Metabolic phenotyping of saliva to identify possible biomarkers of periodontitis using proton nuclear magnetic resonance.

AIM: The aim of this study was to propose biomarker candidates for periodontitis via untargeted metabolomics analysis. MATERIALS AND METHODS: Metabolic profiling was performed using saliva samples from 92 healthy controls (H) and 129 periodontitis patients (P) in the discovery cohort using proton nuclear magnetic resonance spectroscopy. Random forest was applied to identify metabolites that significantly differentiated the control group from the periodontitis group. Candidate metabolites were then validated in an independent validation cohort. RESULTS: In the discovery set, the metabolic profiles of the P group were clearly separated from those of the H group. A total of 31 metabolites were identified in saliva, and 7 metabolites were selected as candidate biomarkers. These metabolites were further confirmed in the validation set. Ethanol, taurine, isovalerate, butyrate, and glucose were finally confirmed as biomarkers. Furthermore, the biomarker panel showed more than 0.9 of the area under curve value in both discovery and validation sets, indicating that panels were more effective than individual metabolites for diagnosing periodontitis. CONCLUSIONS: We identified five metabolite biomarkers that discriminated patients with periodontitis from healthy controls in two independent cohorts. These biomarkers have the potential for periodontal screening, detection of periodontitis, and monitoring of the outcome of periodontal therapy.

Metal Nanozyme with Ester Hydrolysis Activity in the Presence of Ammonia-Borane and Its Use in a Sensitive Immunosensor.

Metal nanoparticle surfaces are used for peroxidase- and oxidase-like nanozymes but not for esterase-like nanozymes. It is challenging to obtain rapid catalytic hydrolysis on a metal surface and even more so without a catalytically labile substrate. Here, we report that metal nanoparticle surfaces rapidly catalyze non-redox ester hydrolysis in the presence of redox H3 N-BH3 (AB). Metal hydrides are readily generated on a Pt nanoparticle (PtNP) from AB, and as a result the PtNP becomes electron-rich, which might assist nucleophilic attack of H2 O on the carbonyl group of an ester. The nanozyme system based on PtNP, AB, and 4-aminonaphthalene-1-yl acetate provides an electrochemical signal-to-background ratio much higher than natural enzymes, due to the rapid ester hydrolysis and redox cycling involving the hydrolysis product. The nanozyme system is applied in a sensitive electrochemical immunosensor for thyroid-stimulating hormone detection. The calculated detection limit is approximately 0.3 pg mL-1 , which indicates the high sensitivity of the immunosensor using the PtNP nanozyme.

Characterization of Fecal Peritonitis-Induced Sepsis in a Porcine Model.

BACKGROUND: Although there are well-established small-animal sepsis models, the longitudinal assessment of hemodynamic variables, laboratory values, and blood culture in a single living sepsis model is limited. Therefore, we aimed to comprehensively characterize fecal peritonitis-induced sepsis in a porcine model. MATERIALS AND METHODS: Autologous feces (1 g/kg) was administered into the peritoneum of 11 male pigs (49 ± 8 kg). The pigs were monitored up to 12 h with full fluid and vasopressor support to maintain the mean arterial pressure at >65 mm Hg. Longitudinal blood culture and laboratory values were obtained at defined time intervals. The cytokine levels in plasma were analyzed. Furthermore, a clinical registry of sepsis patients at a single emergency department was used to compare the Sepsis-related Organ Failure Assessment scores with those of the porcine model. RESULTS: The hyperdynamic phase of increasing cardiac output with decreasing systemic vascular resistance was maintained until 2 h, followed by the reverse (hypodynamic phase). With the escalating requirement for fluid and vasopressor, the lactate level progressively increased while the platelet count, urine output, and serum albumin level consistently decreased. Bacteremia developed 7 h (median) after the administration of feces, and Escherichia coli was the most common pathogen. The pattern of Sepsis-related Organ Failure Assessment scores with prominent cardiovascular failure was comparable to clinical data. CONCLUSIONS: We implemented a porcine fecal peritonitis-induced sepsis model that demonstrates culture-proven bacteremia and multiple organ failure, particularly cardiovascular system failure. This model could facilitate the development of technologies for the early diagnosis of bacterial pathogens in blood.

Effect of Electrical Vagus Nerve Stimulation on Cerebral Blood Flow and Neurological Outcome in Asphyxial Cardiac Arrest Model of Rats.

BACKGROUND: Vagus nerve stimulation (VNS) during post-resuscitation may increase recovery of cerebral blood flow (CBF) and reduce neurological injury. OBJECTIVE: This study was designed to investigate the effect of electrical VNS on neurological outcomes following cardiac arrest (CA). METHODS: Male Sprague-Dawley rats (n = 48) were subjected to the asphyxial CA model and blindly allocated to the VN isolation (CA + VN isolation) or VNS group (CA + VNS group). Cardiopulmonary resuscitation was initiated 450 s after pulseless electrical arrest, and the left cervical vagus nerve was electrically stimulated (0.05 mA, 1 Hz) for 3 h in the CA + VNS group. The neurological deficit score (NDS) and overall performance category (OPC) were assessed at 24 h after resuscitation, and histological injury of the hippocampus was evaluated. Independent experiments were performed to evaluate the effect of VNS on global cortical CBF after resuscitation using laser speckle Doppler imaging through a thinned skull window from pre-arrest to 6 h after resuscitation. RESULTS: The baseline characteristics were not significantly different between the two groups. The NDS was significantly higher, and the OPC was substantially lower in the CA + VNS group (p = 0.022 and p = 0.049, respectively) supported by decrease in histological injury of the hippocampal CA1 region. CBF in the early period of post-return of spontaneous circulation (ROSC) was significantly higher in the CA + VNS group (p < 0.05 at post-ROSC 2 h and 4 h), and 4-hydroxynonenal was significantly lower in the CA + VNS group (p = 0.026). CONCLUSIONS: VNS improved cerebral perfusion and neurological outcomes at 24 h after ROSC in an asphyxial CA model of rats.

Dynamic Changes in Soluble Triggering Receptor Expressed on Myeloid Cells-1 in Sepsis with Respect to Antibiotic Susceptibility.

Purpose: Proper antibiotic administration is crucial for sepsis management. Given the escalating incidence of antimicrobial resistance, there is a pressing need for indicators of antimicrobial susceptibility with short turnaround times. This study aimed to investigate the potential of soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) as an early biomarker for in vivo antibiotic susceptibility in patients with sepsis. Patients and Methods: We conducted a retrospective analysis of plasma samples from patients enrolled in a pre-established study designed to investigate prognostic biomarkers in patients with sepsis or septic shock. Baseline and 6 h sTREM-1 levels were examined using enzyme-linked immunosorbent assays. The primary outcome of the study was the comparison of percentage changes in sTREM-1 levels at the 6 h relative to baseline with respect to antibiotic susceptibility. Results: Of the 596 patients enrolled in the pre-established study, 29 with a median age of 75.8 and a 28-day mortality rate of 17.2% were included in the present analysis. Among these patients, 24 were classified into the susceptible group, whereas the remaining five were classified into the resistant group. The trend in plasma sTREM-1 levels differed with respect to antibiotic susceptibility. Moreover, percentage change in sTREM-1 levels at the 6 h relative to baseline was significantly higher in the resistant group (P = 0.028). Conclusion: The trend in plasma sTREM-1 levels in patients with sepsis differed with respect to antibiotic susceptibility, with a higher percentage change in patients treated with inappropriate antibiotics. These findings indicate the potential utility of sTREM-1 as an early biomarker of antibiotic susceptibility.

Short-Term and Long-Term Risk of Diabetes Mellitus among Patients with Spinal Cord Injury: A Nationwide Retrospective Cohort Study.

OBJECTIVES: Estimating the risk of diabetes mellitus (DM) is important for the proper management of patients with spinal cord injury (SCI). We investigated the short-term and long-term risks of DM among patients with SCI, according to the presence or severity of post-SCI disability and the level of injury. METHODS: We conducted a retrospective cohort study using the Korean National Health Insurance Service (2010-2018) database. After matching by age and sex, 6129 SCI patients and 22,979 controls were included. The primary outcome was incident DM, and risk was evaluated for both the short term (within 1 year after SCI) and the long term (after 1 year of SCI diagnosis). RESULTS: The risk of DM was higher among patients with cervical- and thoracic-level SCI accompanied by disability compared to the controls during follow-up (4.6 ± 2.6 years). The short-term risk of DM was higher among patients with SCI (odds ratio [OR] 2.51, 95% confidence interval [CI] 1.91-3.27) than among the controls and it was even higher among patients with severe disability (OR 5.38, 95% CI 2.91-9.27). According to the level of injury, patients with cervical SCI had the highest short-term risk of DM (with disability, OR 4.93, 95% CI 3.07-7.63). There was no significant increase of DM risk in the long term, after 1 year of SCI diagnosis. CONCLUSIONS: Patients with SCI accompanied by severe disability and cervical-level injury had higher risks of pronounced DM in the short term. The findings of this study emphasize the need for active surveillance of DM among patients with high-level SCI and disability, especially in the short term, in addition to continuous monitoring and proper management of DM in the long term.

Risk of fracture among patients with spinal cord injury: A nationwide cohort study in South Korea.

BACKGROUND: Clinical concerns about preventing and managing fractures after spinal cord injury (SCI) have been growing. OBJECTIVE: This study investigates the risk of fractures among SCI patients according to the presence of disability, disease severity, and level of injury. METHODS: We performed a retrospective cohort study using the Korean National Health Insurance Service (KNHIS 2010-2018) database. We included 5190 SCI patients and 1:3 age- and sex-matched control participants. The primary outcome was fracture, and the cohort was followed until December 31, 2019. RESULTS: SCI patients had a higher fracture risk than the matched controls (adjusted hazard ratio [aHR] 1.33, 95 % CI 1.16-1.54). The risk of fracture was higher in the presence of disability (aHR 1.57, 95 % CI 1.19-2.07), especially among patients with severe disability (aHR 1.65, 95 % CI 1.05-2.60). Higher fracture risks were observed among SCI patients regardless of injury level, but statistical significance was found only with cervical-level injury. When we considered site-specific fractures, vertebral (aHR 1.31, 95 % CI 1.04-1.64) and hip fracture risks (aHR 2.04, 95 % CI 1.39-2.98) were both higher among SCI patients than the controls. SCI patients with disability and cervical-level injury showed the highest hip fracture risk (aHR 3.67, 95 % CI 1.90-7.07). CONCLUSIONS: Compared with the controls, SCI patients were at higher risk of any fracture, particularly hip fracture, especially those with disability and cervical-level injury. Clinicians should be aware of the fracture risk among SCI patients to provide proper management.

Extracorporeal Blood Treatment Using Functional Magnetic Nanoclusters Mitigates Organ Dysfunction of Sepsis in Swine.

Mitigating sepsis-induced severe organ dysfunction with magnetic nanoparticles has shown remarkable advances in extracorporeal blood treatment. Nevertheless, treating large septic animals remains challenging due to insufficient magnetic separation at rapid blood flow rates (>6 L h-1) and limited incubation time in an extracorporeal circuit. Herein, superparamagnetic nanoclusters (SPNCs) coated with red blood cell (RBC) membranes are developed, which promptly capture and magnetically separate a wide range of pathogens at high blood flow rates in a swine sepsis model. The SPNCs exhibited an ultranarrow size distribution of clustered iron oxide nanocrystals and exceptionally high saturation magnetization (≈ 90 emu g-1) close to that of bulk magnetite. It is also revealed that CD47 on the RBCs allows the RBC-SPNCs to remain at a consistent concentration in the blood by evading innate immunity. The uniform size distribution of the RBC-SPNCs greatly enhances their effectiveness in eradicating various pathogenic materials in extracorporeal blood. The use of RBC-SPNCs for extracorporeal treatment of swine infected with multidrug-resistant E. coli is validated and found that severe bacteremic sepsis-induced organ dysfunction is significantly mitigated after 12 h. The findings highlight the potential application of RBC-SPNCs for extracorporeal therapy of severe sepsis in large animal models and potentially humans.

Metabolic effects of diclofenac on the aquatic food chain - 1 H-NMR study of water flea-zebrafish system.

In the environment, aquatic organisms are not only directly exposed to pollutants, but the effects can be exacerbated along the food chain. In this study, we investigated the effect of the food (water flea) on the secondary consumer (zebrafish) with the exposure diclofenac (DCF) Both organisms were exposed to an environmentally relevant concentrations (15 µg/L) of diclofenac for five days, and zebrafish were fed exposed and non-exposed water fleas, respectively. Metabolites of the water fleas were directly analyzed using HRMAS NMR, and for zebrafish, polar metabolite were extracted and analyzed using liquid NMR. Metabolic profiling was performed and statistically significant metabolites which affected by DCF exposure were identified. There were more than 20 metabolites with variable importance (VIP) score greater than 1.0 in comparisons in fish groups, and identified metabolites differed depending on the effect of exposure and the effect of food. Specifically, exposure to DCF significantly increased alanine and decreased NAD + in zebrafish, which means energy demand was increased. Additionally, the effects of exposed food decreased in guanosine, a neuroprotective metabolite, which explained that the neurometabolic pathway was perturbated by the feeding of exposed food. Our results which short-term exposed primary consumers to pollutants indirectly affected the metabolism of secondary consumers suggest that the long-term exposure further study remains to be investigated.

Sensitive electrochemical immunosensor via amide hydrolysis by DT-diaphorase combined with five redox-cycling reactions.

Amide hydrolysis using enzyme labels, such as proteases, occurs at a slower rate than phosphoester and carboxyl ester hydrolysis. Thus, it is not very useful for obtaining high signal amplification in biosensors. However, amide hydrolysis is less sensitive to nonenzymatic spontaneous hydrolysis, allowing for lower background levels. Herein, we report that amide hydrolysis by DT-diaphorase (DT-D) occurs rapidly and that its combination with five redox-cycling reactions allows for the development of a highly sensitive electrochemical immunosensor. DT-D rapidly generates ortho-aminohydroxy-naphthalene (oAN) from its amide substrate via amide hydrolysis, which not even trypsin, a highly catalytic protease, can achieve. NADH, which is required for amide hydrolysis, advantageously acts as a reducing agent for rapid electrooxidation-based redox-cycling reactions. In the presence of oAN, DT-D, and NADH, two redox-cycling reactions rapidly occur. In the additional presence of an electron mediator, Ru(NH3)63+ [Ru(III)], three more redox-cycling reactions occur because Ru(III) reacts rapidly with oAN and DT-D. Although the O2-related redox-cycling reactions and redox reaction decrease electrochemical signals, this signal-decreasing effect is not significant in air-saturated solutions. The slow electrooxidation of NADH at an indium tin oxide electrode and sluggish reaction between NADH and Ru(III) allow for low electrochemical backgrounds. When the developed signal amplification scheme is tested for the sandwich-type electrochemical detection of parathyroid hormone (PTH), a detection limit of ∼1 pg/mL is obtained. The detection method is highly sensitive and can accurately measure PTH in serum samples.

Identification of a Biomarker Panel for Diagnosis of Early Childhood Caries Using Salivary Metabolic Profile.

Several studies have demonstrated that nuclear magnetic resonance (NMR) metabolic profiles can differentiate patients with caries from healthy individuals; however, these studies only identified individual metabolites. The present study aimed to identify a salivary metabolite biomarker panel for the diagnosis of early childhood caries (ECC). Saliva samples from children with and without caries were analyzed using NMR spectroscopy. Multivariate and univariate analyses were performed to identify the discriminating metabolites. Selected metabolites were further evaluated and used to detect ECC. The saliva samples of children with ECC were characterized based on the increased levels of formate, glycerophosphocholine, and lactate and reduced levels of alanine, glycine, isoleucine, lysine, proline, and tyrosine. The levels of these metabolites were significantly different from those in the control in the ECC subgroup according to caries severity and correlated with the number of decayed and filled teeth or surfaces. Subsequently, an optimal salivary metabolite biomarker panel comprising formate, lactate, proline, and glycine was developed. This panel exhibited a better diagnostic performance for ECC than a single metabolite. These results demonstrate that salivary metabolic signatures can reflect oral conditions associated with dental caries, thereby emphasizing the importance of distinct salivary metabolic profiles as potential biomarkers of ECC.

Factors Associated with Long-Term Dietary Supplement Use among Korean Breast Cancer Survivors: A Cross-Sectional Study.

PURPOSE: The factors associated with the dietary supplement (DS) use of Asian breast cancer survivors in consideration of the duration of use and types of DS have not been well established. METHODS: We recruited 693 Korean female breast cancer survivors at two university-affiliated hospitals and collected study data through a self-administered questionnaire and a review of medical records. A multiple logistic regression analysis was conducted to evaluate the multivariable-adjusted association between DS use and study variables. RESULTS: The prevalence of any (≥2 weeks) and long-term (≥6 months) DS use among study participants was 48.2% and 12.0%, respectively. Education level, alcohol use, adequate physical activity (≥150 min/week), and time lapse after cancer diagnosis were positively associated with any DS use. Among DS users, as compared with short-term (≥2 weeks and <6 months) users, long-term users were more likely to have a higher cancer stage, more diverse cancer treatment modalities, a shorter time since cancer diagnosis, and lower fear of cancer recurrence. When we repeated the analysis for each DS type, time lapse after cancer diagnosis showed a consistently inverse association with long-term use of the most frequently consumed DS (multivitamins, followed by vitamin D/calcium, vitamin C, and omega-3). The number of cancer treatment modalities was positively associated with the long-term use of multivitamins and vitamin D/calcium. Alcohol consumption and low bone mineral density were positively associated with long-term vitamin D/calcium use. CONCLUSIONS: The factors associated with DS use differed by the duration of DS use and specific DS type. Long-term DS use was more frequently associated with cancer-related factors.

Differences in the metabolomic profile of the human palatine tonsil between pediatrics and adults.

Palatine tonsils (PT) are B cell-predominant lymphoid organs that provide primary immune responses to airborne and dietary pathogens. Numerous histopathological and immunological studies have been conducted on PT, yet no investigations have been conducted on its metabolic profile. We performed high-resolution magic angle spinning nuclear magnetic resonance spectroscopy-based metabolic profiling in 35 pediatric and 28 adult human palatine tonsillar tissue samples. A total of 36 metabolites were identified, and the levels of 10 metabolites were significantly different depending on age. Among them, partial correlation analysis shows that glucose levels increased with age, whereas glycine, phosphocholine, phosphoethanolamine, and ascorbate levels decreased with age. We confirmed the decrease in immunometabolic activity in adults through metabolomic analysis, which had been anticipated from previous histological and immunological studies on the PT. These results improve our understanding of metabolic changes in the PT with aging and serve as a basis for future tonsil-related metabolomic studies.

MONITORING OF PULMONARY EDEMA USING ULTRASOUND RADIOFREQUENCY SIGNAL.

ABSTRACT: Objectives: Excessive accumulation of extravascular lung water impairs respiratory gas exchange and results in respiratory distress. Real-time radiofrequency signals of ultrasound can continuously and quantitatively monitor excessive lung water. This study aims to evaluate the availability of continuous real-time quantitative pulmonary edema monitoring using ultrasound radiofrequency signals and compare it with Pa o2 (partial pressure of arterial oxygen)/F io2 (fraction of inspired oxygen) (PF) ratio, conventional lung ultrasound, and the Hounsfield unit of chest computed tomography. Methods: Male Yorkshire pigs (40.5 ± 0.5 kg) were anesthetized and mechanically ventilated. A balanced crystalloid was administered to induce hydrostatic pulmonary edema. Three different infusion rates of 2, 4, and 6 mL/kg per minute were tested to determine the infusion rate for the appropriate swine model. The chest computed tomography and ultrasonography with radiofrequency signals were taken every 5 min during the full inspiration. The ultrasonography scans with radiofrequency signals were measured at the intercostal space where the line crossing the two armpits and the right anterior axillary line intersected. Results: The infusion rate of fluid for the pulmonary edema model was determined to be 6 mL/kg per minute, and a total of four pigs were tested at an injection rate of 6 mL/kg. The adjusted R2 values of regression analysis between the radiofrequency signal and computer tomography Hounsfield score were 0.990, 0.993, 0.988, and 0.993 (all P values <0.05). All radiofrequency signal changes preceded changes in PF ratio or lung ultrasound changes. The area under the receiver operating characteristic curve of the radiofrequency signal for predicting PF ratio <300 was 0.88 (95% confidence interval, 0.82-0.93). Conclusion: We evaluated ultrasound radiofrequency signals to assess pulmonary edema in a swine model that can worsen gradually and showed that quantitative ultrasound radiofrequency signal analysis could assess pulmonary edema and its progression before PF ratio or lung ultrasound changes.

Feasibility study using longitudinal bioelectrical impedance analysis to evaluate body water status during fluid resuscitation in a swine sepsis model.

Fluid resuscitation is crucial in the initial management of sepsis; however, little is known about the serial changes and overall distribution of fluids administered into the body. To identify the feasibility of longitudinal bioelectrical impedance analysis during fluid treatment, a preclinical porcine model of Escherichia coli-induced sepsis was used. After sepsis induction, pigs were treated with fluid and vasopressors and monitored for up to 12 h after bacterial infusion or until death. Bipolar electrodes for bioelectrical impedance analysis were attached to the left extremities and measurements were performed every 10 min. Among the 12 subjects, 7 pigs expired during the experiment, and the median survival was 9.5 h. As sepsis progressed with an increase in cumulative fluid balance, R0 [∝ 1/extracellular water (ECW)] decreased, while Ri [∝ 1/intracellular water (ICW)] and ratio of extracellular water to total body water (ECW/TBW) increased. The phase angle constantly decreased throughout the monitoring period, and all non-survivors died when the phase angle decreased by more than 10%. Among the variables, ΔR0 and Δphase angle showed moderate negative correlations, and ΔECW/TBW showed a moderate positive correlation with the hourly fluid balance. Compared to survivors, a greater increase in ΔECW/TBW and a decrease in phase angle were observed in non-survivors over time, with an increase in cumulative fluid balance. Differences in ΔECW/TBW and phase angle emerged at 240 min when the difference in cumulative fluid balance between the two groups (survivors vs non-survivors) exceeded 1000 mL. In conclusion, continuous measurements of bioelectrical impedance analysis in a porcine sepsis model are feasible and may reflect changes in the body water profile during fluid resuscitation.

Vagus Nerve Stimulation Improves Mitochondrial Dysfunction in Post-cardiac Arrest Syndrome in the Asphyxial Cardiac Arrest Model in Rats.

Cerebral mitochondrial dysfunction during post-cardiac arrest syndrome (PCAS) remains unclear, resulting in a lack of therapeutic options that protect against cerebral ischemia-reperfusion injury. We aimed to assess mitochondrial dysfunction in the hippocampus after cardiac arrest and whether vagus nerve stimulation (VNS) can improve mitochondrial dysfunction and neurological outcomes. In an asphyxial cardiac arrest model, male Sprague-Dawley rats were assigned to the vagus nerve isolation (CA) or VNS (CA + VNS) group. Cardiopulmonary resuscitation was performed 450 s after pulseless electrical activity. After the return of spontaneous circulation (ROSC), left cervical VNS was performed for 3 h in the CA + VNS group. Mitochondrial respiratory function was evaluated using high-resolution respirometry of the hippocampal tissue. The neurologic deficit score (NDS) and overall performance category (OPC) were assessed at 24, 48, and 72 h after resuscitation. The leak respiration and oxidative phosphorylation capacity of complex I (OXPHOS CI) at 6 h after ROSC were significantly higher in the CA + VNS group than in the CA group (p = 0.0308 and 0.0401, respectively). Compared with the trends of NDS and OPC in the CA group, the trends of those in the CA + VNS group were significantly different, thus suggesting a favorable neurological outcome in the CA + VNS group (p = 0.0087 and 0.0064 between times × groups interaction, respectively). VNS ameliorated mitochondrial dysfunction after ROSC and improved neurological outcomes in an asphyxial cardiac arrest rat model.

Translocalization of enhanced PKM2 protein into the nucleus induced by cancer upregulated gene 2 confers cancer stem cell-like phenotypes.

Increased mRNA levels of cancer upregulated gene (CUG)2 have been detected in many different tumor tissues using Affymetrix microarray. Oncogenic capability of the CUG2 gene has been further reported. However, the mechanism by which CUG2 overexpression promotes cancer stem cell (CSC)-like phenotypes remains unknown. With recent studies showing that pyruvate kinase muscle 2 (PKM2) is overexpressed in clinical tissues from gastric, lung, and cervical cancer patients, we hypothesized that PKM2 might play an important role in CSC-like phenotypes caused by CUG2 overexpression. The present study revealed that PKM2 protein levels and translocation of PKM2 into the nucleus were enhanced in CUG2-overexpressing lung carcinoma A549 and immortalized bronchial BEAS-2B cells than in control cells. Expression levels of c-Myc, CyclinD1, and PKM2 were increased in CUG2-overexpressing cells than in control cells. Furthermore, EGFR and ERK inhibitors as well as suppression of Yap1 and NEK2 expression reduced PKM2 protein levels. Interestingly, knockdown of ß-catenin expression failed to reduce PKM2 protein levels. Furthermore, reduction of PKM2 expression with its siRNA hindered CSC-like phenotypes such as faster wound healing, aggressive transwell migration, and increased size/number of sphere formation. The introduction of mutant S37A PKM2-green fluorescence protein (GFP) into cells without ability to move to the nucleus did not confer CSC-like phenotypes, whereas forced expression of wild-type PKM2 promoted such phenotypes. Overall, CUG2-induced increase in the expression of nuclear PKM2 contributes to CSC-like phenotypes by upregulating c-Myc and CyclinD1 as a co-activator. [BMB Reports 2022;55(2): 98-103].

Enzyme Nanosheet-Based Electrochemical Aspartate Biosensor for Fish Point-of-Care Applications.

Bacterial infections in marine fishes are linked to mass mortality issues; hence, rapid detection of an infection can contribute to achieving a faster diagnosis using point-of-care testing. There has been substantial interest in identifying diagnostic biomarkers that can be detected in major organs to predict bacterial infections. Aspartate was identified as an important biomarker for bacterial infection diagnosis in olive flounder (Paralichthys olivaceus) fish. To determine aspartate levels, an amperometric biosensor was designed based on bi-enzymes, namely, glutamate oxidase (GluOx) and aspartate transaminase (AST), which were physisorbed on copolymer reduced graphene oxide (P-rGO), referred to as enzyme nanosheets (GluOx-ASTENs). The GluOx-ASTENs were drop casted onto a Prussian blue electrodeposited screen-printed carbon electrode (PB/SPCE). The proposed biosensor was optimized by operating variables including the enzyme loading amount, coreactant (α-ketoglutarate) concentration, and pH. Under optimal conditions, the biosensor displayed the maximum current responses within 10 s at the low applied potential of -0.10 V vs. the internal Ag/AgCl reference. The biosensor exhibited a linear response from 1.0 to 2.0 mM of aspartate concentrations with a sensitivity of 0.8 µA mM-1 cm-2 and a lower detection limit of approximately 500 µM. Moreover, the biosensor possessed high reproducibility, good selectivity, and efficient storage stability.

Quantitative Fluorescence In Situ Hybridization (FISH) of Magnetically Confined Bacteria Enables Early Detection of Human Bacteremia.

The current diagnosis of bacteremia mainly relies on blood culture, which is inadequate for the rapid and quantitative determination of most bacteria in blood. Here, a quantitative, multiplex, microfluidic fluorescence in situ hybridization method (µFISH) is developed, which enables early and rapid (3-h) diagnosis of bacteremia without the need for prior blood culture. This novel technology employs mannose-binding lectin-coated magnetic nanoparticles, which effectively opsonize a broad range of pathogens, magnetically sequestering them in a microfluidic device. Therein, µFISH probes, based on unique 16S rRNA sequences, enable the identification and quantification of sequestered pathogens both in saline and whole blood, which is more sensitive than conventional polymerase chain reaction. Using µFISH, Escherichia coli (E. coli) is detected in whole blood collected from a porcine disease model and from E. coli-infected patients. Moreover, the proportion of E. coli, relative to other bacterial levels in the blood, is accurately and rapidly determined, which is not possible using conventional diagnostic methods. Blood from E. coli-infected patients is differentiated from healthy donors' blood using cutoff values with a 0.05 significance level. Thus, µFISH is a versatile method that can be used to rapidly identify pathogens and determine their levels relative to other culturable and nonculturable bacteria in biological samples.

Changes in Biomarkers and Hemodynamics According to Antibiotic Susceptibility in a Model of Bacteremia.

Proper selection of susceptible antibiotics in drug-resistant bacteria is critical to treat bloodstream infection. Although biomarkers that guide antibiotic therapy have been extensively evaluated, little is known about host biomarkers targeting in vivo antibiotic susceptibility. Therefore, we aimed to evaluate the trends of hemodynamics and biomarkers in a porcine bacteremia model treated with insusceptible antibiotics compared to those in susceptible models. Extended-spectrum ß-lactamase (ESBL)-producing Escherichia coli (E. coli, 5.0 * 10^9 CFU) was intravenously administered to 11 male pigs. One hour after bacterial infusion, pigs were assigned to two groups of antibiotics, ceftriaxone (n = 6) or ertapenem (n = 5). Pigs were monitored up to 7 h after bacterial injection with fluid and vasopressor support to maintain the mean arterial blood pressure over 65 mmHg. Blood sampling for blood culture and plasma acquisition was performed before and every predefined hour after E. coli injection. Cytokine (tumor necrosis factor-α, interleukin [IL]-1ß, IL-6, IL-8, IL-10, C-reactive protein, procalcitonin, presepsin, heparan sulfate, syndecan, and soluble triggering receptor expressed on myeloid cells-1 [sTREM-1]) levels in plasma were analyzed using enzyme-linked immunosorbent assays. Bacteremia developed after intravenous injection of E. coli, and negative conversion was confirmed only in the ertapenem group. While trends of other biomarkers failed to show differences, the trend of sTREM-1 was significantly different between the two groups (P = 0.0001, two-way repeated measures analysis of variance). Among hemodynamics and biomarkers, the sTREM-1 level at post 2 h after antibiotics administration represented a significant difference depending on susceptibility, which can be suggested as a biomarker candidate of in vivo antibiotics susceptibility. Further clinical studies are warranted for validation. IMPORTANCE Early and appropriate antibiotic treatment is a keystone in treating patients with sepsis. Despite its importance, blood culture which requires a few days remains as a pillar of diagnostic method for microorganisms and their antibiotic susceptibility. Whether changes in biomarkers and hemodynamics indicate treatment response of susceptible antibiotic compared to resistant one is not well understood to date. In this study using extended-spectrum ß-lactamase -producing E. coli bacteremia porcine model, we have demonstrated the comprehensive cardiovascular hemodynamics and trends of plasma biomarkers in sepsis and compared them between two groups with susceptible and resistant antibiotics. While other hemodynamics and biomarkers have failed to differ, we have identified that levels of soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) significantly differed between the two groups over time. Based on the data in this study, trends of sTREM-1 obtained before the antibiotics and 2~4 h after the antibiotics could be a novel host biomarker that triggers the step-up choice of antibiotics.