Major clinical events and healthcare resource use among patients with long-chain fatty acid oxidation disorders in the United States: Results from LC-FAOD Odyssey program.

Major clinical events (MCEs) related to long-chain fatty acid oxidation disorders (LC-FAOD) in triheptanoin clinical trials include inpatient or emergency room (ER) visits for three major clinical manifestations: rhabdomyolysis, hypoglycemia, and cardiomyopathy. However, outcomes data outside of LC-FAOD clinical trials are limited. The non-interventional cohort LC-FAOD Odyssey study examines data derived from US medical records and patient reported outcomes to quantify LC-FAOD burden according to management strategy including MCE frequency and healthcare resource utilization (HRU). Thirty-four patients were analyzed of which 21 and 29 patients had received triheptanoin and/or medium chain triglycerides (MCT), respectively. 36% experienced MCEs while receiving triheptanoin versus 54% on MCT. Total mean annualized MCE rates on triheptanoin and MCT were 0.1 and 0.7, respectively. Annualized disease-related inpatient and ER events were lower on triheptanoin (0.2, 0.3, respectively) than MCT (1.2, 1.0, respectively). Patients were managed more in an outpatient setting on triheptanoin (8.9 annualized outpatient visits) vs MCT (7.9). Overall, this shows that those with LC-FAOD in the Odyssey program experienced fewer MCEs and less HRU in inpatient and ER settings during triheptanoin-treated periods compared with the MCT-treated periods. The MCE rate was lower after initiation of triheptanoin, consistent with clinical trials.

Can a physiotherapy student assume the role of an advanced practice physiotherapist in Orthopaedic surgery triage? A prospective observational study.

BACKGROUND: Advanced practice physiotherapists (APP) have helped improve accessibility to orthopaedic outpatient care. Several studies have validated the APP practice model in orthopaedic care, demonstrating high agreement between APPs and orthopaedic surgeons (OS) regarding diagnosis and management. However, as APPs tend to be experienced senior physiotherapists, such a study involving physiotherapy students (PS) has not yet been explored. The objective of this study was to evaluate the agreement for orthopaedic diagnoses and surgical triage between a PS and OSs. METHODS: A prospective study involving a final year PS and seven OSs was conducted in a university hospital, after the PS had undergone a three-week intensive training. Eighty-six adult patients referred to OSs for knee osteoarthritis, hip osteoarthritis or shoulder problem were independently evaluated by the PS, and then re-evaluated by an OS. The diagnoses and surgical triage recommendations of both clinicians were analyzed for agreement using raw percent agreement and Cohen's kappa. Patient satisfaction with the outpatient clinic experience was noted using a modified version of the Visit-Specific Satisfaction Instrument. RESULTS: Our sample consisted of 86 patients (mean age = 63.4 years). Reasons for consultation included shoulder problems (36%), knee osteoarthritis (52%) and hip osteoarthritis (12%). The raw percent agreement for diagnosis was 95.3%. The agreement for surgical triage was high (κ = 0.86, 95% CI: 0.74-0.98) with a raw agreement of 94.2%. Patient satisfaction was high. CONCLUSIONS: The PS and OSs made similar diagnoses and triage recommendations suggesting that clinical experience alone is not a prerequisite for physiotherapists to help increase accessibility to orthopaedic care.

Identifying Neutrophil Extracellular Traps (NETs) in Blood Samples Using Peripheral Smear Autoanalyzers.

Neutrophil Extracellular Traps (NETs) are large neutrophil-derived structures composed of decondensed chromatin, cytosolic, and granule proteins. NETs play an important role in fighting infection, inflammation, thrombosis, and tumor progression processes, yet their fast and reliable identification has been challenging. Smudge cells (SCs) are a subcategory of white cells identified by CellaVision®, a hematology autoanalyzer routinely used in clinical practice that uses digital imaging to generate "manual" differentials of peripheral blood smears. We hypothesize that a proportion of cells identified in the SC category by CellaVision® Hematology Autoanalyzers are actually NETs. We demonstrate that NET-like SCs are not present in normal blood samples, nor are they an artifact of smear preparation. NET-like SCs stain positive for neutrophil markers such as myeloperoxidase, leukocyte alkaline phosphatase, and neutrophil elastase. On flow cytometry, cells from samples with high percent NET-like SCs that are positive for surface DNA are also positive for CD45, myeloperoxidase and markers of neutrophil activation and CD66b. Samples with NET-like SCs have a strong side fluorescent (SFL) signal on the white count and nucleated red cells (WNR) scattergram, representing cells with high nucleic acid content. When compared to patients with low percent SCs, those with a high percentage of SCs have a significantly higher incidence of documented bacterial and viral infections. The current methodology of NET identification is time-consuming, complicated, and cumbersome. In this study, we present data supporting identification of NETs by CellaVision®, allowing for easy, fast, cost-effective, and high throughput identification of NETs that is available in real time and may serve as a positive marker for a bacterial or viral infections.

Decreased S-nitrosylation of tissue transglutaminase contributes to age-related increases in vascular stiffness.

RATIONALE: Although an age-related decrease in NO bioavailability contributes to vascular stiffness, the underlying molecular mechanisms remain incompletely understood. We hypothesize that NO constrains the activity of the matrix crosslinking enzyme tissue transglutaminase (TG2) via S-nitrosylation in young vessels, a process that is reversed in aging. OBJECTIVE: We sought to determine whether endothelium-dependent NO regulates TG2 activity by S-nitrosylation and whether this contributes to age-related vascular stiffness. METHODS AND RESULTS: We first demonstrate that NO suppresses activity and increases S-nitrosylation of TG2 in cellular models. Next, we show that nitric oxide synthase (NOS) inhibition leads to increased surface and extracellular matrix-associated TG2. We then demonstrate that endothelium-derived bioactive NO primarily mediates its effects through TG2, using TG2(-/-) mice chronically treated with the NOS inhibitor l-N(G)-nitroarginine methyl ester (L-NAME). We confirm that TG2 activity is modulated by endothelium-derived bioactive NO in young rat aorta. In aging rat aorta, although TG2 expression remains unaltered, its activity increases and S-nitrosylation decreases. Furthermore, TG2 inhibition decreases vascular stiffness in aging rats. Finally, TG2 activity and matrix crosslinks are augmented with age in human aorta, whereas abundance remains unchanged. CONCLUSIONS: Decreased S-nitrosylation of TG2 and increased TG activity lead to enhanced matrix crosslinking and contribute to vascular stiffening in aging. TG2 appears to be the member of the transglutaminase family primarily contributing to this phenotype. Inhibition of TG2 could thus represent a therapeutic target for age-associated vascular stiffness and isolated systolic hypertension.

Association Between Treatment Progression, Disease Refractoriness, and Burden of Illness Among Hospitalized Patients With Status Epilepticus.

Importance: Status epilepticus (SE) is associated with poor clinical outcomes and high cost. Increased levels of refractory SE require treatment with additional medications and carry increased morbidity and mortality, but the associations between SE refractoriness, clinical outcomes, and cost remain poorly characterized. Objective: To examine differences in clinical outcomes and costs associated with hospitalization for SE of varying refractoriness. Design, Setting, and Participants: A cross-sectional study of 43 988 US hospitalizations from January 1, 2016 to December 31, 2018, was conducted, including patients with primary or secondary International Statistical Classification of Diseases, Tenth Revision, diagnosis specifying "with status epilepticus." Exposure: Patients were categorized by administration of antiseizure drugs given during hospitalization. Low refractoriness denoted treatment with none or 1 intravenous antiseizure drug. Moderate refractoriness denoted treatment with more than 1 intravenous antiseizure drug. High refractoriness denoted treatment with 1 or more intravenous antiseizure drug, more than 1 intravenous anesthetic, and intensive care unit admission. Main Outcomes and Measures: Outcomes included discharge disposition, hospital length of stay, intensive care unit length of stay, hospital-acquired conditions, and cost (total and per diem). Results: Among 43 988 hospitalizations for SE, 22 851 patients (51.9%) were male; mean age was 49.9 years (95% CI, 49.7-50.1 years). There were 14 694 admissions (33.4%) for low refractory, 10 140 (23.1%) for moderate refractory, and 19 154 (43.5%) for highly refractory SE. In-hospital mortality was 11.2% overall, with the highest rates among patients with highly (18.9%) compared with moderate (6.3%) and low (4.6%) refractory SE (P < .001 for all comparisons). Median hospital length of stay was 5 days (interquartile range [IQR], 2-10 days) with greater length of stay in highly (8 days; IQR, 4-15 days) compared with moderate (4 days; IQR, 2-8 days) and low (3 days; IQR, 2-5 days) refractory SE (P < .001 for all comparisons). Patients with highly refractory SE also had greater hospital costs, with median costs of $25 105 (mean [SD], $41 858 [$59 063]) in the high, $10 592 (mean [SD], $18 328 [$30 776]) in the moderate, and $6812 (mean [SD], $11 532 [$17 228]) in the low refractory cohorts (P < .001 for all comparisons). Conclusions and Relevance: Status epilepticus apparently continues to be associated with a large burden on patients and the US health system, with high mortality and costs that increase with disease refractoriness. Interventions that prevent SE from progressing to a more refractory state may have the potential to improve outcomes and lower costs associated with this neurologic condition.

New-Onset Gout as an Independent Risk Factor for Returning to Dialysis After Kidney Transplantation.

BACKGROUND: The causal relationship between gout and renal transplant outcomes is difficult to assess due to multiple interacting covariates. This study sought to estimate the independent effect of new-onset gout on renal transplant outcomes using a methodology that accounted for these interactions. METHODS: This study analyzed data on patients in the US Renal Data System (USRDS) who received a primary kidney transplant between 2008 and 2015. The exposure was new-onset gout, and the primary endpoint was returning to dialysis >12 months postindex date (transplant date). A marginal structural model (MSM) was fitted to determine the relative risk of new-onset gout on return to dialysis. RESULTS: 18 525 kidney transplant recipients in the USRDS met study eligibility. One thousand three hundred ninety-nine (7.6%) patients developed new-onset gout, and 1420 (7.7%) returned to dialysis >12 months postindex. Adjusting for baseline and time-varying confounders via the MSM showed new-onset gout was associated with a 51% increased risk of return to (RR, 1.51; 95% CI, 1.03-2.20). CONCLUSIONS: This finding suggests that new onset gout after kidney transplantation could be a harbinger for poor renal outcomes, and to our knowledge is the first study of kidney transplant outcomes using a technique that accounted for the dynamic relationship between renal dysfunction and gout.

Insulin Resistance Promotes Parkinson's Disease through Aberrant Expression of α-Synuclein, Mitochondrial Dysfunction, and Deregulation of the Polo-Like Kinase 2 Signaling.

Background: Insulin resistance (IR), considered a hallmark of diabetes at the cellular level, is implicated in pre-diabetes, results in type 2 diabetes, and negatively affects mitochondrial function. Diabetes is increasingly associated with enhanced risk of developing Parkinson's disease (PD); however, the underlying mechanism remains unclear. This study investigated the probable culpability of IR in the pathogenesis of PD. Methods: Using MitoPark mice in vivo models, diabetes was induced by a high-fat diet in the in vivo models, and IR was induced by protracted pulse-stimulation with 100 nM insulin treatment of neuronal cells, in vitro to determine the molecular mechanism(s) underlying altered cellular functions in PD, including mitochondrial dysfunction and α-synuclein (SNCA) aberrant expression. Findings: We observed increased SNCA expression in the dopaminergic (DA) neurons of both the wild-type and diabetic MitoPark mice, coupled with enhanced degeneration of DA neurons in the diabetic MitoPark mice. Ex vivo, in differentiated human DA neurons, IR was associated with increased SNCA and reactive oxygen species (ROS) levels, as well as mitochondrial depolarization. Moreover, we demonstrated concomitant hyperactivation of polo-like kinase-2 (PLK2), and upregulated p-SNCA (Ser129) and proteinase K-resistant SNCA proteins level in IR SH-SY5Y cells, however the inhibition of PLK2 reversed IR-related increases in phosphorylated and total SNCA. Similarly, the overexpression of peroxisome proliferator-activated receptor-γ coactivator 1-alpha (PGC)-1α suppressed ROS production, repressed PLK2 hyperactivity, and resulted in downregulation of total and Ser129-phosphorylated SNCA in the IR SH-SY5Y cells. Conclusions: These findings demonstrate that IR-associated diabetes promotes the development and progression of PD through PLK2-mediated mitochondrial dysfunction, upregulated ROS production, and enhanced SNCA signaling, suggesting the therapeutic targetability of PLK2 and/or SNCA as potential novel disease-modifying strategies in patients with PD.

Dual specificity phosphatase 1/CL100 is a direct transcriptional target of E2F-1 in the apoptotic response to oxidative stress.

E2F-1 mediates apoptosis through transcriptional regulation of its targets. We report here that E2F-1 acts as a direct transcriptional regulator of dual specificity phosphatase 1 (DUSP1; CL100), a threonine and tyrosine phosphatase that inhibits mitogen-activated protein (MAP) kinases. We found that DUSP1 is transcriptionally induced by ectopic E2F-1 expression and that extracellular signal-regulated kinase 1/2 are dephosphorylated in the presence of E2F-1 and DUSP1. E2F-1 mediates apoptosis in the cellular response to oxidative stress. DUSP1 levels are significantly increased in an E2F-1-dependent manner following oxidative stress but not other stresses examined. DUSP1 mediates the cellular response to oxidative stress. We found that E2F-1 binds to chromatin encompassing the DUSP1 promoter and greatly stimulates the promoter activity of the DUSP1 gene. In particular, E2F-1 physically binds to an E2F-1 consensus sequence and a palindromic motif in the DUSP1 promoter. Interestingly, E2F-1 is acetylated following oxidative stress. Our findings show that E2F-1 is a transcriptional activator of DUSP1 and that DUSP1 is a link between E2F-1 and MAP kinases.

Understanding the Factors That Control the Quality of Mini-Tablet Compression: Flow, Particle Size, and Tooling Dimension.

Despite the increasing importance of mini-tablet for its advantages as pediatric formulations and in modified-release applications, its popularity is limited due to the lack of formulation and processing knowledge in developing such dosage forms. In this study, common grades of microcrystalline cellulose and roller compacted granules with a range of powder properties were used to evaluate the critical material properties required for the successful manufacturing of 1.7-mm mini-tablets. It was found that blends with small particle size had poor flow properties that did not support consistent die filling and also tended to cause tooling jam and damage. While the granulation process was effective in improving blend flow properties by increasing particle size, it is imperative to avoid very large particles that could also cause inadequate flow by blocking the space within the die. Successful mini-tablet compression could be achieved by removing particles larger than roughly 1/3 of the die diameter or milling the granules using a screen less than 1/3 of the die diameter.

Defective recruitment of motor proteins to autophagic compartments contributes to autophagic failure in aging.

Inability to preserve proteostasis with age contributes to the gradual loss of function that characterizes old organisms. Defective autophagy, a component of the proteostasis network for delivery and degradation of intracellular materials in lysosomes, has been described in multiple old organisms, while a robust autophagy response has been linked to longevity. The molecular mechanisms responsible for defective autophagic function with age remain, for the most part, poorly characterized. In this work, we have identified differences between young and old cells in the intracellular trafficking of the vesicular compartments that participate in autophagy. Failure to reposition autophagosomes and lysosomes toward the perinuclear region with age reduces the efficiency of their fusion and the subsequent degradation of the sequestered cargo. Hepatocytes from old mice display lower association of two microtubule-based minus-end-directed motor proteins, the well-characterized dynein, and the less-studied KIFC3, with autophagosomes and lysosomes, respectively. Using genetic approaches to mimic the lower levels of KIFC3 observed in old cells, we confirmed that reduced content of this motor protein in fibroblasts leads to failed lysosomal repositioning and diminished autophagic flux. Our study connects defects in intracellular trafficking with insufficient autophagy in old organisms and identifies motor proteins as a novel target for future interventions aiming at correcting autophagic activity with anti-aging purposes.

Reduction of organelle motility by removal of potassium and other solutes.

There are surprisingly few studies that describe how the composition of cell culture medium may affect the trafficking of organelles. Here we utilize time lapse multi-channel fluorescent imaging to show that short term exposure of Huh-7 cells to medium lacking potassium, sodium, or chloride strongly reduces but does not eliminate the characteristic back and forth and cell-traversing movement of fluorescent EGF (FL-EGF) containing organelles. We focused on potassium because of its relatively low abundance in media and serum and its energy requiring accumulation into cells. Upon exposure to potassium free medium, organelle motility declined steadily through 90 min and then persisted at a low level. Reduced motility was confirmed in 5 independent cell lines and for organelles of the endocytic pathway (FL-EGF and Lysotracker), autophagosomes (LC3-GFP), and mitochondria (TMRE). As has been previously established, potassium free medium also inhibited endocytosis. We expected that diminished cellular metabolism would precede loss of organelle motility. However, extracellular flux analysis showed near normal mitochondrial oxygen consumption and only a small decrease in extracellular acidification, the latter suggesting decreased glycolysis or proton efflux. Other energy dependent activities such as the accumulation of Lysotracker, TMRE, DiBAC4(3), and the exclusion of propidium iodide remained intact, as did the microtubule cytoskeleton. We took advantage of cell free in vitro motility assays and found that removal of potassium or sodium from the reconstituted cytosolic medium decreased the movement of endosomes on purified microtubules. The results indicate that although changes in proton homeostasis and cell energetics under solute depletion are not fully understood, potassium as well as sodium appear to be directly required by the motile machinery of organelles for optimal trafficking.

Protective effects of aspirin from cardiac hypertrophy and oxidative stress in cardiomyopathic hamsters.

OBJECTIVE: To evaluate the capacity of chronic ASA therapy to prevent cardiac alterations and increased oxidative stress in cardiomyopathic hamsters. METHODS AND RESULTS: Male Syrian cardiomyopathic and age-matched inbred control hamsters received ASA orally from the age of 60 days. Animals were sacrificed at the age of 150, 250, and 350 days to evaluate the time course of cardiac hypertrophy and cardiovascular tissue superoxide anion (O(2)(-)) production. At the age of 150 days, the ventricular weight over body weight ratio, resting heart rate, and cardiovascular O(2)(-) production were much higher in cardiomyopathic hamsters than those in control. At the age of 250 days, in addition to the continual deterioration of these parameters with age, the blood pressure started to fall and the signs of heart failure appeared. In these cardiomyopathic hamsters, chronic ASA treatment (a) completely prevented elevated O(2)(-) production and the NAD(P)H oxidase activity, (b) significantly slowed down the development of the cardiac hypertrophy and fibrosis. CONCLUSIONS: Chronic ASA treatment significantly prevents the deterioration of cardiac function and structure as well as the increased oxidative stress in the cardiomyopathic hamster. Our findings suggest that ASA presents a therapeutic potential to prevent cardiac dysfunction.

Vocal competition in male Xenopus laevis frogs.

Male Xenopus laevis frogs produce underwater advertisement calls that attract gravid females and suppress calling by male competitors. Here we explore whether groups of males establish vocal ranks and whether auditory cues alone suffice for vocal suppression. Tests of male-male pairs within assigned groups reveal linear vocal dominance relations, in which each male has a defined rank. Both the duration over which males interact, as well as the number of competitive opportunities, affect linearity. Linear dominance across the group is stable for about 2 weeks; rank is dynamic. Males engage in physical interactions (clasping) while paired but clasping and vocal rank are not correlated. Playbacks of advertisement calls suppress calling and calls from high- and low-ranking males are equally effective. Thus, auditory cues alone suffice to suppress vocal behavior. Playback intensities equivalent to a nearby male advertising effectively suppress calling while low-intensity playbacks are either ineffective or stimulate vocal behavior. X. laevis advertisement calls are biphasic, composed of alternating fast and slow click trills. Approximately half the males tested are more vocally suppressed by all slow than by all fast trills; thus, these males can distinguish between the two phases. The fully aquatic family Pipidae diverged from terrestrial ancestors approximately 170 mya. Vocal suppression in the X. laevis mating system may represent the translation of an ancient anuran social strategy to underwater life.