Artificial intelligence evaluation of coronary computed tomography angiography for coronary stenosis classification and diagnosis.

BACKGROUND: Ruling out obstructive coronary artery disease (CAD) using coronary computed tomography angiography (CCTA) is time-consuming and challenging. This study developed a deep learning (DL) model to assist in detecting obstructive CAD on CCTA to streamline workflows. METHODS: In total, 2929 DICOM files and 7945 labels were extracted from curved planar reformatted CCTA images. A modified Inception V3 model was adopted. To validate the artificial intelligence (AI) model, two cardiologists labelled and adjudicated the classification of coronary stenosis on CCTA. The model was trained to differentiate the coronary artery into binary stenosis classifications <50% and ≥50% stenosis. Using the quantitative coronary angiography (QCA) consensus results as a reference standard, the performance of the AI model and CCTA radiology readers was compared by calculating Cohen's kappa coefficients at patient and vessel levels. The net reclassification index was used to evaluate the net benefit of the DL model. RESULTS: The diagnostic accuracy of the AI model was 92.3% and 88.4% at the patient and vessel levels, respectively. Compared with CCTA radiology readers, the AI model had a better agreement for binary stenosis classification at both patient and vessel levels (Cohen kappa coefficient: .79 vs. .39 and .77 vs. .40, p < .0001). The AI model also exhibited significantly improved model discrimination and reclassification (Net reclassification index = .350; Z = 4.194; p < .001). CONCLUSIONS: The developed AI model identified obstructive CAD, and the model results correlated well with QCA results. Incorporating the model into the reporting system of CCTA may improve workflows.

Association between Premorbid Renin-Angiotensin-Aldosterone System Blockade and the Risk of Acute Kidney Injury in Critically Ill Patients.

Background: Angiotensin-converting enzyme inhibitors (ACEis) and angiotensin receptor blockers (ARBs) are commonly used for hypertension and cardiovascular diseases. However, whether their use increases the risk of acute kidney injury (AKI) and should be discontinued during acute illness remains controversial. Methods: This retrospective study enrolled 952 dialysis-free patients who were admitted to intensive care units (ICUs) between 2015 and 2017, including 476 premorbid long-term (> 1 month) ACEi/ARB users. Propensity score matching was performed to adjust for age, gender, comorbidities, and disease severity. The primary endpoint was the occurrence of AKI during hospitalization, and the secondary endpoint was mortality or dialysis within 1 year. Results: Compared with non-users, the ACEi/ARB users were not associated with an increased AKI risk during hospitalization [66.8% vs. 70.4%; hazard ratio (HR): 1.13, 95% confidence interval (CI): 0.97-1.32, p = 0.126]. However, the ACEi/ARB users with sepsis (HR: 1.29, 95% CI: 1.04-1.60, p = 0.021) or hypotension (HR: 1.21, 95% CI: 1.02-1.14, p = 0.034) were found to have an increased AKI risk in subgroup analysis. Nevertheless, compared with the non-users, the ACEi/ARB users were associated with a lower incidence of mortality or dialysis within 1 year (log-rank p = 0.011). Conclusions: Premorbid ACEi/ARB usage did not increase the incidence of AKI, and was associated with a lower 1-year mortality and dialysis rate in patients admitted to ICUs. Regarding the results of subgroup analysis, renin-angiotensin-aldosterone system blockade may still be safe and beneficial in the absence of sepsis or circulation failure. Further large-scale studies are needed to confirm our findings.

Obesity paradox in patients with chronic total occlusion of coronary artery.

BACKGROUND: Obesity is associated with metabolic syndrome which increases further risk of coronary artery disease and adverse cardiovascular events. Impact of body mass index (BMI) on long-term outcome in patients with coronary chronic total occlusion (CTO) is less clear. METHOD AND RESULTS: From January 2005 to November 2020, a total of 1301 patients with coronary angiographic confirmed CTO were enrolled in our study. Patients were divided into two groups: low BMI group: 18-24.99 kg/m2 and high BMI group ≥25 kg/m2 . Clinical outcomes were 3-year all-cause mortality, 3-year cardiovascular mortality and 3-year non-fatal myocardial infarct. During the 3-year follow-up period, all-cause mortality was significantly higher in patients with low BMI group compared to those in high BMI groups (14% vs. 6%, p = .0001). Kaplan-Meier analysis showed patients with high BMI groups had significant better survival compared with those in low BMI group (p = .0001). In multivariate analysis, higher BMI was independently associated with decreased risk of 3-year all-cause mortality (Hazard ratio [HR]: 0.534; 95% confidence interval [CI]: 0.349-0.819, p = .004) after controlling for age, renal function, prior history of stroke, coronary artery bypass graft, co-morbidities with peripheral arterial disease, heart failure and revascularization status for CTO. In propensity-matched multivariate analysis, high BMI remained a significant predictor of 3-year all-cause mortality (HR, 0.525; 95% CI, 0.346-0.795, p = .002). CONCLUSION: Higher BMI was associated with better long-term outcome in patients with coronary CTO.

2022 Taiwan lipid guidelines for primary prevention.

Elevated circulating low-density lipoprotein cholesterol (LDL-C) is a major risk factor of atherosclerotic cardiovascular disease (ASCVD). Early control of LDL-C to prevent ASCVD later in life is important. The Taiwan Society of Lipids and Atherosclerosis in association with the other seven societies developed this new lipid guideline focusing on subjects without clinically significant ASCVD. In this guideline for primary prevention, the recommended LDL-C target is based on risk stratification. A healthy lifestyle with recommendations for foods, dietary supplements and alcohol drinking are described. The pharmacological therapies for LDL-C reduction are recommended. The aim of this guideline is to decrease the risk of ASCVD through adequate control of dyslipidemia in Taiwan.

The QseEF Two-Component System-GlmY Small RNA Regulatory Pathway Controls Swarming in Uropathogenic Proteus mirabilis.

Bacterial sensing of environmental signals through the two-component system (TCS) plays a key role in modulating virulence. In the search for the host hormone-sensing TCS, we identified a conserved qseEGF locus following glmY, a small RNA (sRNA) gene in uropathogenic Proteus mirabilis. Genes of glmY-qseE-qseG-qseF constitute an operon, and QseF binding sites were found in the glmY promoter region. Deletion of glmY or qseF resulted in reduced swarming motility and swarming-related phenotypes relative to the wild-type and the respective complemented strains. The qseF mutant had decreased glmYqseEGF promoter activity. Both glmY and qseF mutants exhibited decreased flhDC promoter activity and mRNA level, while increased rcsB mRNA level was observed in both mutants. Prediction by TargetRNA2 revealed cheA as the target of GlmY. Then, construction of the translational fusions containing various lengths of cheA 5'UTR for reporter assay and site-directed mutagenesis were performed to investigate the cheA-GlmY interaction in cheA activation. Notably, loss of glmY reduced the cheA mRNA level, and urea could inhibit swarming in a QseF-dependent manner. Altogether, this is the first report elucidating the underlying mechanisms for modulation of swarming motility by a QseEF-regulated sRNA GlmY, involving expression of cheA, rcsB and flhDC in uropathogenic P. mirabilis.

Gender difference in the association between TyG index and subclinical atherosclerosis: results from the I-Lan Longitudinal Aging Study.

BACKGROUND: Insulin resistance (IR) is a known risk factor for cardiovascular disease (CVD) in non-diabetic patients through the association of hyperglycemia or associated metabolic factors. The triglyceride glucose (TyG) index, which was defined by incorporating serum glucose and insulin concentrations, was developed as a surrogate marker of insulin resistance. We aimed to investigate the association between the TyG index and the early phase of subclinical atherosclerosis (SA) between the sexes. METHODS: The I-Lan Longitudinal Aging Study (ILAS) enrolled 1457 subjects aged 50-80 years. For each subject, demographic data and the TyG index {ln[fasting triglyceride (mg/dL) × fasting plasma glucose (mg/dL)]/2} were obtained. Patients were further stratified according to sex and the 50th percentile of the TyG index (≥ 8.55 or < 8.55). SA was defined as the mean carotid intima-media thickness (cIMT) at the 75th percentile of the entire cohort. Demographic characteristics and the presence of SA were compared between the groups. Logistic regression analysis was performed to assess the relationship between TyG index and SA. RESULTS: Patients with a higher TyG index (≥ 8.55) had a higher body mass index (BMI), hypertension (HTN) and diabetes mellitus (DM). They had higher lipid profiles, including total cholesterol (T-Chol) and low-density lipoprotein (LDL), compared to those with a lower TyG index (< 8.55). Gender disparity was observed in non-diabetic women who had a significantly higher prevalence of SA in the high TyG index group than in the low TyG index group. In multivariate logistic regression analysis, a high TyG index was independently associated with SA in non-diabetic women after adjusting for traditional risk factors [adjusted odds ratio (OR): 1.510, 95% CI 1.010-2.257, p = 0.045] but not in non-diabetic men. The TyG index was not associated with the presence of SA in diabetic patients, irrespective of sex. CONCLUSION: A high TyG index was significantly associated with SA and gender disparity in non-diabetic patients. This result may highlight the need for a sex-specific risk management strategy to prevent atherosclerosis.

Serum creatinine to cystatin C ratio is associated with major adverse cardiovascular events in patients with obstructive coronary artery disease.

BACKGROUND & AIMS: Sarcopenia is a clinical syndrome that features muscle atrophy and weakness, and has been associated with cardiovascular events and poor clinical outcomes. Recently, the sarcopenia index (SI) was developed as a simple screening tool based upon the serum creatinine to cystatin C (CysC) ratio. We investigated the association between SI and the prevalence of major adverse cardiovascular events (MACE) in patients with obstructive CAD. METHODS & RESULTS: Between January 2010 and December 2018, patients with angina pectoris and obstructive CAD requiring coronary artery intervention were enrolled. Serum levels of CysC and other biomarkers were assessed. Patients were divided into two groups according to the SI ([Cr/CysC] x 100). Demographic characteristics and clinical outcomes of the two groups were evaluated. A total of 427 patients (79.6% men, mean age 69.55 ± 12.04 years) were enrolled. Patients with SI < 120 (n = 214, 28%) were older, more likely to be of the female gender, and to have more hypertension and congestive heart failure (all p < 0.05). The prevalence of major adverse cardiovascular events (MACE) composed of myocardial infarction, stroke, and all-cause mortality was higher in patients with lower SI (p = 0.026). After adjusting for potential confounding factors, multivariate Cox regression (hazard ratio 2.08, p = 0.045) and Kaplan-Meier analyses (log-rank p = 0.0371) revealed that lower SI was significantly associated with a higher prevalence of MACE. CONCLUSIONS: Serum creatinine to cystatin C ratio (SI) may be a useful surrogate marker to predict the future prevalence of MACE in patients with obstructive CAD.

Feasibility of the transcatheter mitral valve repair for patients with severe mitral regurgitation and endangered heart failure.

BACKGROUND: The transcatheter edge-to-edge mitral valve repair using MitraClip has been a safe and effective treatment for severe mitral regurgitation (MR). In patients with severe MR and cardiogenic shock under hemodynamic supporting devices, emergent surgical mitral valve interventions carry extremely high risk for peri-operative morbidities and mortalities. The feasibility and efficacy of emergent MitraClip to rescue patients in critical conditions remains elucidate. METHODS: Patients with severe MR and high or prohibitive surgical risks were referred for MitraClip procedures. Emergent MitraClip were conducted in patients with unstable hemodynamics and under mechanical or inotropic support. The hemodynamic measures, transthoracic echocardiography, transesophageal echocardiography, and blood tests were performed before MitraClip procedures. Procedural success was defined as having mild mitral regurgitation immediately after MitraClip, and patients were free from in-hospital mortality. Clinical and echocardiographic outcomes were followed by telephones and clinics. RESULTS: Among 50 consecutive patients (74.7 ± 11.2 years, 74% male), 8 emergent MitraClip procedures were conducted to rescue patients with cardiogenic shock. Extracorporeal membrane oxygenations were used in 2 patients and intra-aortic balloon pump were applied in 4 patients (50%). Compare to those who underwent elective procedures, patients underwent emergent MitraClip had higher surgical risk profile (EuroSCORE II 34.8% vs 5.1% and STS score 19.7% vs 5.1%), poorer renal function and higher right atrial pressure. There was no peri-procedural death, myocardial infarction, stroke or any adverse events requiring emergent cardiac surgery in both groups. Mild mitral regurgitation was achieved in 87.5% patients from the emergent group and 95.2% patients in the elective group (P = 0.514). The Kaplan-Meier analysis showed patients who underwent emergent procedures have poorer long-term survival rate as compare to those who received elective procedures. (P value = 0.008). CONCLUSION: When open-heart surgery is not feasible, trans-catheter mitral valve repair is an alternative way to rescue patients in cardiogenic shock status.

Velar Closing Ratio As a Predictor for the Verlopharyngeal Function After Double Opposing Z-Plasty for Postpalatoplasty Velopharyngeal Insufficiency in Patients With Cleft Palate.

BACKGROUND: This study was designed to identify the potential predictors of postoperative velopharyngeal function after double opposing Z-plasty (DOZP) for the treatment of velopharyngeal insufficiency (VPI) in patients who had prior palatoplasty for cleft palate. METHODS: This retrospective study reviewed the medical records of consecutive patients who received DOZP for VPI after receiving a prior palatoplasty treating cleft palate between 2004 and 2017. The speech outcome of patient was measured using the Pittsburgh Weighted Speech Scale (PWSS) at 6 months following surgery and determined the outcome suggests velopharyngeal competence (PWSS ≤2) or incompetence (PWSS >2). Stepwise logistic regression was used to identify the variables for the prediction of competent surgical outcome. The specific receiver operating characteristic curves with an area under the curve (AUC) was used to evaluate the predictor related to the surgical outcome as competence. RESULTS: The study included 93 patients. Age, relative velar length, velar lengthening, and closure pattern were not significantly associated with postoperative competence status of the patient. The only variable that predicted a successful surgical outcome was preoperative velar closing ratio. However, the accuracy of velar closing ratio in predicting a competent surgical outcome is only moderate (AUC = 70.37). CONCLUSION: The results of this study showed that preoperative velar closing ratio may predict, with moderate accuracy, a successful surgical outcome in patients with postpalatoplasty VPI who undergo DOZP. Therefore, in patients with a low preoperative velar closing ratio, some alternative surgical methods other than DOZP may be considered to avoid unsatisfactory surgical outcome.

Investigation on reliability of red micro-light emitting diodes with atomic layer deposition passivation layers.

In this study, AlGaInP red light emitting diodes with sizes ranging from 5 to 50 micrometers were fabricated and characterized. The atomic layer deposition technology is applied to coat a layer of silicon dioxide for passivation and protection. The top emission area is covered by ITO layer to maximize the optical output. From the optical measurement, the linewidth and emission peaks shift very little among different current levels (from 30 to 150 A/cm2). High current level lifetests are performed and a 15 µm ALD device can last 27 hours of continuous operation at 100 A/cm2 before their diode junction failed. A much shorter lifetime of 5.32 hours was obtained when the driving current is raised to 400 A/cm2. When the same condition was applied to 15 µm PECVD devices, 25 hours and 4.33 hours are registered for 100 A/cm2 and 400 A/cm2 tests, respectively. The cross-sectional SEM reveals the voids, defects, and dark lines developed during the aging tests, and most of them are caused by top contact failure. The surface layers of ITO and SiO2 were melted and the dark lines which were originated from the top surface propagated through the device and led to the eventual failure of the diode. The optical intensity degradation slopes of different sizes of devices indicate a large device can last longer in this accelerated aging test. The efficiencies of the devices are also evaluated by the ABC model and the fitted bimolecular coefficient ranges from 1.35 to 3.40×10-10 cm3/s.

Long term clinical impact of successful recanalization of chronic total occlusion in patients with and without type 2 diabetes mellitus.

BACKGROUND: Diabetes mellitus is one of the risk factors for coronary artery disease and frequently associated with multivessels disease and poor clinical outcomes. Long term outcome of successful revascularization of chronic total occlusions (CTO) in diabetes patients remains controversial. METHODS AND RESULTS: From January 2005 to December 2015, 739 patients who underwent revascularization for CTO in Taipei Veterans General Hospital were included in this study, of which 313 (42%) patients were diabetes patients. Overall successful rate of revascularization was 619 (84%) patients whereas that in diabetics and non-diabetics were 265 (84%) and 354 (83%) respectively. Median follow up was 1095 days (median: 5 years, interquartile range: 1-10 years). During 3 years follow-up period, 59 (10%) in successful group and 18 (15%) patients in failure group died. Although successful revascularization of CTO was non-significantly associated with better outcome in total cohort (hazard ratio (HR): 0.593, 95% confidence interval (CI) 0.349-0.008, P: 0.054), it might be associated with lower risk of all-cause mortality (HR: 0.307, 95% CI 0.156-0.604, P: 0.001) and CV mortality (HR: 0.266, 95% CI 0.095-0.748, P: 0.012) in diabetics (P: 0.512). In contrast, successful CTO revascularization didn't improve outcomes in non-diabetics (all P > 0.05). In multivariate cox regression analysis, successful CTO revascularization remained an independent predictor for 3-years survival in diabetic subgroup (HR: 0.289, 95% CI 0.125-0.667, P: 0.004). The multivariate analysis result was similar after propensity score matching (all-cause mortality, HR: 0.348, 95% CI 0.142-0.851, P: 0.021). CONCLUSIONS: Successful CTO revascularization in diabetes may be related to better long term survival benefit but not in non-diabetic population.

Feasibility and rationale of direct current cardioversion immediately after transcatheter percutaneous edge-to-edge mitral valve repair.

AIMS: Atrial fibrillation (AF) is a frequent comorbidity among patients with severe mitral regurgitation (MR). Direct current (DC) cardioversion is one of the strategies for rhythm control. However, the safety and feasibility of immediate DC cardioversion after MitraClip are not elucidated. METHODS AND RESULTS: In this study, patients with symptomatic severe MR who underwent MitraClip were included. After fixing the MR, synchronized DC cardioversion was attempted for those with AF. A total of consecutive 60 patients, 36 subjects (60%), comorbid with AF. DC cardioversion was performed in 30 patients (mean age of 76.0 ± 9.3 years), and the successful conversion was achieved in 15 patients (50%). There was no any adverse event related to the cardioversion. Subjects with sustained conversion to SR experienced significant improvement in 6MWT (failed: 285 ± 110-308 ± 135 m, P = .278; successful: 269 ± 109 m-328 ± 78, P = .047) and reduction in NT-proBNP level (failed: 4411 ± 7401-3296 ± 4299 ng/mL, P = .217; successful: 4094 ± 2735-2353 ± 2856 ng/mL, P = .026) at 1 month. CONCLUSIONS: Direct current cardioversion seemed to be safe and feasible immediately after the transcatheter edge-to-edge mitral valve repairs. Subjects who maintain SR experienced better functional improvement.

Circulating Activin A Is a Surrogate for the Incidence of Diastolic Dysfunction and Heart Failure in Patients With Preserved Ejection Fraction.

BACKGROUND: Diastolic dysfunction (DD) is a characteristic of heart failure with preserved ejection fraction (HFpEF), which is thought to be caused by cardiac hypertrophy or fibrosis. Activin A is involved in the inflammatory response and myocardial fibrosis, but the relationship between the activin A level and DD remains unclear.Methods and Results:A total of 209 patients with stable angina were enrolled. Serum activin A levels were assessed, and echocardiography and cross-sectional analysis were performed. Among the subjects (65% male; mean age, 70±13 years), 84 (40%) subjects had DD. The subjects were divided into tertiles based on activin A levels. Patients in the high activin A group had enhanced left ventricular mass indexes, medial E/e' ratios, left atrial diameter, and right ventricular systolic pressure compared with those in the lower activin A groups (all P<0.001). Prevalence of DD (P=0.001), HFpEF at enrollment (P=0.007), and the composite endpoints including new-onset heart failure (HF) or death within 3 years (P<0.001) correlated positively with high activin A levels. After adjusting for confounding factors, high activin A levels remained significantly associated with DD (P=0.036) and the composite endpoints (P=0.012). CONCLUSIONS: Enhanced serum activin A levels were associated with the incidence of DD and development of HF.

Fabrication of optical vortex lattices based on holographic polymer-dispersed liquid crystal films.

This Letter demonstrates optical vortex lattices based on holographic polymer-dispersed liquid crystal (HPDLC) films. The fabrication method uses a phase-only reflective spatial light modulator with numerically calculated phase profiles loaded on it to simplify multi-helical-wave interference. The beam profiles of the diffraction beams are simulated using the Fourier transform and compared with experimental results. The topological charges of the 1st-order diffraction beams reconstructed from the HPDLC films are examined using a Michelson interferometer.

A potential diagnostic pitfall in acute chest pain: Massive pulmonary embolism mimicking acute STEMI.

BACKGROUND: Pulmonary embolism (PE) represents a clinical challenge for clinicians because of nonspecific presentations, including dyspnea, chest pain, and tachycardia. The immediate 12-lead electrocardiogram (ECG) is commonly used to facilitate differential diagnosis of acute chest pain. Although relative rare, massive pulmonary embolism could induce ST segment elevation and mimic acute myocardial infarction. CASE PRESENTATION: We present a challenging scenario that ECG showed ST segment elevation, nevertheless, urgent coronary angiogram revealed non-obstructive coronary artery disease. Unfortunately, the patient suffered from cardiac arrest and required extracorporeal membrane oxygenation devices. Finally, massive pulmonary embolism was diagnosed. CONCLUSION: This case illustrates acute PE could mimic ST segment elevation myocardial infarction. ST elevations on ECG should be interpreted after considering clinical presentations before making a decision.

Exploiting members of the BAHD acyltransferase family to synthesize multiple hydroxycinnamate and benzoate conjugates in yeast.

BACKGROUND: BAHD acyltransferases, named after the first four biochemically characterized enzymes of the group, are plant-specific enzymes that catalyze the transfer of coenzyme A-activated donors onto various acceptor molecules. They are responsible for the synthesis in plants of a myriad of secondary metabolites, some of which are beneficial for humans either as therapeutics or as specialty chemicals such as flavors and fragrances. The production of pharmaceutical, nutraceutical and commodity chemicals using engineered microbes is an alternative, green route to energy-intensive chemical syntheses that consume petroleum-based precursors. However, identification of appropriate enzymes and validation of their functional expression in heterologous hosts is a prerequisite for the design and implementation of metabolic pathways in microbes for the synthesis of such target chemicals. RESULTS: For the synthesis of valuable metabolites in the yeast Saccharomyces cerevisiae, we selected BAHD acyltransferases based on their preferred donor and acceptor substrates. In particular, BAHDs that use hydroxycinnamoyl-CoAs and/or benzoyl-CoA as donors were targeted because a large number of molecules beneficial to humans belong to this family of hydroxycinnamate and benzoate conjugates. The selected BAHD coding sequences were synthesized and cloned individually on a vector containing the Arabidopsis gene At4CL5, which encodes a promiscuous 4-coumarate:CoA ligase active on hydroxycinnamates and benzoates. The various S. cerevisiae strains obtained for co-expression of At4CL5 with the different BAHDs effectively produced a wide array of valuable hydroxycinnamate and benzoate conjugates upon addition of adequate combinations of donors and acceptor molecules. In particular, we report here for the first time the production in yeast of rosmarinic acid and its derivatives, quinate hydroxycinnamate esters such as chlorogenic acid, and glycerol hydroxycinnamate esters. Similarly, we achieved for the first time the microbial production of polyamine hydroxycinnamate amides; monolignol, malate and fatty alcohol hydroxycinnamate esters; tropane alkaloids; and benzoate/caffeate alcohol esters. In some instances, the additional expression of Flavobacterium johnsoniae tyrosine ammonia-lyase (FjTAL) allowed the synthesis of p-coumarate conjugates and eliminated the need to supplement the culture media with 4-hydroxycinnamate. CONCLUSION: We demonstrate in this study the effectiveness of expressing members of the plant BAHD acyltransferase family in yeast for the synthesis of numerous valuable hydroxycinnamate and benzoate conjugates.

New aspects of RpoE in uropathogenic Proteus mirabilis.

Proteus mirabilis is a common human pathogen causing recurrent or persistent urinary tract infections (UTIs). The underlying mechanisms for P. mirabilis to establish UTIs are not fully elucidated. In this study, we showed that loss of the sigma factor E (RpoE), mediating extracytoplasmic stress responses, decreased fimbria expression, survival in macrophages, cell invasion, and colonization in mice but increased the interleukin-8 (IL-8) expression of urothelial cells and swarming motility. This is the first study to demonstrate that RpoE modulated expression of MR/P fimbriae by regulating mrpI, a gene encoding a recombinase controlling the orientation of MR/P fimbria promoter. By real-time reverse transcription-PCR, we found that the IL-8 mRNA amount of urothelial cells was induced significantly by lipopolysaccharides extracted from rpoE mutant but not from the wild type. These RpoE-associated virulence factors should be coordinately expressed to enhance the fitness of P. mirabilis in the host, including the avoidance of immune attacks. Accordingly, rpoE mutant-infected mice displayed more immune cell infiltration in bladders and kidneys during early stages of infection, and the rpoE mutant had a dramatically impaired ability of colonization. Moreover, it is noteworthy that urea (the major component in urine) and polymyxin B (a cationic antimicrobial peptide) can induce expression of rpoE by the reporter assay, suggesting that RpoE might be activated in the urinary tract. Altogether, our results indicate that RpoE is important in sensing environmental cues of the urinary tract and subsequently triggering the expression of virulence factors, which are associated with the fitness of P. mirabilis, to build up a UTI.

Serratia marcescens arn, a PhoP-regulated locus necessary for polymyxin B resistance.

Polymyxins, which are increasingly being used to treat infections caused by multidrug-resistant bacteria, perform poorly against Serratia marcescens. To investigate the underlying mechanisms, Tn5 mutagenesis was performed and two mutants exhibiting increased polymyxin B (PB) susceptibility were isolated. The mutants were found to have Tn5 inserted into the arnB and arnC genes. In other bacteria, arnB and arnC belong to the seven-gene arn operon, which is involved in lipopolysaccharide (LPS) modification. LPSs of arn mutants had greater PB-binding abilities than that of wild-type LPS. Further, we identified PhoP, a bacterial two-component response regulator, as a regulator of PB susceptibility in S. marcescens. By the reporter assay, we found PB- and low-Mg2+-induced expression of phoP and arn in the wild-type strain but not in the phoP mutant. Complementation of the phoP mutant with the full-length phoP gene restored the PB MIC and induction by PB and low Mg2+ levels, as in the wild type. An electrophoretic mobility shift assay (EMSA) further demonstrated that PhoP bound directly to the arn promoter. The PB challenge test confirmed that pretreatment with PB and low Mg2+ levels protected S. marcescens from a PB challenge in the wild-type strain but not in the phoP mutant. Real-time reverse transcriptase-PCR also indicated that PB serves as a signal to regulate expression of ugd, a gene required for LPS modification, in S. marcescens through a PhoP-dependent pathway. Finally, we found that PB-resistant clinical isolates displayed greater expression of arnA upon exposure to PB than did susceptible isolates. This is the first report to describe the role of S. marcescens arn in PB resistance and its modulation by PB and Mg2+ through the PhoP protein.

Surface-functionalized hyperbranched poly(amido acid) magnetic nanocarriers for covalent immobilization of a bacterial γ-glutamyltranspeptidase.

In this study, we synthesized water-soluble hyperbranched poly(amido acid)s (HBPAAs) featuring multiple terminal CO2H units and internal tertiary amino and amido moieties and then used them in conjunction with an in situ Fe2+/Fe3+ co-precipitation process to prepare organic/magnetic nanocarriers comprising uniformly small magnetic iron oxide nanoparticles (NP) incorporated within the globular HBPAAs. Transmission electron microscopy revealed that the HBPAA-γ-Fe2O3 NPs had dimensions of 6-11 nm, significantly smaller than those of the pristine γ-Fe2O3 (20-30 nm). Subsequently, we covalently immobilized a bacterial γ-glutamyltranspeptidase (BlGGT) upon the HBPAA-γ-Fe2O3 nanocarriers through the formation of amide linkages in the presence of a coupling agent. Magnetization curves of the HBPAA-γ-Fe2O3/BlGGT composites measured at 300 K suggested superparamagnetic characteristics, with a saturation magnetization of 52 emu g⁻¹. The loading capacity of BlGGT on the HBPAA-γ-Fe2O3 nanocarriers was 16 mg g⁻¹ support; this sample provided a 48% recovery of the initial activity. The immobilized enzyme could be recycled 10 times with 32% retention of the initial activity; it had stability comparable with that of the free enzyme during a storage period of 63 days. The covalent immobilization and stability of the enzyme and the magnetization provided by the HBPAA-γ-Fe2O3 NPs suggests that this approach could be an economical means of depositing bioactive enzymes upon nanocarriers for BlGGT-mediated bio-catalysis.