CRL4(WDR23)-Mediated SLBP Ubiquitylation Ensures Histone Supply during DNA Replication.

To maintain genome integrity and epigenetic information, mammalian cells must carefully coordinate the supply and deposition of histones during DNA replication. Here we report that the CUL4 E3 ubiquitin ligase complex CRL4(WDR23) directly regulates the stem-loop binding protein (SLBP), which orchestrates the life cycle of histone transcripts including their stability, maturation, and translation. Lack of CRL4(WDR23) activity is characterized by depletion of histones resulting in inhibited DNA replication and a severe slowdown of growth in human cells. Detailed analysis revealed that CRL4(WDR23) is required for efficient histone mRNA 3' end processing to produce mature histone mRNAs for translation. CRL4(WDR23) binds and ubiquitylates SLBP in vitro and in vivo, and this modification activates SLBP function in histone mRNA 3' end processing without affecting its protein levels. Together, these results establish a mechanism by which CUL4 regulates DNA replication and possible additional chromatin transactions by controlling the concerted expression of core histones.

Risk factors and health behaviors associated with loneliness among cancer survivors during the COVID-19 pandemic.

Loneliness may exacerbate poor health outcomes particularly among cancer survivors during the COVID-19 pandemic. Little is known about the risk factors of loneliness among cancer survivors. We evaluated the risk factors of loneliness in the context of COVID-19 pandemic-related prevention behaviors and lifestyle/psychosocial factors among cancer survivors. Cancer survivors (n = 1471) seen at Huntsman Cancer Institute completed a survey between August-September 2020 evaluating health behaviors, medical care, and psychosocial factors including loneliness during COVID-19 pandemic. Participants were classified into two groups: 'lonely' (sometimes, usually, or always felt lonely in past month) and 'non-lonely' (never or rarely felt lonely in past month). 33% of cancer survivors reported feeling lonely in the past month. Multivariable logistic regression showed female sex, not living with a spouse/partner, poor health status, COVID-19 pandemic-associated lifestyle factors including increased alcohol consumption and marijuana/CBD oil use, and psychosocial stressors such as disruptions in daily life, less social interaction, and higher perceived stress and financial stress were associated with feeling lonely as compared to being non-lonely (all p < 0.05). A significant proportion of participants reported loneliness, which is a serious health risk among vulnerable populations, particularly cancer survivors. Modifiable risk factors such as unhealthy lifestyle behaviors and psychosocial stress were associated with loneliness. These results highlight the need to screen for unhealthy lifestyle factors and psychosocial stressors to identify cancer survivors at increased risk of loneliness and to develop effective management strategies.

The impact of genomic variation on protein phosphorylation states and regulatory networks.

Genomic variation impacts on cellular networks by affecting the abundance (e.g., protein levels) and the functional states (e.g., protein phosphorylation) of their components. Previous work has focused on the former, while in this context, the functional states of proteins have largely remained neglected. Here, we generated high-quality transcriptome, proteome, and phosphoproteome data for a panel of 112 genomically well-defined yeast strains. Genetic effects on transcripts were generally transmitted to the protein layer, but specific gene groups, such as ribosomal proteins, showed diverging effects on protein levels compared with RNA levels. Phosphorylation states proved crucial to unravel genetic effects on signaling networks. Correspondingly, genetic variants that cause phosphorylation changes were mostly different from those causing abundance changes in the respective proteins. Underscoring their relevance for cell physiology, phosphorylation traits were more strongly correlated with cell physiological traits such as chemical compound resistance or cell morphology, compared with transcript or protein abundance. This study demonstrates how molecular networks mediate the effects of genomic variants to cellular traits and highlights the particular importance of protein phosphorylation.

Early steps in autophagy depend on direct phosphorylation of Atg9 by the Atg1 kinase.

Bulk degradation of cytoplasmic material is mediated by a highly conserved intracellular trafficking pathway termed autophagy. This pathway is characterized by the formation of double-membrane vesicles termed autophagosomes engulfing the substrate and transporting it to the vacuole/lysosome for breakdown and recycling. The Atg1/ULK1 kinase is essential for this process; however, little is known about its targets and the means by which it controls autophagy. Here we have screened for Atg1 kinase substrates using consensus peptide arrays and identified three components of the autophagy machinery. The multimembrane-spanning protein Atg9 is a direct target of this kinase essential for autophagy. Phosphorylated Atg9 is then required for the efficient recruitment of Atg8 and Atg18 to the site of autophagosome formation and subsequent expansion of the isolation membrane, a prerequisite for a functioning autophagy pathway. These findings show that the Atg1 kinase acts early in autophagy by regulating the outgrowth of autophagosomal membranes.

Characterization of the chondrocyte secretome in photoclickable poly(ethylene glycol) hydrogels.

Poly(ethylene glycol) (PEG) hydrogels are highly tunable platforms that are promising cell delivery vehicles for chondrocytes and cartilage tissue engineering. In addition to characterizing the type of extracellular matrix (ECM) that forms, understanding the types of proteins that are secreted by encapsulated cells may be important. Thus, the objectives for this study were to characterize the secretome of chondrocytes encapsulated in PEG hydrogels and determine whether the secretome varies as a function of hydrogel stiffness and culture condition. Bovine chondrocytes were encapsulated in photoclickable PEG hydrogels with a compressive modulus of 8 and 46 kPa and cultured under free swelling or dynamic compressive loading conditions. Cartilage ECM deposition was assessed by biochemical assays and immunohistochemistry. The conditioned medium was analyzed by liquid chromatography-tandem mass spectrometry. Chondrocytes maintained their phenotype within the hydrogels and deposited cartilage-specific ECM that increased over time and included aggrecan and collagens II and VI. Analysis of the secretome revealed a total of 64 proteins, which were largely similar among all experimental conditions. The identified proteins have diverse functions such as biological regulation, response to stress, and collagen fibril organization. Notably, many of the proteins important to the assembly of a collagen-rich cartilage ECM were identified and included collagen types II(α1), VI (α1, α2, and α3), IX (α1), XI (α1 and α2), and biglycan. In addition, many of the other identified proteins have been reported to be present within cell-secreted exosomes. In summary, chondrocytes encapsulated within photoclickable PEG hydrogels secrete many types of proteins that diffuse out of the hydrogel and which have diverse functions, but which are largely preserved across different hydrogel culture environments. Biotechnol. Bioeng. 2017;114: 2096-2108. © 2017 Wiley Periodicals, Inc.

Phosphoproteomic analyses reveal novel cross-modulation mechanisms between two signaling pathways in yeast.

Cells respond to environmental stimuli via specialized signaling pathways. Concurrent stimuli trigger multiple pathways that integrate information, predominantly via protein phosphorylation. Budding yeast responds to NaCl and pheromone via two mitogen-activated protein kinase cascades, the high osmolarity, and the mating pathways, respectively. To investigate signal integration between these pathways, we quantified the time-resolved phosphorylation site dynamics after pathway co-stimulation. Using shotgun mass spectrometry, we quantified 2,536 phosphopeptides across 36 conditions. Our data indicate that NaCl and pheromone affect phosphorylation events within both pathways, which thus affect each other at more levels than anticipated, allowing for information exchange and signal integration. We observed a pheromone-induced down-regulation of Hog1 phosphorylation due to Gpd1, Ste20, Ptp2, Pbs2, and Ptc1. Distinct Ste20 and Pbs2 phosphosites responded differently to the two stimuli, suggesting these proteins as key mediators of the information exchange. A set of logic models was then used to assess the role of measured phosphopeptides in the crosstalk. Our results show that the integration of the response to different stimuli requires complex interconnections between signaling pathways.

The development of a culturally relevant, theoretically driven HPV prevention intervention for urban adolescent females and their parents/guardians.

Human papillomavirus (HPV) is the most common sexually transmitted infection in the United States, accounting for the large majority of cervical cancer and anogenital warts cases. Two HPV vaccines are currently licensed and recommended for women and girls. However, vaccination rates have been suboptimal, with evidence of disparities influencing both uptake and series completion among African American and Hispanic adolescents. There has been a dearth of theory-based, behavioral interventions targeted to prevent HPV infection and increase HPV vaccine uptake among urban adolescents. This article describes the development of two skills-based intervention curricula aimed to increase HPV prevention and vaccination among low-income urban adolescent females 9 to 18 years old. Guided by the theory of planned behavior, elicitation research was conducted to elucidate the social psychological factors that underlie HPV vaccination intentions (N = 141). The findings were subsequently used to identify theoretical mediators of behavioral change to drive the intervention. Culturally relevant strategies to promote HPV vaccination were translated into the curricula content. Both curricula were designed to motivate and empower participants to reduce risk of being infected with HPV. Targeting theoretical mediators of behavioral change, derived from the voices of the community, may prove to be successful in increasing HPV vaccination and preventing HPV.

The Integration of Proteogenomics and Ribosome Profiling Circumvents Key Limitations to Increase the Coverage and Confidence of Novel Microproteins.

There has been a dramatic increase in the identification of non-conical translation and a significant expansion of the protein-coding genome and proteome. Among the strategies used to identify novel small ORFs (smORFs), Ribosome profiling (Ribo-Seq) is the gold standard for the annotation of novel coding sequences by reporting on smORF translation. In Ribo-Seq, ribosome-protected footprints (RPFs) that map to multiple sites in the genome are computationally removed since they cannot unambiguously be assigned to a specific genomic location, or to a specific transcript in the case of multiple isoforms. Furthermore, RPFs necessarily result in short (25-34 nucleotides) reads, increasing the chance of ambiguous and multi-mapping alignments, such that smORFs that reside in these regions cannot be identified by Ribo-Seq. Here, we show that the inclusion of proteogenomics to create a Ribosome Profiling and Proteogenomics Pipeline (RP3) bypasses this limitation to identify a group of microprotein-encoding smORFs that are missed by current Ribo-Seq pipelines. Moreover, we show that the microproteins identified by RP3 have different sequence compositions from the ones identified by Ribo-Seq-only pipelines, which can affect proteomics identification. In aggregate, the development of RP3 maximizes the detection and confidence of protein-encoding smORFs and microproteins.

Profiling mouse brown and white adipocytes to identify metabolically relevant small ORFs and functional microproteins.

Microproteins (MPs) are a potentially rich source of uncharacterized metabolic regulators. Here, we use ribosome profiling (Ribo-seq) to curate 3,877 unannotated MP-encoding small ORFs (smORFs) in primary brown, white, and beige mouse adipocytes. Of these, we validated 85 MPs by proteomics, including 33 circulating MPs in mouse plasma. Analyses of MP-encoding mRNAs under different physiological conditions (high-fat diet) revealed that numerous MPs are regulated in adipose tissue in vivo and are co-expressed with established metabolic genes. Furthermore, Ribo-seq provided evidence for the translation of Gm8773, which encodes a secreted MP that is homologous to human and chicken FAM237B. Gm8773 is highly expressed in the arcuate nucleus of the hypothalamus, and intracerebroventricular administration of recombinant mFAM237B showed orexigenic activity in obese mice. Together, these data highlight the value of this adipocyte MP database in identifying MPs with roles in fundamental metabolic and physiological processes such as feeding.

Identifying individual cell types in heterogeneous cultures using secondary ion mass spectrometry imaging with C60 etching and multivariate analysis.

Tissue engineering approaches fabricate and subsequently implant cell-seeded and unseeded scaffold biomaterials. Once in the body, these biomaterials are repopulated with somatic cells of various phenotypes whose identification upon explantation can be expensive and time-consuming. We show that imaging time-of-flight secondary ion mass spectrometry (TOF-SIMS) can be used to distinguish mammalian cell types in heterogeneous cultures. Primary rat esophageal epithelial cells (REEC) were cultured with NIH 3T3 mouse fibroblasts on tissue culture polystyrene and freeze-dried before TOF-SIMS imaging. Results show that a short etching sequence with C(60)(+) ions can be used to clean the sample surface and improve the TOF-SIMS image quality. Principal component analysis (PCA) and partial least-squares discriminant analysis (PLS-DA) were used to identify peaks whose contributions to the total variance in the multivariate model were due to either the two cell types or the substrate. Using PLS-DA, unknown regions of cellularity that were otherwise unidentifiable by SIMS could be classified. From the loadings in the PLS-DA model, peaks were selected that were indicative of the two cell types and TOF-SIMS images were created and overlaid that showed the ability of this method to distinguish features visually.

The interchangeability of global positioning system and semiautomated video-based performance data during elite soccer match play.

In elite-level soccer, player motion characteristics are commonly generated from match play and training situations using semiautomated video analysis systems and global positioning system (GPS) technology, respectively. Before such data are used collectively to quantify global player load, it is necessary to understand both the level of agreement and direction of bias between the systems so that specific interventions can be made based on the reported results. The aim of this report was to compare data derived from both systems for physical match performances. Six elite-level soccer players were analyzed during a competitive match using semiautomated video analysis (ProZone® [PZ]) and GPS (MinimaxX) simultaneously. Total distances (TDs), high speed running (HSR), very high speed running (VHSR), sprinting distance (SPR), and high-intensity running distance (HIR; >4.0 m·s(-1)) were reported in 15-minute match periods. The GPS reported higher values than PZ did for TD (GPS: 1,755.4 ± 245.4 m; PZ: 1,631.3 ± 239.5 m; p < 0.05); PZ reported higher values for SPR and HIR than GPS did (SPR: PZ, 34.1 ± 24.0 m; GPS: 20.3 ± 15.8 m; HIR: PZ, 368.1 ± 129.8 m; GPS: 317.0 ± 92.5 m; p < 0.05). Caution should be exercised when using match-load (PZ) and training-load (GPS) data interchangeably.

Motion analysis of match-play in elite U12 to U16 age-group soccer players.

The aim of this study was to quantify the motion demands of match-play in elite U12 to U16 age-group soccer players. Altogether, 112 players from two professional soccer clubs at five age-group levels (U12-U16) were monitored during competitive matches (n = 14) using a 5 Hz non-differential global positioning system (NdGPS). Velocity thresholds were normalized for each age-group using the mean squad times for a flying 10 m sprint test as a reference point. Match performance was reported as total distance, high-intensity distance, very high-intensity distance, and sprint distance. Data were reported both in absolute (m) and relative (m · min(-1)) terms due to a rolling substitute policy. The U15 (1.35 ± 0.09 s) and U16 (1.31 ± 0.06 s) players were significantly quicker than the U12 (1.58 ± 0.10 s), U13 (1.52 ± 0.07 s), and U14 (1.51 ± 0.08 s) players in the flying 10 m sprint test (P < 0.001). The U16 age-group covered significantly more absolute total distance (U16 > U12, U13, U14), high-intensity distance (U16 > U12, U13, U14, U15), very high-intensity distance (U16 > U12, U13), and sprint distance (U16 > U12, U13) than their younger counterparts (P < 0.05). When the data are considered relative to match exposure, few differences are apparent. Training prescription for youth soccer players should consider the specific demands of competitive match-play in each age-group.

Exercise and lung cancer surgery: A systematic review of randomized-controlled trials.

Lung cancer patients undergoing surgery are often left physically deconditioned and/or with functional deficits. Exercise interventions may improve pulmonary and physical function before and after lung resection. We conducted a systematic review of randomized-controlled trials (RCTs) testing the impact of pre-, post-, and combined pre-and-post surgery exercise interventions on physical and pulmonary function in lung cancer patients. Exercise pre-surgery seems to substantially improve physical and pulmonary function, which are factors associated with improved ability to undergo surgery while reducing post-surgery complications. Evidence is inconsistent for post-surgery interventions, reporting no or moderate effects. Results from pre-and-post surgery interventions are limited to one study. In conclusion, pre- and post-surgery exercise interventions, individually, have shown beneficial effects for lung cancer patients undergoing surgery. The impact of interventions combining both pre- and post-surgery exercise programs remains unknown. More evidence is needed on the ideal exercise setting, and timing across the lung cancer care continuum.

Generating high quality libraries for DIA MS with empirically corrected peptide predictions.

Data-independent acquisition approaches typically rely on experiment-specific spectrum libraries, requiring offline fractionation and tens to hundreds of injections. We demonstrate a library generation workflow that leverages fragmentation and retention time prediction to build libraries containing every peptide in a proteome, and then refines those libraries with empirical data. Our method specifically enables rapid, experiment-specific library generation for non-model organisms, which we demonstrate using the malaria parasite Plasmodium falciparum, and non-canonical databases, which we show by detecting missense variants in HeLa.

Clinically Integrated Physical Therapist Practice in Cancer Care: A New Comprehensive Approach.

Best practice recommendations in cancer care increasingly call for integrated rehabilitation services to address physical impairments and disability. These recommendations have languished primarily due to a lack of pragmatic, generalizable intervention models. This perspective paper proposes a clinically integrated physical therapist (CI-PT) model that enables flexible and scalable services for screening, triage, and intervention addressing functional mobility. The model is based on (1) a CI-PT embedded in cancer care provider clinics, and (2) rehabilitation across the care continuum determined by the patient's level of functional mobility. The CI-PT model includes regular screening of functional mobility in provider clinics via a patient-reported mobility measure-the Activity Measure for Post-Acute Care, a brief physical therapy evaluation tailored to the specific functional needs of the individual-and a tailored, skilled physical therapist intervention based on functional level. The CI-PT model provides a pragmatic, barrier-free, patient-centric, data-driven approach to integrating rehabilitation as part of standard care for survivors of cancer. The model standardizes CI-PT practice and may be sufficiently agile to provide targeted interventions in widely varying cancer settings and populations. Therefore, it may be ideal for wide implementation among outpatient oncological settings. Implementation of this model requires a shared approach to care that includes physical therapists, rehabilitation administrators, cancer care providers, and cancer center administrators.

The effects of contrast bathing and compression therapy on muscular performance.

UNLABELLED: Contrast bathing (CB) and compression garments (CG) are widely used to promote recovery. PURPOSE: To evaluate CB and CG as regeneration strategies after exercise-induced muscle damage (EIMD). METHODS: Baseline values of muscle soreness, serum creatine kinase (CK) and myoglobin (Mb), joint range of motion, limb girth, 10- or 30-m sprint, countermovement jump (CMJ), and five repetition maximum squat were completed by 26 young men who then undertook a resistance exercise challenge (REC) to induce EIMD: 6 x 10 parallel squats at 100% body weight with 5-s one repetition maximum eccentric squat superimposed onto each set. After the REC, subjects were separated into three intervention groups: CB, CG, and control (CONT). Forty-eight hours after REC, the subjects exercise performance was reassessed. CK and Mb were also measured +1, +24, and +48 h post-REC. RESULTS: CK was elevated at +24 h ( upward arrow140%; upward arrow161%; upward arrow270%), and Mb was elevated at +1 h ( upward arrow523%; upward arrow458%; upward arrow682%) in CB, CG, and CONT. Within-group large effect sizes for loge[CK] were found for CB at +24 h (0.80) and +48 h (0.84). Area under the [Mb] curve was lower in CB compared with CG and CONT (P < or = 0.05). At +48 h, significant differences from baseline were found in all groups for CMJ (CG, downward arrow5.1%; CB, downward arrow4.4%; CONT, downward arrow8.5%) and soreness ( upward arrow213%; upward arrow284%; upward arrow284%). Soreness transiently fell at +1 h compared with post-REC in the CB group. At +48 h, midthigh girth increased in CB ( upward arrow1.4%) and CONT ( upward arrow1.6%), whereas 30-m sprint time increased in CG ( upward arrow2%). CONCLUSION: No hierarchy of recovery effects was found. Neither contrast bathing nor compression acted to promote acute recovery from EIMD any more effectively than passive conditions, although contrast bathing may transiently attenuate postexercise soreness.

Precision-Exercise-Prescription in patients with lung cancer undergoing surgery: rationale and design of the PEP study trial.

INTRODUCTION: Lung cancer is a significant burden on societies worldwide, and the most common cause of death in patients with cancer overall. Exercise intervention studies in patients with lung cancer have consistently shown benefits with respect to physical and emotional functioning. However, to date, exercise training has not been consistently implemented into clinical practice given that interventions have been costly and not aligned with clinical care. METHODS/DESIGN: The Precision-Exercise-Prescription (PEP) study is a prospective randomised controlled trial comparing the effectiveness and feasibility of a personalised intervention exercise programme among patients with lung cancer undergoing surgery. Two-hundred patients who are diagnosed with stage primary or secondary lung cancer and are eligible to undergo surgical treatment at Huntsman Cancer Institute comprise the target population. Patients are randomised to either the (1) outpatient precision-exercise intervention group or (2) delayed intervention group. The intervention approach uses Motivation and Problem Solving, a hybrid behavioural treatment based on motivational interviewing and practical problem solving. The dosage of the exercise intervention is personalised based on the individual's Activity Measure for Post-Acute-Care outpatient basic mobility score, and incorporates four exercise modes: mobility, callisthenics, aerobic and resistance. Exercise is implemented by physical therapists at study visits from presurgery until 6 months postsurgery. The primary endpoint is the level of physical function assessed by 6 min walk distance at 2 months postsurgery. Secondary outcomes include patient-reported outcomes (eg, quality of life, fatigue and self-efficacy) and other clinical outcomes, including length of stay, complications, readmission, pulmonary function and treatment-related costs up to 6 months postsurgery. ETHICS/DISSEMINATION: The PEP study will test the clinical effectiveness and feasibility of a personalised exercise intervention in patients with lung cancer undergoing surgery. Outcomes of this clinical trial will be presented at national and international conferences and symposia and will be published in international, peer-reviewed journals. Ethics approval was obtained at the University of Utah (IRB 00104671). TRIAL REGISTRATION NUMBER: NCT03306992.

The effects of delayed reinforcement on variability and repetition of response sequences.

Four experiments examined the effects of delays to reinforcement on key peck sequences of pigeons maintained under multiple schedules of contingencies that produced variable or repetitive behavior. In Experiments 1, 2, and 4, in the repeat component only the sequence right-right-left-left earned food, and in the vary component four-response sequences different from the previous 10 earned food. Experiments 1 and 2 examined the effects of nonresetting and resetting delays to reinforcement, respectively. In Experiment 3, in the repeat component sequences had to be the same as one of the previous three, whereas in the vary component sequences had to be different from each of the previous three for food. Experiment 4 compared postreinforcer delays to prereinforcement delays. With immediate reinforcement sequences occurred at a similar rate in the two components, but were less variable in the repeat component. Delays to reinforcement decreased the rate of sequences similarly in both components, but affected variability differently. Variability increased in the repeat component, but was unaffected in the vary component. These effects occurred regardless of the manner in which the delay to reinforcement was programmed or the contingency used to generate repetitive behavior. Furthermore, the effects were unique to prereinforcement delays.

A SPOPL/Cullin-3 ubiquitin ligase complex regulates endocytic trafficking by targeting EPS15 at endosomes.

Cullin-3 (CUL3)-based ubiquitin ligases regulate endosome maturation and trafficking of endocytic cargo to lysosomes in mammalian cells. Here, we report that these functions depend on SPOPL, a substrate-specific CUL3 adaptor. We find that SPOPL associates with endosomes and is required for both the formation of multivesicular bodies (MVBs) and the endocytic host cell entry of influenza A virus. In SPOPL-depleted cells, endosomes are enlarged and fail to acquire intraluminal vesicles (ILVs). We identify a critical substrate ubiquitinated by CUL3-SPOPL as EPS15, an endocytic adaptor that also associates with the ESCRT-0 complex members HRS and STAM on endosomes. Indeed, EPS15 is ubiquitinated in a SPOPL-dependent manner, and accumulates with HRS in cells lacking SPOPL. Together, our data indicates that a CUL3-SPOPL E3 ubiquitin ligase complex regulates endocytic trafficking and MVB formation by ubiquitinating and degrading EPS15 at endosomes, thereby influencing influenza A virus infection as well as degradation of EGFR and other EPS15 targets.

Linking the foreign body response and protein adsorption to PEG-based hydrogels using proteomics.

Poly(ethylene glycol) (PEG) hydrogels with their highly tunable properties are promising implantable materials, but as with all non-biological materials, they elicit a foreign body response (FBR). Recent studies, however, have shown that incorporating the oligopeptide RGD into PEG hydrogels reduces the FBR. To better understand the mechanisms involved and the role of RGD in mediating the FBR, PEG, PEG-RGD and PEG-RDG hydrogels were investigated. After a 28-day subcutaneous implantation in mice, a thinner and less dense fibrous capsule formed around PEG-RGD hydrogels, while PEG and PEG-RDG hydrogels exhibited stronger, but similar FBRs. Protein adsorption to the hydrogels, which is considered the first step in the FBR, was also characterized. In vitro experiments confirmed that serum proteins adsorbed to PEG-based hydrogels and were necessary to promote macrophage adhesion to PEG and PEG-RDG, but not PEG-RGD hydrogels. Proteins adsorbed to the hydrogels in vivo were identified using liquid chromatography-tandem mass spectrometry. The majority (245) of the total proteins (≥300) that were identified was present on all hydrogels with many proteins being associated with wounding and acute inflammation. These findings suggest that the FBR to PEG hydrogels may be mediated by the presence of inflammatory-related proteins adsorbed to the surface, but that macrophages appear to sense the underlying chemistry, which for RGD improves the FBR.