Age-related changes in left ventricular vortex and energy loss patterns: from newborns to adults.

Left ventricular vortex formation optimizes the effective transport of blood volume while minimizing energy loss (EL). Vector flow mapping (VFM)-derived EL patterns have not been described in children, especially in those less than 1 yr of age. A prospective cohort of 66 (0 days-22 yr, 14 patients ≤ 2 mo) cardiovascularly normal children was used to determine left ventricular (LV) vortex number, size (mm2), strength (m2/s), and energy loss (mW/m/m2) in systole and diastole and compared across age groups. One early diastolic (ED) vortex at the anterior mitral leaflet and one late diastolic (LD) vortex at the LV outflow tract (LVOT) were seen in all newborns ≤ 2 mo. At >2 mo, two ED vortices and one LD vortex were seen, with 95% of subjects > 2 yr demonstrating this vortex pattern. Peak and average diastolic EL acutely increased in the same 2 mo-2-yr period and then decreased within the adolescent and young adult age groups. Overall, these findings suggest that the growing heart undergoes a transition to adult vortex flow patterns over the first 2 yr of life with a corresponding acute increase in diastolic EL. These findings offer an initial insight into the dynamic changes of LV flow patterns in pediatric patients and can serve to expand our understanding of cardiac efficiency and physiology in children.NEW & NOTEWORTHY This research article demonstrates, for the first time, echocardiographic evidence of a transition in left ventricular vortex patterns from the newborn to the adult period, with an associated change in cardiac efficiency, marked by increased energy loss, during infancy.

Necrotizing enterocolitis in infants with ductal-dependent congenital heart disease.

OBJECTIVE: Infants with congenital heart disease (CHD) receiving prostaglandins (PGEs) may be at an increased risk for necrotizing enterocolitis (NEC). Enteral feeding may further increase the risk of NEC in these patients. We evaluated the incidence of NEC and its association with enteral feeding in infants with ductal-dependent CHD. STUDY DESIGN: We examined a cohort of infants with CHD receiving PGE in neonatal intensive care units managed by the Pediatrix Medical Group (Sunrise, FL) between 1997 and 2010. We used logistic regression to evaluate the association between NEC and enteral feeding, as well as other risk factors, including antacid medications, inotropic and ventilator support, and anatomic characteristics, controlling for gestational age. RESULTS: We identified 6,710 infants with ductal-dependent CHD receiving PGE for 17,158 infant days. NEC occurred in 21 of the 6,710 (0.3%) infants, of whom 12/21 (57%) were < 37 weeks gestational age. The incidence of NEC was 1.2/1,000 infant days while on enteral feeds versus 0.4/1,000 infant days while not on enteral feeds (p = 0.27). Enteral feeding was not associated with a statistically significant increased odds of NEC on the day of diagnosis (odds ratio [OR] 2.08; 95% confidence interval [CI] 0.38, 11.7). Risk factors associated with a significant increased odds of NEC included a diagnosis of single-ventricle heart defect (OR 2.82; 95% CI 1.23, 6.49), although the overall risk in this population remained low (8/1,631, 0.5%). CONCLUSION: The incidence of NEC in our cohort of infants with ductal-dependent CHD on PGE therapy was low and did not increase with enteral feeding.

The Diagnostic Yield of [18F]FDG-PET/CT in a Heterogeneous In-Patient Population with Suspected Infection or Inflammation Is Comparable to Findings in Patients with Classic Fever of Unknown Origin.

INTRODUCTION: Suspected infection or inflammation of unknown origin in in-patients remains challenging. Literature on [18F]FDG-PET/CT is abundant in classic fever of unknown origin (FUO), but evidence is complex and may not always reflect clinical reality. This study explores the application of [18F]FDG-PET/CT in a diverse clinical population of in-patients with suspected infection not defined by stringent FUO-criteria. METHODS: Retrospective chart review of consecutive in-patients who underwent [18F]FDG-PET/CT in the workup of suspected infection or inflammation from 1 July 2022 to 31 December 2022 was conducted. We evaluated indications, diagnostic yield, and clinical impact of [18F]FDG-PET/CT, and compared the findings of [18F]FDG-PET/CT and stand-alone CT. Univariate logistic regression assessed associations between [18F]FDG-PET/CT outcome and clinical parameters. Receiver operating characteristic curve (ROC) analysis evaluated diagnostic performance. RESULTS: 77 patients met the inclusion criteria. [18F]FDG-PET/CT established a diagnosis in 35% of cases, ruled out focal infection in 26%, and thus was helpful in 61% of patients. It prompted 72 additional examinations resulting in seven incidental diagnoses, including two cancers. Antibiotic treatment was changed in 26% of cases. Regression analysis found white blood cell counts (WBC) associated with true positive outcomes. [18F]FDG-PET/CT was compared to stand-alone CT findings, and was concordant in 69% of cases. CONCLUSIONS: Results were comparable to findings in more classic FUO. [18F]FDG-PET/CT was clinically helpful in 61% of cases but also prompted many additional examinations with relatively few clinically important findings. WBC count was a predictor of true positive outcome. CT and [18F]FDG-PET/CT were discordant in 31%, of cases, especially in cases of endocarditis and spondylodiscitis.

Reciprocal regulation of 11ß-hydroxysteroid dehydrogenase 1 and glucocorticoid receptor expression by dexamethasone inhibits human coronary artery smooth muscle cell proliferation in vitro.

The actions of glucocorticoids are mediated, in part, by 11ß-hydroxysteroid dehydrogenase 1 (11ß-HSD1), which amplifies their effects at the pre-receptor level by converting cortisone to cortisol. Glucocorticoids, such as dexamethasone, inhibit vascular smooth muscle cell proliferation; however, the role of 11ß-HSD1 in this response remains unknown. Accordingly, we treated human coronary artery smooth muscle cells (HCSMC) with dexamethasone (10(-9)-10(-6) mol/l) and found that after 72 h dexamethasone increased 11ß-HSD1 expression (14.16 ± 1.6-fold, P < 0.001) and activity (6.21 ± 1.2-fold, P < 0.001) in a dose- and time-dependent manner, which was dependent upon glucocorticoid receptor (GR) activation and C/EBPß and C/EBPδ signaling. As glucocorticoids are known to negatively regulate GR expression, we examined the effect of decreasing 11ß-HSD1 expression on GR expression. In HCSMC transfected with 11ß-HSD1 siRNA, GR expression was increased; this effect was associated with protein kinase A activation and CREB phosphorylation. To examine the role of 11ß-HSD1 in HCSMC proliferation, we decreased 11ß-HSD1 expression and stimulated cells with platelet-derived growth factor (PDGF) (10 ng/ml). Decreased 11ß-HSD1 expression was associated with increased cell proliferation in the absence of PDGF compared to scrambled control-transfected cells (236.10 ± 13.11%, n = 4, P < 0.001) and this effect was augmented by PDGF. Furthermore, the inhibitory effect of dexamethasone on cellular proliferation was abrogated in 11ß-HSD1 siRNA-transfected HCSMC. Downregulation of 11ß-HSD1 was associated with decreased p27(kip1) expression and increased phosphorylated retinoblastoma protein, consistent with a proliferative response. These findings suggest that 11ß-HSD1 plays a role in the effects of glucocorticoids on vascular smooth muscle cell phenotype.

Nanoparticle-based capillary electroseparation of proteins in polymer capillaries under physiological conditions.

Totally porous lipid-based liquid crystalline nanoparticles were used as pseudostationary phase for capillary electroseparation with LIF detection of proteins at physiological conditions using unmodified cyclic olefin copolymer capillaries (Topas, 6.7 cm effective length). In the absence of nanoparticles, i.e. in CE mode, the protein samples adsorbed completely to the capillary walls and could not be recovered. In contrast, nanoparticle-based capillary electroseparation resolved green fluorescent protein from several of its impurities within 1 min. Furthermore, a mixture of native green fluorescent protein and two of its single-amino-acid-substituted variants was separated within 2.5 min with efficiencies of 400 000 plates/m. The nanoparticles prevent adsorption by introducing a large interacting surface and by obstructing the attachment of the protein to the capillary wall. A one-step procedure based on self-assembly of lipids was used to prepare the nanoparticles, which benefit from their biocompatibility and suspension stability at high concentrations. An aqueous tricine buffer at pH 7.5 containing lipid-based nanoparticles (2% w/w) was used as electrolyte, enabling separation at protein friendly conditions. The developed capillary-based method facilitates future electrochromatography of proteins on polymer-based microchips under physiological conditions and enables the initial optimization of separation conditions in parallel to the chip development.

Circulating MicroRNA Profiling in Non-ST Elevated Coronary Artery Syndrome Highlights Genomic Associations with Serial Platelet Reactivity Measurements.

Changes in platelet physiology are associated with simultaneous changes in microRNA concentrations, suggesting a role for microRNA in platelet regulation. Here we investigated potential associations between microRNA and platelet reactivity (PR), a marker of platelet function, in two cohorts following a non-ST elevation acute coronary syndrome (NSTE-ACS) event. First, non-targeted microRNA concentrations and PR were compared in a case (N = 77) control (N = 76) cohort within the larger TRILOGY-ACS trial. MicroRNA significant in this analysis plus CVD-associated microRNAs from the literature were then quantified by targeted rt-PCR in the complete TRILOGY-ACS cohort (N = 878) and compared with matched PR samples. Finally, microRNA significant in the non-targeted & targeted analyses were verified in an independent post NSTE-ACS cohort (N = 96). From the non-targeted analysis, 14 microRNAs were associated with PR (Fold Change: 0.91-1.27, p-value: 0.004-0.05). From the targeted analysis, five microRNAs were associated with PR (Beta: -0.09-0.22, p-value: 0.004-0.05). Of the 19 significant microRNAs, three, miR-15b-5p, miR-93 and miR-126, were consistently associated with PR in the TRILOGY-ACS and independent Singapore post-ACS cohorts, suggesting the measurement of circulating microRNA concentrations may report on dynamic changes in platelet biology following a cardiovascular ischemic event.

Hydrophobic interaction capillary electrochromatography of protein mutants. Use of lipid-based liquid crystalline nanoparticles as pseudostationary phase.

Nanoparticle-based hydrophobic interaction-capillary electrochromatography was utilized for separation of proteins with similar mass-to-charge ratio at neutral pH without organic modifier. Lipid-based liquid crystalline nanoparticles were prepared and used as pseudostationary phase,benefiting from their high biocompatibility, ease of preparation,and suspension stability at high concentrations.Use of laser-induced fluorescence enabled detection at high nanoparticle concentrations. Green fluorescent protein(GFP) and mutants of GFP harboring single or double amino acid substitutions with the same charge were separated in the described system but not in conventional capillary electrophoresis. Separation was achieved by increasing the salt concentration to promote hydrophobic interactions by shielding of the repulsive electrostatic interactions. In addition, the method was adapted to a capillary with an effective length of 6.7 cm, enabling fast separations and future applications on chip.

A double role for a strictly conserved serine: further insights into the dUTPase catalytic mechanism.

Ser72 at the active site of the Escherichia coli dUTPase has been mutated to an alanine, and the properties of the mutant have been investigated. The serine is absolutely conserved among the monomeric and trimeric dUTPases (including the bifunctional dCTP deaminase:dUTPases), and it has been proposed to promote catalysis by balancing negative charge at the oxygen that bridges the alpha- and beta-phosphorus of the substrate. In all reported complexes of dUTPases with the substrate analogue alpha,beta-imido-dUTP.Mg, the serine beta-OH is indeed hydrogen bonded to the alpha,beta-bridging nitrogen of the analogue. However, in the complex of the Asp90 --> Asn mutant dUTPase with the true substrate dUTP.Mg, the serine beta-OH points in the opposite direction and may form a hydrogen bond to Asn84 at the bottom of the pyrimidine pocket. Here we show that the replacement of the beta-OH by hydrogen reduces k cat from 5.8 to 0.008 s (-1) but also k -1 , the rate of substrate dissociation, from 6.2 to 0.1 s (-1) ( K M = 6 x 10 (-9) M). We conclude that the serine beta-OH exercises both ground state (GS) destabilization and transition state (TS) stabilization, effects not usually linked to a single residue. With experimental support, we argue that the beta-OH destabilizes the GS by imposing conformational constraints on the enzyme and that formation of the TS depends on a rotation of the serine side chain that not only relieves the constraints but brings the beta-OH into a position where it can electrostatically stabilize the TS. This rotation would also allow the beta-OH to promote both deamination and hydrolysis in the bifunctional deaminases. We find that the E. coli dUTPase does not catalyze the hydrolysis of the alpha,beta-imido-dUTP.Mg, suggesting that the analogue provides the hydrogen in the bond to the serine beta-OH.

Factor IXa inhibitors as novel anticoagulants.

Currently available anticoagulants are limited by modest therapeutic benefits, narrow clinical applications, increased bleeding risk, and drug-induced thrombophilia. Because factor IX plays a pivotal role in tissue factor (TF)-mediated thrombin generation, it may represent a promising target for drug development. Several methods of attenuating factor IX activity, including monoclonal antibodies, synthetic active site-blocked competitive inhibitors, oral inhibitors, and RNA aptamers, have undergone investigation. This review summarizes present knowledge of factor IX inhibitors with emphasis on biology, pharmacology, preclinical data, and early-phase clinical experience in humans.

Probing protein surface accessibility of amino acid substitutions using hydrophobic interaction chromatography.

Hydrophobic interaction chromatography (HIC) has been used to determine the influence of amino acid substitutions on protein retention and thereby their accessibility on the protein surface. The retentions of mutants of green fluorescent protein (GFPuv) and human hemoglobin (Hb) were studied on multimodal HIC media and compared to the hydrophobicities from known hydrophobicity scales with respect to the accessible surface area. For GFPuv, the theoretical and experimental results of three hydrophobicity scales correlated well (R(2)>0.85), which clearly indicate that the results can be used for protein retention prediction as well as probing surface properties of protein variants.

Multipurpose peptide tags for protein isolation.

A multifunctional peptide tag (HYDHYD) consisting of histidine, tyrosine and aspartate residues was fused to the N-terminal ends of green fluorescent protein (GFP), lactate dehydrogenase (LDH) and human hemoglobin (Hb), proteins which were subjected to ion-exchange chromatography (IEC), aqueous two-phase system partition, immobilized metal-ion affinity chromatography (IMAC), and hydrophobic interaction chromatography (HIC). Tagged GFP was retained significantly longer (>1 column volume) in both HIC and IEC. It exhibited 3x greater partition in favor of the hydrophobic phase in a two-phase system and 96% could be bound to an IMAC column which did not bind native GFP.

Nucleic acid aptamers as adjuncts to vaccine development.

Nucleic acid 'aptamers', a term derived from the Latin word aptus, 'to fit', are RNA or DNA oligonucleotides that conform to the three-dimensional structure of a selected protein, peptide or small molecules' functional moiety. The 'lock and key' relationship between aptamers and their binding partner permits distinction between closely related but non-identical members of a protein family, or between different functional or conformational states of the same protein. This, along with other properties, separates aptamers from antibodies--the most popular class of molecular recognition tool for the past three decades. Despite the chemical, biological and manufacturing advantages offered by nucleic acid aptamers in a wide variety of conditions, and their generation against a range of clinically relevant targets, including growth factors, transcription factors and coagulation proteins, by two dozen or more companies devoted to the technology platform, only one aptamer, developed for the treatment of wet age-related macular degeneration, is currently available for use in humans. Nevertheless, phase I and II clinical trials for several indications are proceeding with considerable enthusiasm. The potential application of nucleic acid aptamers in novel arenas, including molecular imaging, vaccine development, immunomodulation, decoys for natural RNA-binding events, antiviral therapeutics and both cancer prophylaxis and treatment, is emerging with a pioneering mentality destined to change the paradigm of patient care.

Drug Dosing and Pharmacokinetics in Children With Obesity: A Systematic Review.

IMPORTANCE: Obesity affects nearly one-sixth of US children and results in alterations to body composition and physiology that can affect drug disposition, possibly leading to therapeutic failure or toxic side effects. The depth of available literature regarding obesity's effect on drug safety, pharmacokinetics, and dosing in obese children is unknown. OBJECTIVE: To perform a systematic literature review describing the current evidence of the effect of obesity on drug disposition in children. EVIDENCE REVIEW: We searched the MEDLINE, Cochrane, and EMBASE databases (January 1, 1970-December 31, 2012) and included studies if they contained data on drug clearance, volume of distribution, or drug concentration in obese children (aged ≤18 years). We compared exposure and weight-normalized volume of distribution and clearance between obese and nonobese children. We explored the association between drug physicochemical properties and clearance and volume of distribution. FINDINGS: Twenty studies met the inclusion criteria and contained pharmacokinetic data for 21 drugs. The median number of obese children studied per drug was 10 (range, 1-112) and ages ranged from newborn to 29 years (1 study described pharmacokinetics in children and adults together). Dosing schema varied and were either a fixed dose (6 [29%]) or based on body weight (10 [48%]) and body surface area (4 [19%]). Clinically significant pharmacokinetic alterations were observed in obese children for 65% (11 of 17) of the studied drugs. Pharmacokinetic alterations resulted in substantial differences in exposure between obese and nonobese children for 38% (5 of 13) of the drugs. We found no association between drug lipophilicity or Biopharmaceutical Drug Disposition Classification System class and changes in volume of distribution or clearance due to obesity. CONCLUSIONS AND RELEVANCE: Consensus is lacking on the most appropriate weight-based dosing strategy for obese children. Prospective pharmacokinetic trials in obese children are needed to ensure therapeutic efficacy and enhance drug safety.

Use of the complete blood cell count in early-onset neonatal sepsis.

BACKGROUND: Early-onset sepsis (EOS) is an important cause of morbidity and mortality in neonates, and its diagnosis remains challenging. The complete blood cell count and differential have been previously evaluated as diagnostic tools for EOS in small, single-center reports. We evaluated the diagnostic accuracy of the complete blood cell count and differential in EOS in a large, multicenter population of neonates admitted to the neonatal intensive care unit. METHODS: Using a cohort of 166,092 neonates with suspected EOS with cultures admitted to 293 neonatal intensive care units, we calculated odds ratios and receiver operating characteristic curves for complete blood cell count indices and prediction of a positive culture. We determined sensitivity, specificity and likelihood ratios for various commonly used cutoff values from the complete blood cell count. RESULTS: Low white blood cell counts, low absolute neutrophil counts and high immature-to-total neutrophil ratios were associated with increasing odds of infection (highest odds ratios: 5.38, 6.84 and 7.97, respectively). Specificity and negative predictive values were high (73.7%-99.9% and >99.8%). However, sensitivities were low (0.3%-54.5%) for all complete blood cell count indices analyzed. CONCLUSION: Low white blood cell count, absolute neutrophil count and high immature-to-total neutrophil ratio were associated with increasing odds of infection, but no complete blood cell count-derived index possesses the sensitivity to rule out reliably EOS in neonates.

Use of the complete blood cell count in late-onset neonatal sepsis.

BACKGROUND: Late-onset sepsis is an important cause of morbidity and mortality in infants. Diagnosis of late-onset sepsis can be challenging. The complete blood cell count and differential have been previously evaluated as diagnostic tools for late-onset sepsis in small, single-center reports. OBJECTIVE: We evaluated the diagnostic accuracy of the complete blood cell count and differential in late-onset sepsis in a large multicenter population. STUDY DESIGN: Using a cohort of all infants with cultures and complete blood cell count data from a large administrative database, we calculated odds ratios for infection, as well as sensitivity, specificity, positive and negative predictive values and likelihood ratios for various commonly used cut-off values. RESULTS: High and low white blood cell counts, high absolute neutrophil counts, high immature-to-total neutrophil ratios and low platelet counts were associated with late-onset sepsis. Associations were weaker with increasing postnatal age at the time of the culture. Specificity was highest for white blood cell counts <1000/mm and >50,000/mm (>99%). Positive likelihood ratios were highest for white blood cell counts <1000/mm (4.1) and platelet counts <50,000/mm (3.5). CONCLUSION: No complete blood cell count index possessed adequate sensitivity to reliably rule out late-onset sepsis in this population.

Factor IXa as a target for pharmacologic inhibition in acute coronary syndrome.

Anticoagulant therapy, combined with platelet-directed inhibitors, represents a standard-of-care in the management of patients with acute coronary syndrome, particularly those who require percutaneous coronary interventions. While a vast clinical experience, coupled with large clinical trials have collectively provided guidance, an optimal anticoagulant drug and applied strategy, defined as one that reduces thrombotic and hemorrhagic events consistently, with minimal off-target effects and active control of systemic anticoagulation according to patient and clinical-setting specific need, remains at large. An advancing knowledge of coagulation, hemostasis, and thrombosis suggests that factor IXa, a protease that governs thrombin generation in common thrombotic disorders may represent a prime target for pharmacologic inhibition.

Characterization of multimodal hydrophobic interaction chromatography media useful for isolation of green fluorescent proteins with small structural differences.

Hydrophobic interaction chromatography (HIC) has been developed as a powerful technique for separating and purifying proteins. In this study, we have characterized the ability of new multimodal pH-HIC media to resolve proteins with only small differences in their primary structures. This was done by determining the retention times of different green fluorescent protein (GFP) mutants prepared from Escherichia coli extracts. The mutants, modified with single or double hydrophobic amino acid substitutions in two positions, N212 and T230, could be resolved successfully, up to 2.1 column volumes in retention difference for single substitutions and 2.6 column volumes for double substitutions, at two pH and on two media with varying ligand density. The retention times also correlated well with calculated theoretical retentions (R2=0.91) using a hydrophobic descriptor. This medium can therefore be very useful in a final polishing step during purification and the protein library prepared represents a good screening set in validating and characterizing new future media due to the accessible, but yet, extremely small differences in protein structure.

Antidote-controlled antithrombotic therapy targeting factor IXa and von Willebrand factor.

Thrombotic disorders and their common clinical phenotypes of acute myocardial infarction, ischemic stroke, and venous thromboembolism are the proximate cause of substantial morbidity, mortality, and health care expenditures worldwide. Accordingly, therapies designed to attenuate thrombus initiation and propagation, reflecting integrated platelet-mediated and coagulation protease-mediated events, respectively, represent a standard of care. Unfortunately, there are numerous inherent limitations of existing therapies that include target nonselectivity, variable onset and offset of pharmacodynamic effects, a narrow efficacy-safety profile, and the absence of a safe and reliable platform for either accurate titration, based on existing patient-specific, disease-specific, and clinical conditions, or active reversibility. Herein, we summarize our experience with oligonucleotide antithrombotic agents and their complementary antidotes, targeting the platelet adhesive protein von Willebrand factor and the pivotal coagulation protease factor IXa.

Metal ion accessibility of histidine-modified superfolder green fluorescent protein expressed in Escherichia coli.

Green fluorescent protein (GFP) is frequently utilized for metal ion detection and quantification. To improve the metal binding potential of GFP, three residues (N146, F165, and L201) were substituted to histidines. Each variant responded differently upon interaction with metal ions. More than 80% of N146H, having the most accessible surface area, could bind to immobilized metal ions. However, only F165H exhibited significant differences in quenching by soluble metal ions (22% fluorescence decrease) in comparison with the template protein (12%). These findings can be utilized for designing GFP variants for metal binding and sensor applications.