Variables Affecting Basal Cell Carcinoma and Melanoma In Situ Excision Clearance: A Multi-institutional Retrospective Study.

BACKGROUND: The primary endpoint for surgical excision of skin cancer is the positive margin status. Tumor characteristics may explain much of this risk, but other important factors can include physician specialty. OBJECTIVE: To determine the variables affecting the success of a basal cell carcinoma (BCC) or melanoma in situ (MIS) excision. METHODS/MATERIALS: An 8-year, multicenter, retrospective study of 5,800 BCC or MIS excisions performed at 13 different Kaiser Permanente medical centers. The margin status was determined by searching final pathology diagnosis texts for phrases associated with positive margins. RESULTS: An incomplete excision rate was found in 23% of all specimens (BCC-22%, MIS-25%). Per specialty, the proportion of specimens with positive tumor margins was 24% for dermatology, 26% for plastic surgery, 28% for otolaryngology, and 12% for general surgery. General surgeons most often excised large tumors and tumors from truncal regions, 2 variables conferring lower odds of an incomplete excision. For non-Mohs procedures, dermatologists were no different than otolaryngologists or plastic surgeons in performing an incomplete BCC or MIS excision in all multivariate models (all p > .05). CONCLUSION: Intrinsic tumor characteristics may influence the success of achieving tumor-free resection margins more than the specialty of the provider.

Protein depalmitoylases.

Protein depalmitoylation describes the removal of thioester-linked long chain fatty acids from cysteine residues in proteins. For many S-palmitoylated proteins, this process is promoted by acyl protein thioesterase enzymes, which catalyze thioester hydrolysis to solubilize and displace substrate proteins from membranes. The closely related enzymes acyl protein thioesterase 1 (APT1; LYPLA1) and acyl protein thioesterase 2 (APT2; LYPLA2) were initially identified from biochemical assays as G protein depalmitoylases, yet later were shown to accept a number of S-palmitoylated protein and phospholipid substrates. Leveraging the development of isoform-selective APT inhibitors, several studies report distinct roles for APT enzymes in growth factor and hormonal signaling. Recent crystal structures of APT1 and APT2 reveal convergent acyl binding channels, suggesting additional factors beyond acyl chain recognition mediate substrate selection. In addition to APT enzymes, the ABHD17 family of hydrolases contributes to the depalmitoylation of Ras-family GTPases and synaptic proteins. Overall, enzymatic depalmitoylation ensures efficient membrane targeting by balancing the palmitoylation cycle, and may play additional roles in signaling, growth, and cell organization. In this review, we provide a perspective on the biochemical, structural, and cellular analysis of protein depalmitoylases, and outline opportunities for future studies of systems-wide analysis of protein depalmitoylation.

Tunable Heteroaromatic Sulfones Enhance in-Cell Cysteine Profiling.

Heteroaromatic sulfones react with cysteine via nucleophilic aromatic substitution, providing a mechanistically selective and irreversible scaffold for cysteine conjugation. Here we evaluate a library of heteroaromatic sulfides with different oxidation states, heteroatom substitutions, and a series of electron-donating and electron-withdrawing substituents. Select substitutions profoundly influence reactivity and stability compared to conventional cysteine conjugation reagents, increasing the reaction rate by >3 orders of magnitude. The findings establish a series of synthetically accessible electrophilic scaffolds tunable across multiple centers. New electrophiles and their corresponding alkyne conjugates were profiled directly in cultured cells, achieving thiol saturation in a few minutes at submillimolar concentrations. Direct addition of desthiobiotin-functionalized probes to cultured cells simplified enrichment and elution to enable the mass spectrometry discovery of >3000 reactive and/or accessible thiols labeled in their native cellular environments in a fraction of the standard analysis time. Surprisingly, only half of the annotated cysteines were identified by both iodoacetamide-desthiobiotin and methylsulfonylbenzothiazole-desthiobiotin in replicate experiments, demonstrating complementary detection by mass spectrometry analysis. These probes offer advantages over existing cysteine alkylation reagents, including accelerated reaction rates, improved stability, and robust ionization for mass spectrometry applications. Overall, heteroaromatic sulfones provide modular tunability, shifted chromatographic elution times, and superior in-cell cysteine profiling for in-depth proteome-wide analysis and covalent ligand discovery.

A pilot clinical trial of a near-infrared laser vaccine adjuvant: safety, tolerability, and cutaneous immune cell trafficking.

Many vaccines require adjuvants to enhance immunogenicity, but there are few safe and effective intradermal (i.d.) adjuvants. Murine studies have validated the potency of laser illumination of skin as an adjuvant for i.d. vaccination with advantages over traditional adjuvants. We report a pilot clinical trial of low-power, continuous-wave, near-infrared laser adjuvant treatment, representing the first human trial of the safety, tolerability, and cutaneous immune cell trafficking changes produced by the laser adjuvant. In this trial we demonstrated a maximum tolerable energy dose of 300 J/cm2 to a spot on the lower back. The irradiated spot was biopsied 4 h later, as was a control spot. Paired biopsies were submitted for histomorphologic and immunohistochemical evaluation in a blinded fashion as well as quantitative PCR analysis for chemokines and cytokines. Similar to prior murine studies, highly significant reductions in CD1a+ Langerhans cells in the dermis and CD11c+ dermal dendritic cells were observed, corresponding to the increased migratory activity of these cells; changes in the epidermis were not significant. There was no evidence of skin damage. The laser adjuvant is a safe, well-tolerated adjuvant for i.d. vaccination in humans and results in significant cutaneous immune cell trafficking.-Gelfand, J. A., Nazarian, R. M., Kashiwagi, S., Brauns, T., Martin, B., Kimizuka, Y., Korek, S., Botvinick, E., Elkins, K., Thomas, L., Locascio, J., Parry, B., Kelly, K. M., Poznansky, M. C. A pilot clinical trial of a near-infrared laser vaccine adjuvant: safety, tolerability, and cutaneous immune cell trafficking.

The readability of online patient information about laser resurfacing therapy.

Because there are important distinctions between ablative and non-ablative laser resurfacing, accurate and effective patient education is paramount. However, as more patients use the internet as a resource for medical information, little is known about the content and readability of these sources. Thus, we sought to evaluate the readability of major online resources about laser resurfacing while recognizing the recommendations by the American Medical Association and National Institutes of Health. An internet search for the term "Laser Resurfacing" was performed. The first 9 results were identified, patient information from each of these 9 sites were downloaded, and a total of 25 articles were examined. Readability was analyzed using 7 different established tests. Analysis demonstrated an average grade level of at least 9th grade, with all articles exceeding the recommended 6th grade reading level, emphasizing that these resources are too challenging for many patients to read and comprehend. Such materials may hamper appropriate decision-making in patients considering the use of a laser for their dermatologic conditions. The potential detrimental effect on the opinion, participation, and satisfaction of laser resurfacing should spur dermatologists to be more critical of online patient materials and motivated to produce more appropriate resources.

Dimethyl Itaconate Is Not Metabolized into Itaconate Intracellularly.

Itaconic acid is an important metabolite produced by macrophages after stimulation with LPS. The role of itaconate in the inflammatory cascade is unclear. Here we used [13C]itaconate and dimethyl [13C]itaconate (DMI) to probe itaconate metabolism, and find that [13C]DMI is not metabolized to itaconate. [13C]Itaconate in the cell culture medium leads to elevated intracellular levels of unlabeled succinate, with no evidence of intracellular uptake. The goal of this study is to encourage the development of effective pro-drug strategies to increase the intracellular levels of itaconate, which will enable more conclusive analysis of its action on macrophages and other cell and tissue types.

DIA-SIFT: A Precursor and Product Ion Filter for Accurate Stable Isotope Data-Independent Acquisition Proteomics.

Quantitative mass spectrometry-based protein profiling is widely used to measure protein levels across different treatments or disease states, yet current mass spectrometry acquisition methods present distinct limitations. While data-independent acquisition (DIA) bypasses the stochastic nature of data-dependent acquisition (DDA), fragment spectra derived from DIA are often complex and challenging to deconvolve. In-line ion mobility separation (IMS) adds an additional dimension to increase peak capacity for more efficient product ion assignment. As a similar strategy to sequential window acquisition methods (SWATH), IMS-enabled DIA methods rival DDA methods for protein annotation. Here we evaluate IMS-DIA quantitative accuracy using stable isotope labeling by amino acids in cell culture (SILAC). Since SILAC analysis doubles the sample complexity, we find that IMS-DIA analysis is not sufficiently accurate for sensitive quantitation. However, SILAC precursor pairs share common retention and drift times, and both species cofragment to yield multiple quantifiable isotopic y-ion peak pairs. Since y-ion SILAC ratios are intrinsic for each quantified precursor, combined MS1 and y-ion ratio analysis significantly increases the total number of measurements. With increased sampling, we present DIA-SIFT ( SILAC Intrinsic Filtering Tool), a simple statistical algorithm to identify and eliminate poorly quantified MS1 and/or MS2 events. DIA-SIFT combines both MS1 and y-ion ratios, removes outliers, and provides more accurate and precise quantitation (<15% CV) without removing any proteins from the final analysis. Overall, pooled MS1 and MS2 quantitation increases sampling in IMS-DIA SILAC analyses for accurate and precise quantitation.

Free Radical Initiated Peptide Sequencing for Direct Site Localization of Sulfation and Phosphorylation with Negative Ion Mode Mass Spectrometry.

Tandem mass spectrometry (MS/MS) is the primary method for discovering, identifying, and localizing post-translational modifications (PTMs) in proteins. However, conventional positive ion mode collision induced dissociation (CID)-based MS/MS often fails to yield site-specific information for labile and acidic modifications due to low ionization efficiency in positive ion mode and/or preferential PTM loss. While a number of alternative methods have been developed to address this issue, most require specialized instrumentation or indirect detection. In this work, we present an amine-reactive TEMPO-based free radical initiated peptide sequencing (FRIPS) approach for negative ion mode analysis of phosphorylated and sulfated peptides. FRIPS-based fragmentation generates sequence informative ions for both phosphorylated and sulfated peptides with no significant PTM loss. Furthermore, FRIPS is compared to positive ion mode CID, electron transfer dissociation (ETD), as well as negative ion mode electron capture dissociation (niECD) and CID, both in terms of sequence coverage and fragmentation efficiency for phospho- and sulfo-peptides. Because FRIPS-based fragmentation has no particular instrumentation requirements and shows limited PTM loss, we propose this approach as a promising alternative to current techniques for analysis of labile and acidic PTMs.

Activated G Protein Gαs Samples Multiple Endomembrane Compartments.

Heterotrimeric G proteins are localized to the plasma membrane where they transduce extracellular signals to intracellular effectors. G proteins also act at intracellular locations, and can translocate between cellular compartments. For example, Gαs can leave the plasma membrane and move to the cell interior after activation. However, the mechanism of Gαs translocation and its intracellular destination are not known. Here we use bioluminescence resonance energy transfer (BRET) to show that after activation, Gαs rapidly associates with the endoplasmic reticulum, mitochondria, and endosomes, consistent with indiscriminate sampling of intracellular membranes from the cytosol rather than transport via a specific vesicular pathway. The primary source of Gαs for endosomal compartments is constitutive endocytosis rather than activity-dependent internalization. Recycling of Gαs to the plasma membrane is complete 25 min after stimulation is discontinued. We also show that an acylation-deacylation cycle is important for the steady-state localization of Gαs at the plasma membrane, but our results do not support a role for deacylation in activity-dependent Gαs internalization.

HDAC8 Substrates Identified by Genetically Encoded Active Site Photocrosslinking.

The histone deacetylase family comprises 18 enzymes that catalyze deacetylation of acetylated lysine residues; however, the specificity and substrate profile of each isozyme remains largely unknown. Due to transient enzyme-substrate interactions, conventional co-immunoprecipitation methods frequently fail to identify enzyme-specific substrates. Additionally, compensatory mechanisms often limit the ability of knockdown or chemical inhibition studies to achieve significant fold changes observed by acetylation proteomics methods. Furthermore, measured alterations do not guarantee a direct link between enzyme and substrate. Here we present a chemical crosslinking strategy that incorporates a photoreactive, non-natural amino acid, p-benzoyl-l-phenylalanine, into various positions of the structurally characterized isozyme histone deacetylase 8 (HDAC8). After covalent capture, co-immunoprecipitation, and mass spectrometric analysis, we identified a subset of HDAC8 substrates from human cell lysates, which were further validated for catalytic turnover. Overall, this chemical crosslinking approach identified novel HDAC8-specific substrates with high catalytic efficiency, thus presenting a general strategy for unbiased deacetylase substrate discovery.

Targeted Annotation of S-Sulfonylated Peptides by Selective Infrared Multiphoton Dissociation Mass Spectrometry.

Protein S-sulfinylation (R-SO2-) and S-sulfonylation (R-SO3-) are irreversible oxidative post-translational modifications of cysteine residues. Greater than 5% of cysteines are reported to occupy these higher oxidation states, which effectively inactivate the corresponding thiols and alter the electronic and physical properties of modified proteins. Such higher oxidation states are reached after excessive exposure to cellular oxidants, and accumulate across different disease states. Despite widespread and functionally relevant cysteine oxidation across the proteome, there are currently no robust methods to profile higher order cysteine oxidation. Traditional data-dependent liquid chromatography/tandem mass spectrometry (LC/MS/MS) methods generally miss low-occupancy modifications in complex analyses. Here, we present a data-independent acquisition (DIA) LC/MS-based approach, leveraging the high IR absorbance of sulfoxides at 10.6 µm, for selective dissociation and discovery of S-sulfonated peptides. Across peptide standards and protein digests, we demonstrate selective infrared multiphoton dissociation (IRMPD) of S-sulfonated peptides in the background of unmodified peptides. This selective DIA IRMPD LC/MS-based approach allows identification and annotation of S-sulfonated peptides across complex mixtures while providing sufficient sequence information to localize the modification site.

Variable-Velocity Traveling-Wave Ion Mobility Separation Enhancing Peak Capacity for Data-Independent Acquisition Proteomics.

High mass accuracy, data-dependent acquisition is the current standard method in mass spectrometry-based peptide annotation and quantification. In high complexity samples, limited instrument scan speeds often result in under-sampling. In contrast, all-ion data-independent acquisition methods bypass precursor selection, alternating high and low collision energies to analyze product and precursor ions across wide mass ranges. Despite capturing data for all events, peptide annotation is limited by inadequate alignment algorithms or overlapping ions. Ion mobility separation can add an orthogonal analytical dimension, reducing ion interference to improve reproducibility, peak capacity, and peptide identifications to rival modern hybrid quadrupole orbitrap systems. Despite the advantages of ion mobility separation in complex proteomics analyses, there has been no quantitative measure of ion mobility resolution in a complex proteomic sample. Here, we present TWIMExtract, a data extraction tool to export defined slices of liquid chromatography/ion mobility/mass spectrometry (LC-IM-MS) data, providing a route to quantify ion mobility resolution from a commercial traveling-wave ion mobility time-of-flight mass spectrometer. Using standard traveling-wave ion mobility parameters (600 m/s, 40 V), 90% of the annotated peptides occupied just 23% of the ion mobility drift space, yet inclusion of ion mobility nearly doubled the overall peak capacity. Relative to fixed velocity traveling-wave ion mobility settings, ramping the traveling-wave velocity increased drift space occupancy, amplifying resolution by 16%, peak capacity by nearly 50%, and peptide/protein identifications by 40%. Overall, variable-velocity traveling-wave ion mobility-mass spectrometry significantly enhances proteomics analysis in all-ion fragmentation acquisition.

Profiling Protein S-Sulfination with Maleimide-Linked Probes.

Cysteine residues are susceptible to oxidation to form S-sulfinyl (R-SO2 H) and S-sulfonyl (R-SO3 H) post-translational modifications. Here we present a simple bioconjugation strategy to label S-sulfinated proteins by using reporter-linked maleimides. After alkylation of free thiols with iodoacetamide, S-sulfinated cysteines react with maleimide to form a sulfone Michael adduct that remains stable under acidic conditions. Using this sequential alkylation strategy, we demonstrate differential S-sulfination across mouse tissue homogenates, as well as enhanced S-sulfination following pharmacological induction of endoplasmic reticulum stress, lipopolysaccharide stimulation, and inhibitors of the electron transport chain. Overall, this study reveals a broadened profile of maleimide reactivity across cysteine modifications, and outlines a simple method for profiling the physiological role of cysteine S-sulfination in disease.

State-of-the-art lasers and light treatments for vascular lesions: from red faces to vascular malformations.

Notable milestones in the treatment of vascular lesions have been achieved over the past century. Many cutaneous vascular lesions can be successfully treated with lightbased devices. In this review, we will discuss the treatment of port-wine birthmarks, lymphatic malformations, infantile hemangiomas, rosacea, venous lakes, pyogenic granulomas, cherry angiomas, and angiofibromas using lasers, total reflection amplification of spontaneous emission of radiation, intense pulsed light, and photodynamic therapy. In addition, for several of these diagnoses, we will review medical therapies that can be combined with light-based devices to provide enhanced results.

Apremilast for the treatment of psoriatic arthritis.

Psoriatic arthritis (PsA) is a chronic inflammatoryarthropathy that affects joints and entheses andis associated with psoriasis (PsO). There are fiveclinical patterns of PsA: symmetrical polyarthritis,distal interphalangeal arthropathy, asymmetricaloligoarthritis, arthritis mutilans, and spondylitis, withor without sacroiliitis. Concerning PsA, the goals oftherapy are to control inflammation, prevent articulardamage, and reduce discomfort in the affected joints.Although there are many therapeutic options forthe treatment of PsAs, physicians most often beginwith nonsteroidal anti-inflammatory drugs (NSAIDs)for mild disease. Disease-modifying anti-rheumaticdrugs (DMARDS) are reserved for moderate to severedisease. Apremilast may be a useful option for somepatients.

Harnessing Redox Cross-Reactivity To Profile Distinct Cysteine Modifications.

Cysteine S-nitrosation and S-sulfination are naturally occurring post-translational modifications (PTMs) on proteins induced by physiological signals and redox stress. Here we demonstrate that sulfinic acids and nitrosothiols react to form a stable thiosulfonate bond, and leverage this reactivity using sulfinate-linked probes to enrich and annotate hundreds of endogenous S-nitrosated proteins. In physiological buffers, sulfinic acids do not react with iodoacetamide or disulfides, enabling selective alkylation of free thiols and site-specific analysis of S-nitrosation. In parallel, S-nitrosothiol-linked probes enable enrichment and detection of endogenous S-sulfinated proteins, confirming that a single sulfinic acid can react with a nitrosothiol to form a thiosulfonate linkage. Using this approach, we find that hydrogen peroxide addition increases S-sulfination of human DJ-1 (PARK7) at Cys106, whereas Cys46 and Cys53 are fully oxidized to sulfonic acids. Comparative gel-based analysis of different mouse tissues reveals distinct profiles for both S-nitrosation and S-sulfination. Quantitative proteomic analysis demonstrates that both S-nitrosation and S-sulfination are widespread, yet exhibit enhanced occupancy on select proteins, including thioredoxin, peroxiredoxins, and other validated redox active proteins. Overall, we present a direct, bidirectional method to profile select redox cysteine modifications based on the unique nucleophilicity of sulfinic acids.

A mechanism regulating G protein-coupled receptor signaling that requires cycles of protein palmitoylation and depalmitoylation.

Reversible attachment and removal of palmitate or other long-chain fatty acids on proteins has been hypothesized, like phosphorylation, to control diverse biological processes. Indeed, palmitate turnover regulates Ras trafficking and signaling. Beyond this example, however, the functions of palmitate turnover on specific proteins remain poorly understood. Here, we show that a mechanism regulating G protein-coupled receptor signaling in neuronal cells requires palmitate turnover. We used hexadecyl fluorophosphonate or palmostatin B to inhibit enzymes in the serine hydrolase family that depalmitoylate proteins, and we studied R7 regulator of G protein signaling (RGS)-binding protein (R7BP), a palmitoylated allosteric modulator of R7 RGS proteins that accelerate deactivation of Gi/o class G proteins. Depalmitoylation inhibition caused R7BP to redistribute from the plasma membrane to endomembrane compartments, dissociated R7BP-bound R7 RGS complexes from Gi/o-gated G protein-regulated inwardly rectifying K(+) (GIRK) channels and delayed GIRK channel closure. In contrast, targeting R7BP to the plasma membrane with a polybasic domain and an irreversibly attached lipid instead of palmitate rendered GIRK channel closure insensitive to depalmitoylation inhibitors. Palmitate turnover therefore is required for localizing R7BP to the plasma membrane and facilitating Gi/o deactivation by R7 RGS proteins on GIRK channels. Our findings broaden the scope of biological processes regulated by palmitate turnover on specific target proteins. Inhibiting R7BP depalmitoylation may provide a means of enhancing GIRK activity in neurological disorders.

Substrate-Competitive Activity-Based Profiling of Ester Prodrug Activating Enzymes.

Understanding the mechanistic basis of prodrug delivery and activation is critical for establishing species-specific prodrug sensitivities necessary for evaluating preclinical animal models and potential drug-drug interactions. Despite significant adoption of prodrug methodologies for enhanced pharmacokinetics, functional annotation of prodrug activating enzymes is laborious and often unaddressed. Activity-based protein profiling (ABPP) describes an emerging chemoproteomic approach to assay active site occupancy within a mechanistically similar enzyme class in native proteomes. The serine hydrolase enzyme family is broadly reactive with reporter-linked fluorophosphonates, which have shown to provide a mechanism-based covalent labeling strategy to assay the activation state and active site occupancy of cellular serine amidases, esterases, and thioesterases. Here we describe a modified ABPP approach using direct substrate competition to identify activating enzymes for an ethyl ester prodrug, the influenza neuraminidase inhibitor oseltamivir. Substrate-competitive ABPP analysis identified carboxylesterase 1 (CES1) as an oseltamivir-activating enzyme in intestinal cell homogenates. Saturating concentrations of oseltamivir lead to a four-fold reduction in the observed rate constant for CES1 inactivation by fluorophosphonates. WWL50, a reported carbamate inhibitor of mouse CES1, blocked oseltamivir hydrolysis activity in human cell homogenates, confirming CES1 is the primary prodrug activating enzyme for oseltamivir in human liver and intestinal cell lines. The related carbamate inhibitor WWL79 inhibited mouse but not human CES1, providing a series of probes for analyzing prodrug activation mechanisms in different preclinical models. Overall, we present a substrate-competitive activity-based profiling approach for broadly surveying candidate prodrug hydrolyzing enzymes and outline the kinetic parameters for activating enzyme discovery, ester prodrug design, and preclinical development of ester prodrugs.

Global profiling of dynamic protein palmitoylation.

The reversible thioester linkage of palmitic acid on cysteines, known as protein S-palmitoylation, facilitates the membrane association and proper subcellular localization of proteins. Here we report the metabolic incorporation of the palmitic acid analog 17-octadecynoic acid (17-ODYA) in combination with stable-isotope labeling with amino acids in cell culture (SILAC) and pulse-chase methods to generate a global quantitative map of dynamic protein palmitoylation events in cells. We distinguished stably palmitoylated proteins from those that turn over rapidly. Treatment with a serine lipase-selective inhibitor identified a pool of dynamically palmitoylated proteins regulated by palmitoyl-protein thioesterases. This subset was enriched in oncoproteins and other proteins linked to aberrant cell growth, migration and cancer. Our method provides a straightforward way to characterize global palmitoylation dynamics in cells and confirms enzyme-mediated depalmitoylation as a critical regulatory mechanism for a specific subset of rapidly cycling palmitoylated proteins.