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Abstract Background Periprosthetic joint infection (PJI) is often managed using a combination of medical and surgical treatments, including irrigation and debridement or joint resection, spacer placement, and then revision to reimplant the arthroplasty components. The timing between surgical stages may vary, and little information exists about the clinical and microbiological factors impacting these temporal relationships. Methods A single center cohort study was conducted. The cohort included all cases of PJI for which resection was performed at the University of Iowa Health Care (UIHCs) between January 1, 2009, and December 31, 2016. Using a 1-year follow-up period, we constructed Kaplan-Meier estimates of the time to revision after resection—overall and then stratified by joint location (knee vs. hip), organism identification (known vs unknown), and C-reactive protein level (CRP >10 mg/L vs. CRP ≤10 mg/L). We then used Cox proportional hazard modeling to identify factors associated with revision within 1 year. Results Among a total of 109 cases of prosthetic joint resections (n = 67 knee, n = 42 hip), 56.9% underwent revision surgery during the follow-up period (n = 46 knee, n= 16 hip). Most revisions occurred between 2.5 and 3.0 months after resection. Knee arthroplasties were significantly more likely to undergo revision during the study period (hazard ratio, HR = 2.53, 95% confidence interval, CI: 1.38, 4.94) compared to hip arthroplasties. Identification of one or more organism involved in the PJI was also associated with revision during the study window (HR = 3.56, 95% CI: 1.94, 6.47), whereas elevated CRP was associated with lower rates of revision during the study period (HR = 0.21, 95% CI: 0.09, 0.46 for CRP >10 mg/L compared to CRP ≤10 mg/L). Figure 1. Kaplan-Meier curves for revision within 1-year after resection for the (A) cohort overall and then stratified by (B) joint location (hip vs knee), (C) organism identification (known vs unknown), and (D) C-reactive protein level (CRP >10 mg/L vs. CRP ≤10 mg/L). Conclusion For patients with surgically managed PJIs, joint location, organism identification, and lower CRP level were all associated with revision procedures within 1-year after resection. These findings may reflect mechanical and microbiological factors influencing the decision to proceed with revision. The variable length between stages—and thus, the variable time during which a spacer is retained—may be important to measure in future observational and clinical studies examining PJI outcomes. Disclosures All Authors: No reported disclosures

Abstract Background HIV knowledge and prevention strategies, like PrEP/PEP is a core element in current medical practice but represents a challenge for medical students’ curricula. In countries like Dominican Republic (DR), the stigma towards HIV infection restricts the delivery of HIV related information to the general population. Our study aims to understand the level of knowledge between different academical years pose regarding HIV/AIDS, PrEP and PEP and how their curricula content is covered across the years. Methods We survey medical students in the DR to measure knowledge on HIV, PrEP/PEP. Students who agreed to participate filled in a survey with 3 sections. Each section had 10 true or false questions. Responses were compared mean knowledge for each topic based on a score out of 10. We analyzed mean scores using ANOVA with Post Hoc analysis, assuming significance when p< 0.05. Results A total of 503 participants participated in the survey. ANOVA results showed that there was a statistically significant difference in mean HIV knowledge (F (3, 499) = [25,58], p< 0.001) and PEP knowledge (F (3, 499) = [13,77], p< 0.001) between the academical years. Post-hoc analysis revealed that MS4 had a higher mean score for HIV knowledge (M= 9.3) than MS2 (8.7, p< 0.01) and MS1 (m= 8.2, p< 0.01). Similarly, MS3 had a higher mean HIV knowledge than MS2 (p< 0.01) and MS1 (p< 0.01). Likewise, MS2 had an increased mean HIV knowledge when compared to MS1 (p< 0.01). When we compare mean PEP knowledge, we found that MS3 (m= 7.45) had increased knowledge than MS1 (6.4, p< 0.01) and MS2 (m= 6.9, p< 0.05). Similarly, we found that MS4 had increased mean PEP Knowledge (m= 7.5) when compared to MS1 and MS2 (p< 0.001). When we studied mean PrEP knowledge, ANOVA analysis found no significant difference (F (3, 499) = [1,24], p= 0.29). Conclusion Our study suggests that the mean knowledge of HIV and PEP increases throughout the academic progression of medical school. Although there is no difference in PEP knowledge, this may suggest that medical schools are teaching PEP during MS3-MS4 cycle rather than the M1-M2 cycle. Also, the homogenous mean PrEP knowledge could suggest that the students are learning from non-academic sources. This emphasizes the curricular designers of medical schools to review the contents on prevention of communicable diseases. Disclosures All Authors: No reported disclosures

Background Caveolin‐1 is a structural protein of caveolae which forms a signaling platform for several cell surface receptors including leptin and insulin receptor. Caveolin‐1 expression is increased with obesity which is implicated in impaired adipose tissue leptin signaling. Considering the overlap between insulin and leptin cellular signaling pathways, we investigated known insulin‐sensitizing drugs which may reduce caveolin‐1 expression in adipose tissue and consequently improve leptin signaling. Methods We used an in‐vitro approach to identify insulin‐sensitizing drugs which may reduce caveolin‐1 expression. Differentiated human white preadipocytes (dHWP) were treated with pioglitazone (5–30 μM), troglitazone (10–30 μM), metformin (1–5 mM), or glimepiride (20–100 μM) for 24 hours. Caveolin‐1 expression was determined by Western blot. We further tested the responses in human adipose tissue from obese individuals (n=12, age: 45.4 ± 9.2 years, BMI: 48.9 ± 7.3 kg/m 2 ) undergoing surgery. Tissue was incubated with drugs for 24 h and caveolin‐1 expression was determined. Furthermore, the effect of drugs on leptin‐dependent activation of ERK and STAT3 was examined by western blot. Results Among the drugs investigated, only treatment with increasing concentrations of metformin resulted in a dose‐dependent decease in caveolin‐1 protein (p=0.01) expression in dHWP. Treatment with increasing concentrations of troglitazone upregulated caveolin‐1 (p=0.01), while pioglitazone (p=0.50) and glimepiride (p=0.09) had no effect of caveolin‐1 protein expression. Given these findings, only metformin was tested in human adipose tissue. Overall, metformin did not alter caveolin‐1 expression in adipose tissue from obese individuals (n=12, control: 0.040 ± 0.056, metformin: 0.024 ± 0.030, p=0.57), although a metformin dependent decrease in caveolin‐1 was evident in adipose tissue from 7 of the 12 obese subjects (control: 0.062 ± 0.066, metformin: 0.014 ± 0.013, p=0.02). Notably, changes in caveolin‐1 expression induced by metformin treatment were positively correlated with BMI (p=0.05, r 2 =0.33, n=12). Also, leptin dependent activation of ERK (control: 0.26 ± .098, leptin: 0.28 ± .08) and STAT3 (control: 0.028 ± 0.027, leptin: 0.032 ± 0.023) was impaired in obese adipose tissue and did not change with metformin treatment even in the 7 individuals with metformin associated decreases in caveolin‐1. Conclusions Metformin reduces caveolin‐1 expression in differentiated HWP. However, metformin decreases caveolin‐1 expression in adipose tissue of some but not all obese subjects, and does not have any effects on leptin signaling. The heterogeneous effects of metformin on caveolin‐1 expression in adipose tissue from obese individuals may be related to BMI and needs further investigation. Support or Funding Information MAP and KRS were supported by the American Physiological Society Stride Summer Undergraduate Research Fellowship NHLBI 1 R25 HL115473‐01. PS was supported by AHA scientist development grant 11SDG7260046.

Monoamine Oxidase (MAO), an outer mitochondrial enzyme, catalyzes the oxidative deamination of neurotransmitters and biological amines, and in the process, produces a reactive aldehyde and H2O2. Our lab has recently shown that MAO activity (i.e., H2O2 production) in human right atrial myocardium is associated with postoperative atrial fibrillation. Furthermore, our lab has shown that diabetic patients have higher MAO‐A and ‐B content and rates of activity in this tissue, compared to nondiabetics. While recent studies have explored the connection between MAO and cardiac dysfunction, the chamber‐specific content and activity of MAO within the heart is still unknown. Therefore, we characterized the regional enzymatic activity of MAO‐A and ‐B based upon the hypothesis that the atria would have the highest MAO activity due to the greater density of local sympathetic nerve innervation in this region. For this study, porcine hearts were isolated from domestic pigs, as this model closely recapitulates the physiology of the human heart. One group of pigs were fed a high fat, high sucrose diet for 16 weeks to induce obesity/metabolic syndrome (n=4). Three MAO substrates (dopamine, norepinephrine, serotonin) and two isoform‐selective inhibitors (clorgyline=MAO‐A, deprenyl=MAO‐B) were used to characterize MAO activities. Enzyme content was measured using ELISA and confirmed with immunoblots. Kinetic analysis showed that, across all substrates, when normalized to tissue protein, the left atrium has the highest maximal MAO‐A and –B activity while the other three chambers are similar. Furthermore, maximal MAO activity in the left ventricle of pigs with metabolic syndrome is significantly greater (P<0.05) than left ventricle of control group. Our findings suggest that MAO activity differs by location in the heart (i.e., chamber heterogeneity). This information will be useful to inform future experimental and clinical pharmacology applications where MAO inhibitors might be used. Moreover, increased MAO in left ventricle of pigs with obesity/metabolic syndrome is important as it provides a rationale for exploring MAO inhibitors as therapeutic strategy to mitigate cardiac complications of diabetes. Support or Funding Information R01HL122863

Mesoporous silica nanoparticles coated with a lipid bilayer (protocells) are a stable, versatile and biocompatible delivery platform that allows surface engineering to target specific cells and intracellular cargo delivery. The endo‐lysosomal escape of this type of drug carriers is important to enhance the therapeutic efficacy of their cargo. Motoneurons can be specifically targeted by surface‐coating protocells with the subunit B of cholera toxin (CTB‐protocells). The central hypothesis was that CTB‐protocells escape endo‐lysosomal compartments after internalization by motoneurons. In a first study, fixed NSC‐34 motoneuron‐like cells were treated with fluorescently labeled CTB‐protocells (for 1, 3, 6 and 24 hours), immunostained for lysosomes (with anti‐LAMP1) or early endosomes (with anti‐EEA1) and imaged using multicolor confocal microscopy. In a second study, NSC‐34 cells treated with CTB‐protocells and labeled with the acidic vesicle tracker LysoTracker were visualized using time lapse confocal imaging. In these two studies, colocalization of CTB‐protocells with lysosomes or endosomes was analyzed using ImageJ and found to remain less than 20% up to 24 hours. The fraction of CTB‐protocells in endo‐lysosomes increased only slightly between 1 and 24 hours. In a third study, transmission electron microscopy was used to confirm the intracellular localization of CTB‐protocells in NSC‐34 motoneuron‐like cells. CTB‐protocells were visualized in the cytosol and were not detected within larger vacuole compartments after 24‐hour incubation. In conclusion, CTB‐protocells escape lysosomal degradation in motoneurons making them a suitable vehicle to target drug delivery to motoneurons.

Cholera toxin B modified protocells (CTB‐protocells) are a promising, customizable approach for targeting and delivering drugs to motoneurons. Understanding the endocytic mechanism and intracellular fate of CTB‐protocells is essential for safe therapeutic application and cargo delivery efficiency, and also to elucidate characteristics of motoneuron physiology that are not well understood. To assess the energy dependent uptake of CTB‐protocells by motoneurons, intracellular protocell fluorescence was measured at 4°C and 37°C. Confocal analysis demonstrated that after 3 h at 4°C, there is a 40% reduction in CTB‐protocell uptake compared to the uptake observed at 37°C (p<0.001). Pharmacological endocytic pathway inhibitors were used to elucidate the preferential endocytotic mechanism. Results showed that amiloride (macropinocytosis inhibitor) and methyl‐β‐cyclodextrin (inhibitor of cholesterol dependent endocytic processes) significantly reduced the uptake of CTB‐protocells (p<0.001), consistent with endocytosis being dependent on the integrity of lipid rafts. To track intracellular fate, immunofluorescence techniques and live confocal microscopy were used to demonstrate colocalization of CTB‐protocells with lysosomes, endoplasmic reticulum (ER) and Golgi apparatus at 1 h and 3 h. Colocalization of CTB‐protocells with lysosomes was less than 20%. Both, ER and Golgi colocalization increased from ~20 to ~40% in a 2‐h period (from 1 h to 3 h). To evaluate retrograde transport in motoneurons in vivo , CTB‐protocells were intrapleurally injected in rats. Evidence of protocells in phrenic nerve axons measured by protocell fluorescence intensity supported retrograde transport. In conclusion, uptake of CTB‐protocells is energy‐dependent and mediated principally by macropinocytosis. Additionally, CTB‐protocells are able to escape lysosomal degradation, and are trafficked to relevant subcellular compartments after endocytosis.

The diaphragm muscle (DIAm) must be able to generate sufficient forces to sustain ventilatory and non‐ventilatory behaviors throughout the lifespan. Aging‐related decreases in DIAm fiber cross‐sectional area and specific force, i.e., sarcopenia, may impair the ability to generate transdiaphragmatic pressures (Pdi) necessary to accomplish expulsive, non‐ventilatory behaviors essential for airway clearance. We examined the effects of aging on DIAm function in mice at 6, 18, and 24 months of age. At each age, DIAm‐phrenic nerve preparations were used to determine maximal force by ex vivo contractility of the DIAm as well as neuromuscular transmission failure by repetitive nerve and muscle stimulation. By 18 months, animals had reduced Pdi during maximal forces (e.g., during tracheal occlusion) and increased DIAm neuromuscular transmission failure compared to 6 months, but no change in DIAm maximum specific force. By 24 months, Pdi remained reduced during occlusion, DIAm neuromuscular transmission failure remained increased compared to 6 months, and DIAm maximum specific force was decreased compared to both 6 and 18 months. Using this global measure of neuromuscular transmission, it is evident that impaired transmission precedes muscle weakness. Support or Funding Information Supported by R01‐AG044615.

Neuromotor control of respiratory muscles such as the diaphragm muscle (DIAm), the main inspiratory muscle in mammals, is organized around motor units. Motor units comprise a motoneuron and the muscle fibers they innervate. Diversity in the contractile and fatigue properties of DIAm motor units permits generation of the wide range of behaviors necessary to sustain ventilation and maintain airways patent (e.g., sighs, which generate ~70% of maximal force in rats). Descending inputs to phrenic motoneurons innervating the DIAm is thought to be primarily unilateral in rats. After disrupting ipsilateral inputs by unilateral spinal cord hemisection at C2 (C2SH), latent, bilateral projections underlie recovery of ipsilateral DIAm activity. We hypothesized that DIAm EMG activity reflects bilateral descending premotor drive to motoneurons that varies across motor behaviors and conditions (anesthetized vs. awake). Using chronic DIAm EMG recordings in adult male Sprague‐Dawley rats, the bilateral vs. ipsilateral nature of premotor drive for each behavior and condition was evaluated by comparing the impact of C2SH. Three days prior to the C2SH surgery, a pair of DIAm electrodes was implanted bilaterally and DIAm EMG activity was assessed immediately prior to and after C2SH as well as at 3 days post‐C2SH in spontaneously‐breathing awake, restrained animals and following induction of ketamine‐xylazine anesthesia. DIAm EMG recordings comprised ~5 min of eupnea and exposure to hypoxia (10% O 2 )‐hypercapnia (5% CO 2 ). The completeness of C2SH surgery was confirmed by the absence of ipsilateral diaphragm EMG activity during eupnea at the time of C2SH surgery and at 3 days post‐C2SH in anesthetized animals, consistent with previous studies. In addition, histological assessment of disruption of anterolateral white matter tracts was conducted to verify the C2SH injury. Ipsilateral RMS EMG amplitude in anesthetized rats pre‐C2SH was ~30% and 45% of pre‐injury anesthetized sigh levels during eupnea and hypoxia‐hypercapnia, respectively. During awake conditions pre‐C2SH, ipsilateral RMS EMG amplitude was significantly higher (~50%, 70% and 200% of pre injury anesthetized sigh levels during eupnea, hypoxia‐hypercapnia and sighs, respectively). There were no differences across diaphragm muscle sides pre‐C2SH in RMS EMG amplitude. At 3 days post‐C2SH, ipsilateral RMS EMG activity was evident in anesthetized animals during hypoxia‐hypercapnia and sighs (~10% and 50% of pre‐injury anesthetized sigh levels) and in awake animals during eupnea, hypoxia‐hypercapnia and sighs (~25%, 50% and 125% of pre‐injury anesthetized sigh levels, respectively). These findings represent considerable differences in DIAm activity across motor behaviors and anesthetized vs. awake conditions and highlight physiologically‐relevant differences in the relative contribution of ipsilateral vs. bilateral inputs to phrenic motoneurons. Support or Funding Information NIH grant R01 HL96750.

Autophagy is a series of catabolic processes that involves degradation of cytoplasmic components through lysosomal pathways. There is an important balance that must be met, with impaired autophagy contributing to cell dysfunction and excessive autophagy leading to cell death likely via apoptosis. Indeed autophagy is a dynamic multi‐step process that is modulated positively and negatively at several steps. The role of autophagy in aging‐related motoneuron dysfunction or death is not known. Therefore, it is important to differentiate between the accumulation of autophagosomes due to increased induction of autophagy during conditions of cell stress common across aging‐related conditions, and the accumulation of autophagosomes due to inefficient clearance of damaged organelles and other sequestered cargoes, which reflects impairments in autophagy that would lead to cell dysfunction. We hypothesized that with aging, there is autophagy imbalance causing accumulation of autophagosomes and ultimately neuromuscular dysfunction. Male and female mice at 6 and 24 months of age were used to assess markers of autophagy in the spinal cord. Protein expression of autophagy‐related genes LC3A and LC3B increased in the cervical and lumbar spinal cord of old compared to young mice, whereas protein expression of Atg5, Atg7, and Bcn1 did not change with age. Quantitative real‐time RT‐PCR of microdissected phrenic motoneurons showed an increase in LC3 mRNA expression with age. Overall autophagic flux, which assesses inclusion (or exclusion) of cargo within autophagosomes, delivery of cargo to lysosomes, and subsequent breakdown and release of macromolecules back into the cytosol was evaluated using an mCherry‐GFP‐LC3 sensor. This genetic sensor is designed to monitor LC3 flux through autophagy pathways since the GFP signal is sensitive to the acidic/proteolytic conditions of the lysosome lumen, whereas mCherry fluorescence persists under these environments. We used motoneuron‐like NSC‐34 cells transfected with mCherry‐GFP‐LC3 plasmid to detect changes in the autophagic degradation rate in cells treated with tunicamycin (which leads to cell stress via induction of the unfolded protein response). We observed an increase in yellow vesicles indicating an accumulation of autophagosomes due to inefficient clearance of sequestered cargoes. This genetic sensor indicates impaired autophagy flux in motoneurons in conditions of elevated stress likely present in old age. Taken together, these results suggest impaired autophagy in motoneurons, including phrenic motoneurons, with an associated increase in markers of early autophagosome formation (LC3). Support or Funding Information Supported by R01‐AG044615.