVE: To analyze safety Quality Indicators (IQs) and follow up on prolonged HD with 4% sodium citrate solution in a Genius® hybrid system. METHODS: This is a retrospective cohort conducted in an intensive care unit. RESULTS: 53 random sessions of prolonged HD with 4% sodium citrate solution of critically ill patients with AKI assessed. Evaluated safety indicators were dysnatremia and metabolic alkalosis, observed in 15% and 9.4% of the sessions, respectively. Indicators of effectiveness were system clotting which occurred in 17.3%, and the minimum completion of the prescribed HD time, which was 75.5%. CONCLUSION: The assessment of the indicators showed that the use of RCA with a 4% sodium citrate solution in prolonged HD with the Genius system in critically ill patients with AKI can be performed in a simple, safe, and effective way.

Sodium Citrate Nanoparticles Induce Dual-Path Pyroptosis for Enhanced Antitumor Immunotherapy through Synergistic Ion Overload and Metabolic Disturbance.

Metabolic reprogramming, as one of the characteristics of cancer, is associated with tumorigenesis, growth, or migration, and the modulation of metabolic pathways has emerged as a novel approach for cancer therapy. However, the conventional metabolism-mediated apoptosis process in tumor cells exhibits limited immunogenicity and inadequate activation of antitumor immunity. Herein, phospholipid-coated sodium citrate nanoparticles (PSCT NPs) are successfully prepared, which dissolve in tumor cells and then release significant amounts of citrate ions and Na+ ions. Massive quantities of ions lead to increased intracellular osmotic pressure, which activates the caspase-1/gasdermin D (GSDMD) mediated pyroptosis pathway. Simultaneously, citrate induces activation of the caspase-8/gasdermin C (GSDMC) pathway. The combined action of these two pathways synergistically causes intense pyroptosis, exhibiting remarkable antitumor immune responses and tumor growth inhibition. This discovery provides new insight into the potential of nanomaterials in modulating metabolism and altering cell death patterns to enhance antitumor immunotherapy.

Use of Buffers in Specific Contexts: Highly Trained Female Athletes, Extreme Environments and Combined Buffering Agents-A Narrative Review.

This narrative review evaluated the evidence for buffering agents (sodium bicarbonate, sodium citrate and beta-alanine), with specific consideration of three discrete scenarios: female athletes, extreme environments and combined buffering agents. Studies were screened according to exclusion and inclusion criteria and were analysed on three levels: (1) moderating variables (supplement dose and timing, and exercise test duration and intensity), (2) design factors (e.g., use of crossover or matched group study design, familiarisation trials) and (3) athlete-specific factors (recruitment of highly trained participants, buffering capacity and reported performance improvements). Only 19% of the included studies for the three buffering agents reported a performance benefit, and only 10% recruited highly trained athletes. This low transferability of research findings to athletes' real-world practices may be due to factors including the small number of sodium citrate studies in females (n = 2), no studies controlling for the menstrual cycle (MC) or menstrual status using methods described in recently established frameworks, and the limited number of beta-alanine studies using performance tests replicating real-world performance efforts (n = 3). We recommend further research into buffering agents in highly trained female athletes that control or account for the MC, studies that replicate the demands of athletes' heat and altitude camps, and investigations of highly trained athletes' use of combined buffering agents. In a practical context, we recommend developing evidence-based buffering protocols for individual athletes which feature co-supplementation with other evidence-based products, reduce the likelihood of side-effects, and optimise key moderating factors: supplement dose and timing, and exercise duration and intensity.

From micro to macro: Physical-chemical characterization of wheat starch-based films modified with PEG200, sodium citrate, or citric acid.

Needing to extend the shelf-life of packaged food and the evolving consumer demands led researchers to seek innovative, eco-friendly, and biocompatible packaging solutions. Starch is among the most promising natural and renewable alternatives to non-degradable plastics. Here, we deeply study the structural features of starch films modified by adding citric acid (CA) or sodium citrate (SC) as a cross-linker and polyethylene glycol 200 (PEG200) as a plasticizer and obtained through solvent casting. The substances' influence on starch films was evaluated through Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) and Solid-state Nuclear Magnetic Resonance (ss-NMR) spectroscopies. Films' macroscopic properties, such as swelling index, solubility, thermo-mechanical features, and moisture absorption, were also assessed to foresee potential applications. Proper amounts of CA, CS, and PEG200 improve film properties and inhibit starch chains' retrogradation and recrystallization. Besides, the chemical neighbourhood of nuclei observed through ss-NMR significantly changed alongside the polymer chains' mobility. The latter result indicates a different polymer chain structural organization that could justify the film's higher resistance to thermal degradation and elongation at the break. This methodological approach is effective in predicting the macroscopic behaviour of a polymeric material and could be helpful for the application of such products in food preservation.

A genetic selection for Mycobacterium smegmatis mutants tolerant to killing by sodium citrate defines a combined role for cation homeostasis and osmotic stress in cell death.

Mycobacteria can colonize environments where the availability of metal ions is limited. Biological or inorganic chelators play an important role in limiting metal availability, and we developed a model to examine Mycobacterium smegmatis survival in the presence of the chelator sodium citrate. We observed that instead of restricting M. smegmatis growth, concentrated sodium citrate killed M. smegmatis. RNAseq analysis during sodium citrate treatment revealed transcriptional signatures of metal starvation and hyperosmotic stress. Notably, metal starvation and hyperosmotic stress, individually, do not kill M. smegmatis under these conditions. A forward genetic transposon selection was conducted to examine why sodium citrate was lethal, and several sodium-citrate-tolerant mutants were isolated. Based on the identity of three tolerant mutants, mgtE, treZ, and fadD6, we propose a dual stress model of killing by sodium citrate, where sodium citrate chelate metals from the cell envelope and then osmotic stress in combination with a weakened cell envelope causes cell lysis. This sodium citrate tolerance screen identified mutants in several other genes with no known function, with most conserved in the pathogen M. tuberculosis. Therefore, this model will serve as a basis to define their functions, potentially in maintaining cell wall integrity, cation homeostasis, or osmotolerance. IMPORTANCE Bacteria require mechanisms to adapt to environments with differing metal availability. When Mycobacterium smegmatis is treated with high concentrations of the metal chelator sodium citrate, the bacteria are killed. To define the mechanisms underlying killing by sodium citrate, we conducted a genetic selection and observed tolerance to killing in mutants of the mgtE magnesium transporter. Further characterization studies support a model where killing by sodium citrate is driven by a weakened cell wall and osmotic stress, that in combination cause cell lysis.

Potassium Chloride, Sodium Lactate and Sodium Citrate Impaired the Antimicrobial Resistance and Virulence of Pseudomonas aeruginosa NT06 Isolated from Fish.

Sodium chloride (NaCl) is a commonly used additive in minimally processed fish-based products. The addition of NaCl to fish products and packaging in a modified atmosphere is usually efficient with regard to limiting the occurrence of the aquatic environmental pathogen Pseudomonas aeruginosa. Given the negative effects of excess NaCl in the diet, there is a growing demand to reduce NaCl in food products with safer substituents, but the knowledge of their impact on antibiotic resistant P. aeruginosa is limited. This study aimed to evaluate the physiological and transcriptome characteristics of P. aeruginosa NT06 isolated from fish and to determine the effect of selected concentrations of alternative NaCl compounds (KCl/NaL/NaC) on the P. aeruginosa NT06 virulence phenotype and genotype. In the study, among the isolated microorganisms, P. aeruginosa NT06 showed the highest antibiotic resistance (to ampicillin, ceftriaxone, nalidixic acid, and norfloxacin) and the ability to grow at 4 °C. The Comprehensive Antibiotic Resistance Database (CARD) and the Virulence Factor Database (VFDB) revealed the presence of 24 and 134 gene products assigned to AMR and VF in the P. aeruginosa NT06 transcriptome, respectively. KCl, KCl/NaL and KCl/NaL/NaC inhibited pyocyanin biosynthesis, elastase activity, and protease activity from 40 to 77%. The above virulence phenotypic observations were confirmed via RT-qPCR analyses, which showed that all tested AMR and VF genes were the most downregulated due to KCl/NaL/NaC treatment. In conclusion, this study provides insight into the potential AMR and VF among foodborne P. aeruginosa and the possible impairment of those features by KCl, NaL, and NaC, which exert synergistic effects and can be used in minimally processed fish-based products.

Reductant-dependent DNA-templated silver nanoparticle formation kinetics.

DNA molecules have been demonstrated to be good templates for producing silver nanoparticles (AgNPs), with the advantages of well-controlled sizes, shapes, and properties. Revealing the formation kinetics of DNA-templated AgNPs is crucial for their efficient synthesis. Herein, using magnetic tweezers, we studied the reduction kinetics of the Ag+-DNA structure and the subsequent nucleation kinetics by adding NaBH4, L-ascorbic acid, and sodium citrate solutions. At [Ag+] = 0.01 mM, the addition of NaBH4 solution with the same concentration resulted in the restoration of DNA. In contrast, by increasing the [NaBH4]/[Ag+] ratio (r) to 10 and 100, the DNA extension initially decreased rapidly and then increased, indicating nucleation-dissolution kinetics. With AgNO3 solutions of higher concentrations (0.1 mM and 1 mM), direct particle nucleation and growth kinetics were observed by adding a tenfold (r = 10) or a hundredfold (r = 100) amount of NaBH4, which were evidenced by a significant reduction in DNA extension. The reductant dependence of the kinetics was further investigated. Addition of L-ascorbic acid to the DNA-Ag+ solution yielded an increase-decrease kinetics that was different from that caused by NaBH4, suggesting that nucleation was not initially favored due to the lack of sufficient Ag atoms; while sodium citrate showed a weak nucleation-promoting ability to form AgNPs. We discussed the findings within the framework of classical nucleation theory, in which the supersaturation of the Ag atom is strongly influenced by multiple factors (including the reducing ability of the reductant), resulting in different kinetics.