Austin A. Pitcher

Membrane proteomics of phagosomes suggests a connection to autophagy

Phagocytosis is the central process by which macrophage cells internalize and eliminate infectious microbes as well as apoptotic cells. During maturation, phagosomes containing engulfed particles fuse with various endosomal compartments through the action of regulatory molecules on the phagosomal membrane. In this study, we performed a proteomic analysis of the membrane fraction from latex bead-containing (LBC) phagosomes isolated from macrophages. The profile, which comprised 546 proteins, suggests diverse functions of the phagosome and potential connections to secretory processes, toll-like receptor signaling, and autophagy. Many identified proteins were not previously known to reside in the phagosome. We characterized several proteins in LBC phagosomes that change in abundance on induction of autophagy, a process that has been previously implicated in the host defense against microbial pathogens. These observations suggest crosstalk between autophagy and phagocytosis that may be relevant to the innate immune response of macrophages.

Organelle Membrane Proteomics Reveals Differential Influence of Mycobacterial Lipoglycans on Macrophage Phagosome Maturation and Autophagosome Accumulation

The mycobacterial cell wall component lipoarabinomannan (LAM) has been described as one of the key virulence factors of Mycobacterium tuberculosis. Modification of the terminal arabinan residues of this lipoglycan with mannose caps in M. tuberculosis or with phosphoinositol caps in Mycobacterium smegmatis results in distinct host immune responses. Given that M. tuberculosis typically persists in the phagosomal vacuole after being phagocytosed by macrophages, we performed a proteomic analysis of that organelle after treatment of macrophages with LAMs purified from the two mycobacterial species. The quantitative changes in phagosomal proteins suggested a distinct role for mannose-capped LAM in modulating protein trafficking pathways that contribute to the arrest of phagosome maturation. Enlightened by our proteomic data, we performed further experiments to show that only the LAM from M. tuberculosis inhibits accumulation of autophagic vacuoles in the macrophage, suggesting a new function for this virulence-associated lipid.

Mapping Yeast N-Glycosites with Isotopically Recoded Glycans

Asparagine-linked glycosylation is a common post-translational modification of proteins; in addition to participating in key macromolecular interactions, N-glycans contribute to protein folding, trafficking, and stability. Despite their importance, few N-glycosites have been experimentally mapped in the Saccharomyces cerevisiae proteome. Factors including glycan heterogeneity, low abundance, and low occupancy can complicate site mapping. Here, we report a novel mass spectrometry-based strategy for detection of N-glycosites in the yeast proteome. Our method imparts N-glycopeptide mass envelopes with a pattern that is computationally distinguishable from background ions. Isotopic recoding is achieved via metabolic incorporation of a defined mixture of N-acetylglucosamine isotopologs into N-glycans. Peptides bearing the recoded envelopes are specifically targeted for fragmentation, facilitating high confidence site mapping. This strategy requires no chemical modification of the N-glycans or stringent sample enrichment. Further, enzymatically simplified N-glycans are preserved on peptides. Using this approach, we identify 133 N-glycosites spanning 58 proteins, nearly doubling the number of experimentally observed N-glycosites in the yeast proteome.

Isotopic Signature Transfer and Mass Pattern Prediction (IsoStamp): An Enabling Technique for Chemically-Directed Proteomics

Directed proteomics applies mass spectrometry analysis to a subset of information-rich proteins. Here we describe a method for targeting select proteins by chemical modification with a tag that imparts a distinct isotopic signature detectable in a full-scan mass spectrum. Termed isotopic signature transfer and mass pattern prediction (IsoStamp), the technique exploits the perturbing effects of a dibrominated chemical tag on a peptide’s mass envelope, which can be detected with high sensitivity and fidelity using a computational method. Applying IsoStamp, we were able to detect femtomole quantities of a single tagged protein from total mammalian cell lysates at signal-to-noise ratios as low as 2.5:1. To identify a tagged-peptide’s sequence, we performed an inclusion list-driven shotgun proteomics experiment where peptides bearing a recoded mass envelope were targeted for fragmentation, allowing for direct site mapping. Using this approach, femtomole quantities of several targeted peptides were identified in total mammalian cell lysate, while traditional data-dependent methods were unable to identify as many peptides. Additionally, the isotopic signature imparted by the dibromide tag was detectable on a 12-kDa protein, suggesting applications in identifying large peptide fragments, such as those containing multiple or large posttranslational modifications (e.g., glycosylation). IsoStamp has the potential to enhance any proteomics platform that employs chemical labeling for targeted protein identification, including isotope coded affinity tagging, isobaric tagging for relative and absolute quantitation, and chemical tagging strategies for posttranslational modification.

The effect of patient gender on outcomes after reverse total shoulder arthroplasty

BACKGROUND Gender differences may exist for patients undergoing shoulder arthroplasty. Limited data suggest that women may have worse preoperative disability and outcomes. Our objective was to determine whether gender influences preoperative disability and patient-reported outcomes after reverse total shoulder arthroplasty. METHODS Data were prospectively collected for patients who underwent reverse total shoulder arthroplasty for rotator cuff arthropathy or osteoarthritis with a rotator cuff tear at a single institution between 2009 and 2015. Range of motion, visual analog scale, 12-Item Short Form Health Survey (SF-12), and American Shoulder and Elbow Surgeons (ASES) scores were collected at the preoperative, 1-year, and 2-year postoperative time points. Data were analyzed using multivariate mixed-effect regression analysis. RESULTS There were 117 patients included. Men and women had similar demographics, preoperative range of motion, pain, and function. Length of stay was similar (men, 2.32 days; women, 2.58 days; P = .18). Controlling for patient variables, men achieved higher ASES function (P = .009) and SF-12 Physical Component Summary (P = .008) scores compared with women. There was no difference between men and women in ASES pain and SF-12 Mental Component Summary scores, visual analog scale score, or range of motion. CONCLUSION Improvements in pain and range of motion were similar in men and women; however, men achieved higher ASES function and SF-12 Physical Component Summary scores. Women may be more functionally impaired on the basis of differences in activities of daily living. These results may be used to guide discussion about expected benefits after reverse shoulder arthroplasty.

Recurrent breast cancer in the subpectoral space after implant reconstruction

Breast reconstruction after mastectomy is most commonly performed with a prosthetic implant placed beneath the pectoralis major. Recurrence may rarely be identified in the subpectoral space where the implant was placed. We report a case of recurrent breast cancer after implant‐based reconstruction with isolated subpectoral recurrence discovered 5 years later during secondary revision of her reconstructed breast. J. Surg. Oncol. 2014 109:431–433.

Excision of Elephantiasis Nostras Verrucosa Lesions in a Patient With Hereditary Lymphedema: Case Report and Review of the Literature

Elephantiasis nostras verrucosa (ENV) is a rare cutaneous sequela of chronic lymphedema. Treatment of ENV remains poorly elucidated but has historically involved conservative management aimed at relieving the underlying lymphedema, with a few cases managed by surgical intervention. We report a case of a 27-year-old male with primary lymphedema complicated by large painful ENV lesions on his left foot that we excised surgically with good functional and cosmetic results as validated by the patient. To our knowledge, this is the first report of a case of ENV with a pedunculated morphology and the presence of a deep invasive stalk.

Mental health, preoperative disability, and postoperative outcomes in patients undergoing shoulder arthroplasty

BACKGROUND Mental health conditions are associated with poor outcomes in patients with chronic disease as well as various orthopedic conditions. The purpose of this study was to describe the relationship between mental health, preoperative disability, and postoperative outcomes in patients undergoing shoulder arthroplasty. METHODS Data, including mental health diagnoses, were prospectively collected from patients undergoing total shoulder arthroplasty or reverse total shoulder arthroplasty from 2009 to 2015 at a single academic institution. Shoulder range of motion, visual analog scale, 12-Item Short Form Health Survey, and American Shoulder and Elbow Surgeons scores were collected preoperatively and at 1 and 2 years postoperatively. Data were analyzed using multivariate mixed-effect regression analysis. RESULTS The study included 280 patients, 105 (37.5%) of whom had a mental health diagnosis of depression, anxiety, schizophrenia, or bipolar disorder. Both groups of patients had similar shoulder range of motion, pain, and function before shoulder arthroplasty. Hospital length of stay, discharge destination, and readmissions were similar for both groups. There were similar improvements in pain, function, and range of motion after shoulder arthroplasty in patients with and without diagnosed mental health conditions. CONCLUSIONS Overall, the presence of a psychiatric diagnosis was not predictive of outcomes. Although psychiatric conditions are often considered surgical comorbidities, mental health diagnoses should not be a barrier to performing clinically indicated shoulder arthroplasty, because both groups of patients appear to benefit from pain relief and improved shoulder function.

Suprascarpal fat pad thickness may predict venous drainage patterns in abdominal wall flaps

Abdominal wall flaps are routinely used in reconstructive procedures. In some patients inadequate venous drainage from the deep vein may cause fat necrosis or flap failure. Occasionally the superficial inferior epigastric vessels (SIEV) are of sufficient size to allow for microvascular revascularization. This study looked at the ratio of the sub‐ and suprascarpal fat layers, the number of deep system perforators, and SIEV diameter to determine any correlation of the fat topography and SIEV.