Defining principles that influence antimicrobial peptide activity against capsulated Klebsiella pneumoniae.

The extracellular polysaccharide capsule of Klebsiella pneumoniae resists penetration by antimicrobials and protects the bacteria from the innate immune system. Host antimicrobial peptides are inactivated by the capsule as it impedes their penetration to the bacterial membrane. While the capsule sequesters most peptides, a few antimicrobial peptides have been identified that retain activity against encapsulated K. pneumoniae, suggesting that this bacterial defense can be overcome. However, it is unclear what factors allow peptides to avoid capsule inhibition. To address this, we created a peptide analog with strong antimicrobial activity toward several K. pneumoniae strains from a previously inactive peptide. We characterized the effects of these two peptides on K. pneumoniae, along with their physical interactions with K. pneumoniae capsule. Both peptides disrupted bacterial cell membranes, but only the active peptide displayed this activity against capsulated K. pneumoniae Unexpectedly, the active peptide showed no decrease in capsule binding, but did lose secondary structure in a capsule-dependent fashion compared with the inactive parent peptide. We found that these characteristics are associated with capsule-peptide aggregation, leading to disruption of the K. pneumoniae capsule. Our findings reveal a potential mechanism for disrupting the protective barrier that K. pneumoniae uses to avoid the immune system and last-resort antibiotics.

Enhanced Characterization of Cardiolipins via Hybrid 193 nm Ultraviolet Photodissociation Mass Spectrometry.

Cardiolipins (CLs) constitute a structurally complex class of glycerophospholipids with a unique tetraacylated structure accompanied by distinctive functional roles. Aberrations in the composition of this lipid class have been associated with disease states, spurring interest in the development of new approaches to differentiate the structures of diverse CLs in complex mixtures. The structural characterization of these complex lipids using conventional methods, however, suffers from limited resolution and frequently proves unable to discern subtle yet biologically significant features such as unsaturation sites or acyl chain position assignments. Here, we describe the synergistic use of chemical derivatization and hybrid dissociation techniques to characterize CL from complex biological mixtures with both double bond and sn positional isomer resolution in a shotgun mass spectrometry strategy. Utilizing (trimethylsilyl)diazomethane (TMSD), CL phosphate groups were methylated to promote positive-mode ionization by the production of metal-cationized lipids, enabling structural interrogation via hybrid higher-energy collisional activation/ultraviolet photodissociation (HCD/UVPD). This combination of TMSD derivatization and HCD/UVPD fragmentation results in diagnostic product ions that permit distinction and relative quantitation of sn-stereoisomers and the localization of double bonds. Applying this strategy to a total lipid extract from a thyroid carcinoma revealed a previously unreported 18:2/18:1 motif, elucidating a structural feature unique to the lipid class.

Relative Quantitation of Unsaturated Phosphatidylcholines Using 193 nm Ultraviolet Photodissociation Parallel Reaction Monitoring Mass Spectrometry.

Structural characterization of glycerophospholipids beyond the fatty acid level has become a major endeavor in lipidomics, presenting an opportunity to advance the understanding of the intricate relationship between lipid metabolism and disease state. Distinguishing subtle lipid structural features, however, remains a major challenge for high-throughput workflows that implement traditional tandem mass spectrometry (MS/MS) techniques, stunting the molecular depth of quantitative strategies. Here, reversed phase liquid chromatography is coupled to parallel reaction mass spectrometry utilizing the double bond localization capabilities of ultraviolet photodissociation (UVPD) mass spectrometry to produce double bond isomer specific responses that are leveraged for relative quantitation. The strategy provides lipidomic characterization at the double bond level for phosphatidylcholine phospholipids from biological extracts. In addition to quantifying monounsaturated lipids, quantitation of phospholipids incorporating isomeric polyunsaturated fatty acids is also achieved. Using this technique, phosphatidylcholine isomer ratios are compared across human normal and tumor breast tissue to reveal significant structural alterations related to disease state.

Characterization of Isomers of Lipid A from Pseudomonas aeruginosa PAO1 by Liquid Chromatography with Tandem Mass Spectrometry with Higher-Energy Collisional Dissociation and Ultraviolet Photodissociation.

Lipopolysaccharides (LPS) constitute the outermost layer of Gram-negative bacteria and consequently play an important role in bacterial infections. In order to address public health issues posed by Gram-negative bacteria, it is necessary to elucidate the structure of the molecular actors at the forefront of infections. LPS virulence and toxicity are partially modulated by lipid A, a hydrophobic saccharolipid that anchors LPS to the bacterial outer membrane. Understanding the lipid A structure is inherently intertwined with understanding its role as an endotoxin. Accordingly, several successful strategies incorporating tandem mass spectrometry have been applied toward the structural analysis of lipid A. Herein, a shotgun HCD strategy was applied toward the characterization of the lipid A profile of Pseudomonas aeruginosa PAO1. This analysis was enhanced by the development of an LC-MS/MS approach to eliminate isomeric signals in the MS/MS spectra that confounded characterization. Importantly, combining reverse phase chromatography with HCD and ultraviolet photodissociation analyses of the lipid A profile revealed the presence of previously unreported lipid A acyl chain positional isomers. Altogether, these strategies provide the most in-depth structural and molecular characterization of PAO1 lipid A to date.

Double Bond Characterization of Free Fatty Acids Directly from Biological Tissues by Ultraviolet Photodissociation.

Free fatty acids (FA) are a vital component of cells and are critical to cellular structure and function, so much so that alterations in FA are often associated with cell malfunction and disease. Analysis of FA from biological samples can be achieved by mass spectrometry (MS), but these analyses are often not capable of distinguishing the fine structural alterations within FA isomers and often limited to global profiling of lipids without spatial resolution. Here, we present the use of ultraviolet photodissociation (UVPD) for the characterization of double bond positional isomers of charge inverted dication·FA complexes and the subsequent implementation of this method for online desorption electrospray ionization (DESI) MS imaging of FA isomers from human tissue sections. This method allows relative quantification of FA isomers from heterogeneous biological tissue sections, yielding spatially resolved information about alterations in double bond isomers within these samples. Applying this method to the analysis of the monounsaturated FA 18:1 within breast cancer subtypes uncovered a correlation between double bond positional isomer abundance and the hormone receptor status of the tissue sample, an important factor in the prognosis and treatment of breast cancer patients. This result further validates similar studies that suggest FA synthase activity and FA isomer abundances are significantly altered within breast cancer tissue.

Localization of Double Bonds in Bacterial Glycerophospholipids Using 193 nm Ultraviolet Photodissociation in the Negative Mode.

The need for detailed structural characterization of glycerophospholipids (GPLs) for many types of biologically motivated applications has led to the development of novel mass spectrometry-based methodologies that utilize alternative ion activation methods. Ultraviolet photodissociation (UVPD) has shown great utility for localizing sites of unsaturation within acyl chains and to date has predominantly been used for positive mode analysis of GPLs. In the present work, UVPD is used to localize sites of unsaturation in GPL anions. Similar to UVPD mass spectra of GPL cations, UVPD of deprotonated or formate-adducted GPLs yields diagnostic fragment ions spaced 24 Da apart. This method was integrated into a liquid chromatography workflow and used to evaluate profiles of sites of unsaturation of lipids in Escherichia coli (E. coli) and Acinetobacter baumannii (A. baumannii). When assigning sites of unsaturation, E. coli was found to contain all unsaturation elements at the same position relative to the terminal methyl carbon of the acyl chain; the first carbon participating in a site of unsaturation was consistently seven carbons along the acyl chain when counting carbons from the terminal methyl carbon. GPLs from A. baumannii exhibited more variability in locations of unsaturation. For GPLs containing sites of unsaturation in both acyl chains, an MS3 method was devised to assign sites to specific acyl chains.

Hybrid 193 nm Ultraviolet Photodissociation Mass Spectrometry Localizes Cardiolipin Unsaturations.

Developing alternative MS/MS strategies to distinguish isomeric lipids has become a high impact goal in shotgun lipidomics. Novel approaches have been developed to resolve structural features that are not discernible by traditional shotgun methods and have consequently promoted the discovery of new disease biomarkers. However, these methods have largely been limited to characterizing lipids with low structural complexity. Here, ultraviolet photodissociation (UVPD) strategies for phospholipid characterization are expanded for analysis of cardiolipins (CL), a class of phospholipids that exhibits a higher degree of structural complexity. A hybrid collision induced dissociation/193 nm UVPD (CID/UVPD) approach was implemented to pinpoint the location of both double bond and cyclopropyl unsaturations on the four acyl chains of CLs. This strategy was complemented with CID for the de novo elucidation of unknown CLs in biological extracts.

Expanding the Scope of Cross-Link Identifications by Incorporating Collisional Activated Dissociation and Ultraviolet Photodissociation Methods.

With the advent of new cross-linking chemistries, analytical technologies, and search algorithms, cross-linking has become an increasingly popular strategy for evaluating tertiary and quaternary structures of proteins. Collisional activated dissociation remains the primary MS/MS method for identifications of peptide cross-links in high throughput workflows. Ultraviolet photodissociation (UVPD) at 193 nm has emerged as an alternative ion activation method well-suited for characterization of peptides and has been found in some cases to identify different peptides or provide distinctive sequence information than obtained by collisional activation methods. Complementary high energy collision dissociation (HCD) and UVPD were used in the present study to characterize protein cross-linking for bovine serum albumin, hemoglobin, and E. coli ribosome. Cross-links identified by HCD and UVPD using bis(sulfosuccinimidyl)suberate (BS3), a homobifunctional amine-to-amine cross-linker, and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM), a heterofunctional amine-to-carboxylic acid cross-linker, were evaluated in the present study. While more unique BS3 cross-links were identified by HCD, UVPD, and HCD provided a complementary panel of DMTMM cross-links which extended the degree of structural insight obtained for the proteins.

Seroma Rates Are Not Increased When Combining Liposuction With Progressive Tension Suture Abdominoplasty: A Retrospective Cohort Study of 619 Patients.

BACKGROUND: Several studies have demonstrated that progressive tension sutures (PTS) reduce seroma. Many fear that adding liposuction to abdominoplasty will increase seroma rates and avoid drainless abdominoplasty when performing concomitant liposuction. OBJECTIVES: We sought to identify whether liposuction increases seroma in PTS and non-PTS abdominoplasty. METHODS: In this retrospective study, 619 patients underwent abdominoplasty between 2009 and 2017, of which 299 patients had drainless abdominoplasty with PTS and 320 had drain-based abdominoplasty. We compared complications among PTS patients with and without liposuction and among drain-based abdominoplasty patients with and without liposuction. RESULTS: Demographics were similar between PTS and drain patients and between liposuction and nonliposuction PTS. Mean liposuction volume with PTS was 1592 ± 1048 mL. Seroma in the PTS group was found to be 2.6%, which is consistent with previous data. PTS without liposuction had a rate of seroma of 6.67% compared to a rate of 2.2% with liposuction; these rates were not significantly different (P = 0.20). A total of 207 patients had drain-based abdominoplasty with liposuction, and 113 had it without liposuction. Seroma with liposuction was 9.17% and without liposuction was 6.19%, although these differences were not significant (P = 0.52). PTS lipoabdominoplasty had less seroma compared with drain-based lipoabdominoplasty (P = 0.01). CONCLUSIONS: Liposuction was performed in 80% of the patients, and patients with lipoabdominoplasty were not at a higher risk of seroma, in the drain group or the PTS group. More patients may allow validation that liposuction may actually be protective with PTS. Regardless, there is no increase in seroma with the addition of liposuction to PTS drainless abdominoplasty.

The Ideal Thigh: A Crowdsourcing-Based Assessment of Ideal Thigh Aesthetic and Implications for Gluteal Fat Grafting.

BACKGROUND: As the popularity of aesthetic gluteoplasty continues to grow, there is renewed focus on defining the ideal buttocks. However, the literature lacks studies characterizing an ideal thigh, despite the impact of thigh contour on overall gluteal aesthetic. OBJECTIVES: The authors performed the first population analysis of the characteristics of perception of attractive thighs, to identify a role for fat grafting of the thigh in gluteoplasty. METHODS: Survey images were digitally modified to create thighs of varying widths and angles relative to fixed buttocks. Thigh-to-buttock ratios and the buttock-thigh junction were studied. Data were stratified and analyzed according to age, gender, and ethnicity of the respondents. Amazon Mechanical Turk was used as a novel crowdsourcing platform for surveying aesthetic preferences. RESULTS: A total of 1034 responses were included of whom 54.4% were male, and 45.6% were female. All age groups and ethnicities were represented. Overall, 43.8% of respondents preferred the widest buttock-thick junction angle on posterior view. There was no clear preference between larger or smaller thigh-to-hip ratios on lateral view. CONCLUSIONS: Characteristics of the ideal thigh include wider thighs with greater horizontal projection, creating a more natural contour from the augmented buttock. These findings represent a paradigm shift from the traditionally assumed preference for slender thighs. Plastic surgeons should carefully consider thigh anatomy in their gluteal augmentation patients, as simultaneous thigh augmentation may lead to a more aesthetically pleasing outcome. Further research is needed into best practices and techniques to attain ideal thigh proportions.

Decrease in Seroma Rate After Adopting Progressive Tension Sutures Without Drains: A Single Surgery Center Experience of 451 Abdominoplasties Over 7 Years.

BACKGROUND: Abdominoplasty is a common aesthetic procedure in the United States. Pollock and Pollock described their progressive tension technique in 2000 and published a series of 597 patients in 2012 of their experience. The reported seroma rate in the literature ranges from 2% to 26% with drains and 0.1% to 4% with progressive tension sutures (PTS) without drains. OBJECTIVES: Given these data, we decided to use PTS and forego drains in abdominoplasty. Here we present our experience with the transition. METHODS: This is a retrospective chart review of 451 abdominoplasties performed at our outpatient surgery center over a 7-year period (2009-2015). We gathered data on patient demographics, concomitant liposuction, and complications and length of follow up. RESULTS: Five main differences were examined in PTS vs traditional abdominoplasty using drains groups. These included rate of seroma, wound complication, scar revision, hematoma, and follow up. We found a decreased rate of seroma in the PTS group, 2% vs 9%. Wound complications were similar. Scar revision was slightly higher in the PTS group at 17% vs 10% in traditional abdominoplasty, this association had a P value of .048. The rates of hematoma were similar (0% vs 1%). The mean follow up was 6 months in PTS and 9 months in traditional abdominoplasty. Addition of liposuction did not increase the rate of seroma. CONCLUSIONS: PTS without drains significantly decreased the seroma rate in our practice. Our experience adds to the mounting evidence that surgeons should consider using the PTS technique and abandon the use of drains in abdominoplasty. A well powered, multicenter, randomized controlled study is needed in order to definitively lay this question to rest. LEVEL OF EVIDENCE: 4 Therapeutic.

Inverted nipple repair revisited: a 7-year experience.

BACKGROUND: Nipple inversion in females can be congenital or acquired. Women who desire treatment for this condition often report difficulty with breastfeeding and interference with their sexuality. However, data are limited on the demographics of patients who undergo surgery to repair inverted nipples and the associated recurrence rates and complications. OBJECTIVES: The authors assessed outcomes of a 7-year experience with an integrated approach to the correction of nipple inversion that minimizes ductal disruption. METHODS: A retrospective chart review was performed for 103 consecutive patients who underwent correction of nipple inversion. (The correction technique was initially reported in 2004 and entailed an integrated approach.) Complication rates, breastfeeding status, and patient demographics were documented. RESULTS: Among the 103 patients, 191 nipple corrections were performed. Nine patients had undergone previous nipple-correction surgery. Recurrence was experienced by 12.6% of patients, 3 of whom had bilateral recurrence. Other complications were partial nipple necrosis (1.05%), breast cellulitis (1.57%), and delayed healing (0.5%). The overall complication rate was 15.74%. Fifty-seven percent of the patients had a B-cup breast size, and 59% were 21 to 30 years of age. CONCLUSIONS: Results of the authors' 7-year experience demonstrate the safety and effectiveness of their technique to correct inverted nipples. LEVEL OF EVIDENCE 4: Therapeutic.

Superior pedicle technique of reduction mammaplasty: a stepwise approach.

Numerous surgical options for breast reduction have been described, but in the current healthcare environment, efficiency is of the utmost importance. In this Featured Operative Technique, the authors describe an efficient, reproducible, and simple method for minimal to moderate reduction mammaplasty that utilizes a superior pedicle. The surgical maneuvers were developed and conveyed to the senior author (W.G.S.) by Dr John Bostwick. This approach preserves superior and medial breast fullness while providing appropriate resection of the breast parenchyma to ameliorate symptoms and produce a smaller, lifted breast with a more youthful appearance. The surgical technique maintains a reliable blood supply to the nipple-areola complex (NAC) from the internal mammary artery and its perforators, and involves minimal transposition of the NAC. The authors reviewed the charts of 62 consecutive patients who underwent this procedure and found the complication rate to be 11.3%. Complications included 1 hematoma, 1 standing cone deformity, 3 soft-tissue infections, 8 incisional breakdowns, and 1 unilateral necrosis of the NAC.

Secondary augmentation-mastopexy: indications, preferred practices, and the treatment of complications.

Increasing the volume of the breast while simultaneously decreasing the skin envelope equates to surgery involving opposing forces. Increasing patient demand and the evolving perceptions of surgeons have led to the growing popularity of the combined augmentation-mastopexy operation. In turn, secondary augmentation-mastopexies and revisional surgeries of primary augmentation-mastopexies also have increased in popularity. In this article, the authors describe indications for secondary augmentation-mastopexy, techniques for performing this combined procedure safely and effectively, adjunctive procedures, potential pitfalls, and the treatment of complications.

One-Stage Augmentation Mastopexy: A Review of 1192 Simultaneous Breast Augmentation and Mastopexy Procedures in 615 Consecutive Patients.

BACKGROUND: Despite the increasing popularity of the combined augmentation mastopexy procedure among patients, the safety and efficacy of this surgery have been questioned by many surgeons. OBJECTIVE: The authors investigated the safety and efficacy of the combined augmentation mastopexy procedure. METHODS: The authors retrospectively reviewed the medical records of 615 consecutive patients who underwent combined augmentation mastopexy procedures at a single outpatient surgery center from 1992 through 2011. Patient demographics, operative and implant details, and long-term outcomes were analyzed. Rates of complications and revisions were calculated. RESULTS: The most common complications were poor scarring (5.7%), wound-healing problems (2.9%), and deflation of saline implants (2.4%). Of the 615 patients evaluated, 104 (16.9%) elected to undergo revision surgery: 54 revision procedures were secondary to implant-related complications, and 50 were secondary to tissue-related complications. Our data compare favorably with previously reported revision rates for breast augmentation alone and mastopexy alone. CONCLUSIONS: With a skilled surgeon and proper patient selection, the combined augmentation mastopexy procedure can be safe and effective. LEVEL OF EVIDENCE: 4.

Characterization of Acinetobacter baumannii core oligosaccharide synthesis reveals novel aspects of lipooligosaccharide assembly.

A fundamental feature of Gram-negative bacteria is their outer membrane that protects the cell against environmental stressors. This defense is predominantly due to its asymmetry, with glycerophospholipids located in the inner leaflet and lipopolysaccharide (LPS) or lipooligosaccharide (LOS) confined to the outer leaflet. LPS consists of a lipid A anchor, a core oligosaccharide, and a distal O-antigen while LOS lacks O-antigen. While LPS/LOS is typically essential for growth, this is not the case for Acinetobacter baumannii. Despite this unique property, the synthesis of the core oligosaccharide of A. baumannii LOS is not well-described. Here, we characterized the LOS chemotypes of A. baumannii strains with mutations in a predicted core oligosaccharide locus via tandem mass spectrometry. This allowed for an extensive identification of genes required for core assembly that can be exploited to generate precise structural LOS modifications in many A. baumannii strains. We further investigated two chemotypically identical yet phenotypically distinct mutants, ∆2903 and ∆lpsB, that exposed a possible link between LOS and the peptidoglycan cell wall-two cell envelope components whose coordination has not yet been described in A. baumannii. Selective reconstruction of the core oligosaccharide via expression of 2903 and LpsB revealed that these proteins rely on each other for the unusual tandem transfer of two residues, KdoIII and N-acetylglucosaminuronic acid. The data presented not only allow for better usage of A. baumannii as a tool to study outer membrane integrity but also provide further evidence for a novel mechanism of core oligosaccharide assembly. IMPORTANCE: Acinetobacter baumannii is a multidrug-resistant pathogen that produces lipooligosaccharide (LOS), a glycolipid that confers protective asymmetry to the bacterial outer membrane. The core oligosaccharide is a ubiquitous component of LOS that typically follows a well-established model of synthesis. In addition to providing an extensive analysis of the genes involved in the synthesis of the core region, we demonstrate that this organism has evidently diverged from the long-held archetype of core synthesis. Moreover, our data suggest that A. baumannii LOS assembly is important for cell division and likely intersects with the synthesis of the peptidoglycan cell wall, another essential component of the Gram-negative cell envelope. This connection between LOS and cell wall synthesis provides an intriguing foundation for a unique method of outer membrane biogenesis and cell envelope coordination.

RNA targeting and cleavage by the type III-Dv CRISPR effector complex.

CRISPR-Cas are adaptive immune systems in bacteria and archaea that utilize CRISPR RNA-guided surveillance complexes to target complementary RNA or DNA for destruction1-5. Target RNA cleavage at regular intervals is characteristic of type III effector complexes6-8. Here, we determine the structures of the Synechocystis type III-Dv complex, an apparent evolutionary intermediate from multi-protein to single-protein type III effectors9,10, in pre- and post-cleavage states. The structures show how multi-subunit fusion proteins in the effector are tethered together in an unusual arrangement to assemble into an active and programmable RNA endonuclease and how the effector utilizes a distinct mechanism for target RNA seeding from other type III effectors. Using structural, biochemical, and quantum/classical molecular dynamics simulation, we study the structure and dynamics of the three catalytic sites, where a 2'-OH of the ribose on the target RNA acts as a nucleophile for in line self-cleavage of the upstream scissile phosphate. Strikingly, the arrangement at the catalytic residues of most type III complexes resembles the active site of ribozymes, including the hammerhead, pistol, and Varkud satellite ribozymes. Our work provides detailed molecular insight into the mechanisms of RNA targeting and cleavage by an important intermediate in the evolution of type III effector complexes.

Etiology of breast masses after autologous breast reconstruction.

BACKGROUND: Determining the nature of a breast mass after autologous reconstruction can be difficult. METHODS: A retrospective review of all autologous breast reconstructions was performed over 10 years. All postoperative breast masses were identified. Tumor characteristics, adjuvant treatment, timing of the development of the mass, and correlation with radiology were reviewed. RESULTS: A total of 365 flaps were performed on 272 patients [253 deep inferior epigastric perforator (DIEP), 35 superficial inferior epigastric artery (SIEA), 22 muscle-sparing free transverse rectus abdominis myocutaneous (free MS-TRAM), 25 latissimus, and 30 pedicled TRAM]. Breast masses were identified in 66 breasts (18 %). The majority of these were from fat necrosis, occurring in 54 breasts (15 % overall; DIEP 13.4 %, SIEA 5.7 %, free MS-TRAM 15 %, latissimus 0 %, pedicled TRAM 47 %), first identified at a mean of 3 months. Recurrent carcinoma was diagnosed in 13 breasts (3.6 %). Factors associated with the postreconstruction mass representing recurrent carcinoma were later time period after reconstruction (mean 24 months), closer surgical margins, and lymphovascular invasion. Radiographic imaging accurately diagnosed recurrent carcinoma in 11 (92 %) of 12 patients in whom it was utilized and suggested a benign diagnosis in all 16 patients with fat necrosis in whom it was utilized. CONCLUSIONS: Breast masses frequently present after autologous reconstruction. Fat necrosis is the most common cause. Recurrent carcinoma can occur in the reconstructed breast and presents later. A higher index of suspicion for recurrence should accompany any mass in which prior lymphovascular invasion was present or if original margins were <1 cm. Radiographic imaging accurately identifies the cause of these masses.