The surfaceome of multiple myeloma cells suggests potential immunotherapeutic strategies and protein markers of drug resistance.

The myeloma surface proteome (surfaceome) determines tumor interaction with the microenvironment and serves as an emerging arena for therapeutic development. Here, we use glycoprotein capture proteomics to define the myeloma surfaceome at baseline, in drug resistance, and in response to acute drug treatment. We provide a scoring system for surface antigens and identify CCR10 as a promising target in this disease expressed widely on malignant plasma cells. We engineer proof-of-principle chimeric antigen receptor (CAR) T-cells targeting CCR10 using its natural ligand CCL27. In myeloma models we identify proteins that could serve as markers of resistance to bortezomib and lenalidomide, including CD53, CD10, EVI2B, and CD33. We find that acute lenalidomide treatment increases activity of MUC1-targeting CAR-T cells through antigen upregulation. Finally, we develop a miniaturized surface proteomic protocol for profiling primary plasma cell samples with low inputs. These approaches and datasets may contribute to the biological, therapeutic, and diagnostic understanding of myeloma.

Practice Diversity and Burn Center Growth: A 10-Year Profile of a State's Only Burn Center.

BACKGROUND: Growth and sustainability of burn practices can be impaired by irregular patterns of patient presentations, resulting in uneven utilization of facilities and staff. Burn care itself may not engage the full capacities of members of burn care teams. To address these problems, we organized a burn and reconstruction center to provide statewide acute care as Mississippi's only burn unit, to fully integrate reconstructive surgery into management of burn patients, and to diversify practice based on plastic surgery scope of practice. The first 10 years of this unit were reviewed to evaluate the performance of this scheme. METHODS: Burn admissions to and surgical procedures at this unit between July 2009 and June 2019 were analyzed to quantify acute burn care, secondary reconstructive burn care, and categories of practice growth. RESULTS: The unit admitted 5469 acute burn patients with a mortality rate of 1.49%. Comparing year 10 to year 1 of practice, acute burn admissions increased 58%. Total operations increased 276%. Acute burn procedures increased 176%. Secondary burn procedures increased 405%. Nonburn procedures increased 352%, with the subset of nonburn hand surgery increasing 1062%. CONCLUSION: Acute burn admissions and procedures increased over this period, but greater growth was seen in secondary burn procedures and nonburn procedures, especially hand cases. Expansion of practice into areas within the overall skill sets of burn team members was an effective growth strategy.

LC3 and STRAP regulate actin filament assembly by JMY during autophagosome formation.

During autophagy, actin filament networks move and remodel cellular membranes to form autophagosomes that enclose and metabolize cytoplasmic contents. Two actin regulators, WHAMM and JMY, participate in autophagosome formation, but the signals linking autophagy to actin assembly are poorly understood. We show that, in nonstarved cells, cytoplasmic JMY colocalizes with STRAP, a regulator of JMY's nuclear functions, on nonmotile vesicles with no associated actin networks. Upon starvation, JMY shifts to motile, LC3-containing membranes that move on actin comet tails. LC3 enhances JMY's de novo actin nucleation activity via a cryptic actin-binding sequence near JMY's N terminus, and STRAP inhibits JMY's ability to nucleate actin and activate the Arp2/3 complex. Cytoplasmic STRAP negatively regulates autophagy. Finally, we use purified proteins to reconstitute LC3- and JMY-dependent actin network formation on membranes and inhibition of network formation by STRAP. We conclude that LC3 and STRAP regulate JMY's actin assembly activities in trans during autophagy.

Cryo-EM structure of the bacterial actin AlfA reveals unique assembly and ATP-binding interactions and the absence of a conserved subdomain.

Bacterial actins are an evolutionarily diverse family of ATP-dependent filaments built from protomers with a conserved structural fold. Actin-based segregation systems are encoded on many bacterial plasmids and function to partition plasmids into daughter cells. The bacterial actin AlfA segregates plasmids by a mechanism distinct from other partition systems, dependent on its unique dynamic properties. Here, we report the near-atomic resolution electron cryo-microscopy structure of the AlfA filament, which reveals a strikingly divergent filament architecture resulting from the loss of a subdomain conserved in all other actins and a mode of ATP binding. Its unusual assembly interfaces and nucleotide interactions provide insight into AlfA dynamics, and expand the range of evolutionary variation accessible to actin quaternary structure.

Cold urticaria: a 20-year follow-up study.

BACKGROUND: Chronic cold urticaria results in significant morbidity, yet information on its natural history is limited. OBJECTIVE: We examined the natural history of chronic cold urticaria and its impact on quality of life. METHODS: We analysed the characteristics of patients diagnosed with cold urticaria at a community-based specialist allergy practice in the Australian Capital Territory (ACT) between 1995 and 2015. Follow-up data were obtained using a mailed questionnaire. Possible predictive factors of disease severity and symptom duration were evaluated. RESULTS: A total of 99 patients were assessed with a median age of 42 (range 5-81 years); 63% were female and the median age of onset of symptoms was 22 years. Of 41 questionnaire responders (14 ± 10.9 years follow-up; median 12 years), 5- and 10-year resolution rates were 17.9% ± 6.2% and 24.5% ± 7.2%, respectively. Whereas 22% reported resolution and 23% described improvement, the remaining 55% reported stable or worsening disease. Most individuals relied on lifestyle modification to ameliorate symptoms rather than medication. Risk factors for persistent disease were intercurrent atopic disease (P = 0.025) and those with longer duration of symptoms at the time of initial assessment (P < 0.001). Secondary causes of cold urticaria were identified in only two patients, both with B-cell malignancy. CONCLUSION: In a subset of patients, cold urticaria has low rates of spontaneous resolution and results in lifestyle changes and impaired quality of life.

Treatment of oral dysplasia with 5% imiquimod cream: short communication.

We report what we think is the first treatment of oral dysplasia with 5% imiquimod cream. A 60-year-old man presented with varying degrees of dysplasia on the soft palate. A cover plate was fabricated and the patient was prescribed 5% imiquimod cream, a topical imunomodulator, for six weeks. The lesion improved and histological examination of an incisional biopsy found no features of dysplasia. This case highlights the efficacy of imiquimod cream in the treatment of dysplasia, and the need for development of a preparation suitable for the oral mucosa.

Actomyosin dynamics drive local membrane component organization in an in vitro active composite layer.

The surface of a living cell provides a platform for receptor signaling, protein sorting, transport, and endocytosis, whose regulation requires the local control of membrane organization. Previous work has revealed a role for dynamic actomyosin in membrane protein and lipid organization, suggesting that the cell surface behaves as an active composite composed of a fluid bilayer and a thin film of active actomyosin. We reconstitute an analogous system in vitro that consists of a fluid lipid bilayer coupled via membrane-associated actin-binding proteins to dynamic actin filaments and myosin motors. Upon complete consumption of ATP, this system settles into distinct phases of actin organization, namely bundled filaments, linked apolar asters, and a lattice of polar asters. These depend on actin concentration, filament length, and actin/myosin ratio. During formation of the polar aster phase, advection of the self-organizing actomyosin network drives transient clustering of actin-associated membrane components. Regeneration of ATP supports a constitutively remodeling actomyosin state, which in turn drives active fluctuations of coupled membrane components, resembling those observed at the cell surface. In a multicomponent membrane bilayer, this remodeling actomyosin layer contributes to changes in the extent and dynamics of phase-segregating domains. These results show how local membrane composition can be driven by active processes arising from actomyosin, highlighting the fundamental basis of the active composite model of the cell surface, and indicate its relevance to the study of membrane organization.

Design of reversible, cysteine-targeted Michael acceptors guided by kinetic and computational analysis.

Electrophilic probes that covalently modify a cysteine thiol often show enhanced pharmacological potency and selectivity. Although reversible Michael acceptors have been reported, the structural requirements for reversibility are poorly understood. Here, we report a novel class of acrylonitrile-based Michael acceptors, activated by aryl or heteroaryl electron-withdrawing groups. We demonstrate that thiol adducts of these acrylonitriles undergo ß-elimination at rates that span more than 3 orders of magnitude. These rates correlate inversely with the computed proton affinity of the corresponding carbanions, enabling the intrinsic reversibility of the thiol-Michael reaction to be tuned in a predictable manner. We apply these principles to the design of new reversible covalent kinase inhibitors with improved properties. A cocrystal structure of one such inhibitor reveals specific noncovalent interactions between the 1,2,4-triazole activating group and the kinase. Our experimental and computational study enables the design of new Michael acceptors, expanding the palette of reversible, cysteine-targeted electrophiles.

CEP290 gene transfer rescues Leber congenital amaurosis cellular phenotype.

Mutations in CEP290 are the most common cause of Leber congenital amaurosis (LCA), a severe inherited retinal degenerative disease for which there is currently no cure. Autosomal recessive CEP290-associated LCA is a good candidate for gene replacement therapy, and cells derived from affected individuals give researchers the ability to study human disease and therapeutic gene correction in vitro. Here we report the development of lentiviral vectors carrying full-length CEP290 for the purpose of correcting the CEP290 disease-specific phenotype in human cells. A lentiviral vector containing CMV-driven human full-length CEP290 was constructed. Following transduction of patient-specific, iPSC-derived, photoreceptor precursor cells, reverse transcriptase-PCR analysis and western blotting revealed vector-derived expression. As CEP290 is important in ciliogenesis, the ability of fibroblast cultures from CEP290-associated LCA patients to form cilia was investigated. In cultures derived from these patients, fewer cells formed cilia compared with unaffected controls. Cilia that were formed were shorter in patient-derived cells than in cells from unaffected individuals. Importantly, lentiviral delivery of CEP290 rescued the ciliogenesis defect. The successful construction and viral transfer of full-length CEP290 brings us closer to the goal of providing gene- and cell-based therapies for patients affected with this common form of LCA.

Subtotal epiglottectomy for the management of epiglottic retroversion in a dog.

A six-year-old male neutered Yorkshire terrier was evaluated for severe, acute-onset, inspiratory dyspnoea. Laryngoscopy revealed retroversion of the epiglottis with intermittent occlusion of the rima glottidis during inspiration. The dog underwent both temporary and permanent epiglottopexy procedures that were unsuccessful. Subtotal epiglottectomy was performed and resulted in permanent resolution of dyspnoea without evidence of dysphagia or aspiration. This case highlights potential complications associated with management of this condition as well as describing the successful use of subtotal epiglottectomy in the dog.

Promoting cervical screening after introduction of the human papillomavirus vaccine: the effect of repeated mass media campaigns.

OBJECTIVES: In 2007, Australia implemented an ongoing, school-based human papillomavirus (HPV) vaccination programme for school-aged girls, and a catch-up programme for 18-26-year-old women that ran until the end of 2009. The availability of what is widely known as the 'cervical cancer vaccine' means there is the potential for women to believe, incorrectly, that they no longer require regular cervical screening, and this risk needs to be addressed. The current study aimed to assess the effect of three mass media campaigns to promote cervical screening on the rate of cervical screening tests in the Australian state of Victoria, after HPV vaccine became available. METHODS: Seasonal time series assessed the effect of media campaigns broadcast in 2007, 2009 and 2010 on the rate of weekly cervical screening tests in Victorian women from 2006 to 2010, stratified by time since last screening test. RESULTS: The 2007 and 2009 media campaigns significantly increased the number of cervical screening tests per week. The 2007 campaign had a significant impact on lapsed screeners (>36 months since last test), overdue screeners (28-36 months since last test), and women never previously screened. The 2009 campaign significantly increased screening tests for overdue screeners, and the 2010 media campaign was associated with a significant increase in screening tests for lapsed screeners. CONCLUSIONS: A well-researched and carefully pretested television advertising campaign with accurate, actionable messages can elicit appropriate screening behaviour among some of the appropriate groups even in a changed environment of complex, and potentially competing, messages.

Visualization of actin filaments and monomers in somatic cell nuclei.

In addition to its long-studied presence in the cytoplasm, actin is also found in the nuclei of eukaryotic cells. The function and form (monomer, filament, or noncanonical oligomer) of nuclear actin are hotly debated, and its localization and dynamics are largely unknown. To determine the distribution of nuclear actin in live somatic cells and evaluate its potential functions, we constructed and validated fluorescent nuclear actin probes. Monomeric actin probes concentrate in nuclear speckles, suggesting an interaction of monomers with RNA-processing factors. Filamentous actin probes recognize discrete structures with submicron lengths that are excluded from chromatin-rich regions. In time-lapse movies, these actin filament structures exhibit one of two types of mobility: 1) diffusive, with an average diffusion coefficient of 0.06-0.08 µm(2)/s, or (2) subdiffusive, with a mobility coefficient of 0.015 µm(2)/s. Individual filament trajectories exhibit features of particles moving within a viscoelastic mesh. The small size of nuclear actin filaments is inconsistent with a role in micron-scale intranuclear transport, and their localization suggests that they do not participate directly in chromatin-based processes. Our results instead suggest that actin filaments form part of a large, viscoelastic structure in the nucleoplasm and may act as scaffolds that help organize nuclear contents.

Arp2/3 complex ATP hydrolysis promotes lamellipodial actin network disassembly but is dispensable for assembly.

We examined the role of ATP hydrolysis by the Arp2/3 complex in building the leading edge of a cell by studying the effects of hydrolysis defects on the behavior of the complex in the lamellipodial actin network of Drosophila S2 cells and in a reconstituted, in vitro, actin-based motility system. In S2 cells, nonhydrolyzing Arp2 and Arp3 subunits expanded and delayed disassembly of lamellipodial actin networks and the effect of mutant subunits was additive. Arp2 and Arp3 ATP hydrolysis mutants remained in lamellipodial networks longer and traveled greater distances from the plasma membrane, even in networks still containing wild-type Arp2/3 complex. In vitro, wild-type and ATP hydrolysis mutant Arp2/3 complexes each nucleated actin and built similar dendritic networks. However, networks constructed with Arp2/3 hydrolysis-defective mutants were more resistant to disassembly by cofilin. Our results indicate that ATP hydrolysis on both Arp2 and Arp3 contributes to dissociation of the complex from the actin network but is not strictly necessary for lamellipodial network disassembly.

Differential remodeling of actin cytoskeleton architecture by profilin isoforms leads to distinct effects on cell migration and invasion.

Dynamic actin cytoskeletal reorganization is integral to cell motility. Profilins are well-characterized regulators of actin polymerization; however, functional differences among coexpressed profilin isoforms are not well defined. Here, we demonstrate that profilin-1 and profilin-2 differentially regulate membrane protrusion, motility, and invasion; these processes are promoted by profilin-1 and suppressed by profilin-2. Compared to profilin-1, profilin-2 preferentially drives actin polymerization by the Ena/VASP protein, EVL. Profilin-2 and EVL suppress protrusive activity and cell motility by an actomyosin contractility-dependent mechanism. Importantly, EVL or profilin-2 downregulation enhances invasion in vitro and in vivo. In human breast cancer, lower EVL expression correlates with high invasiveness and poor patient outcome. We propose that profilin-2/EVL-mediated actin polymerization enhances actin bundling and suppresses breast cancer cell invasion.

Recombinant human thrombin: safety and immunogenicity in pediatric burn wound excision.

BACKGROUND: Previous studies of recombinant human thrombin (rThrombin) enrolled adult and adolescent patients. This phase 4, open-label, single-group study was conducted in pediatric patients undergoing synchronous burn wound excision and skin grafting to provide information regarding the safety and immunogenicity of rThrombin (primary and secondary endpoints) in this population. METHODS: Topical rThrombin was applied as a hemostatic aid during a surgical procedure (day 1). Adverse events and clinical laboratory abnormalities were recorded during the study. Immunogenicity samples were collected on days 1 and 29 (study end). Study results were summarized with descriptive statistics. RESULTS: Thirty patients enrolled and 28 completed the study. Mean age was 6.9 years (range, 0.9-17.8 years); 40.0% of patients were girls. Flame and scald were the most common burn types (33.3% each, n = 10/30). Mean graft size was 3.6% total body surface area. Procedural pain (50.0% patients), pruritus (43.3%), and anemia (30.0%) were the most commonly reported adverse events. All adverse events and clinical laboratory abnormalities were considered unrelated to treatment. No patients developed anti-rThrombin product antibodies at day 29. CONCLUSIONS: In pediatric patients undergoing burn wound excision and skin grafting, rThrombin was well tolerated and did not lead to the formation of anti-rThrombin product antibodies.

Architecture and assembly of a divergent member of the ParM family of bacterial actin-like proteins.

Eubacteria and archaea contain a variety of actin-like proteins (ALPs) that form filaments with surprisingly diverse architectures, assembly dynamics, and cellular functions. Although there is much data supporting differences between ALP families, there is little data regarding conservation of structure and function within these families. We asked whether the filament architecture and biochemical properties of the best-understood prokaryotic actin, ParM from plasmid R1, are conserved in a divergent member of the ParM family from plasmid pB171. Previous work demonstrated that R1 ParM assembles into filaments that are structurally distinct from actin and the other characterized ALPs. They also display three biophysical properties thought to be essential for DNA segregation: 1) rapid spontaneous nucleation, 2) symmetrical elongation, and 3) dynamic instability. We used microscopic and biophysical techniques to compare and contrast the architecture and assembly of these related proteins. Despite being only 41% identical, R1 and pB171 ParMs polymerize into nearly identical filaments with similar assembly dynamics. Conservation of the core assembly properties argues for their importance in ParM-mediated DNA segregation and suggests that divergent DNA-segregating ALPs with different assembly properties operate via different mechanisms.

Structural polymorphism of the ParM filament and dynamic instability.

Segregation of the R1 plasmid in bacteria relies on ParM, an actin homolog that segregates plasmids by switching between cycles of polymerization and depolymerization. We find similar polymerization kinetics and stability in the presence of either ATP or GTP and a 10-fold affinity preference for ATP over GTP. We used electron cryo-microscopy to evaluate the heterogeneity within ParM filaments. In addition to variable twist, ParM has variable axial rise, and both parameters are coupled. Subunits in the same ParM filaments can exist in two different structural states, with the nucleotide-binding cleft closed or open, and the bound nucleotide biases the distribution of states. The interface between protomers is different between these states, and in neither state is it similar to F-actin. Our results suggest that the closed state of the cleft is required but not sufficient for ParM polymerization, and provide a structural basis for the dynamic instability of ParM filaments.

The structure and assembly dynamics of plasmid actin AlfA imply a novel mechanism of DNA segregation.

Bacterial cytoskeletal proteins participate in a variety of processes, including cell division and DNA segregation. Polymerization of one plasmid-encoded, actin-like protein, ParM, segregates DNA by pushing two plasmids in opposite directions and forms the current paradigm for understanding active plasmid segregation. An essential feature of ParM assembly is its dynamically instability, the stochastic switching between growth and disassembly. It is unclear whether dynamic instability is an essential feature of all actin-like protein-based segregation mechanisms or whether bacterial filaments can segregate plasmids by different mechanisms. We expressed and purified AlfA, a plasmid-segregating actin-like protein from Bacillus subtilis, and found that it forms filaments with a unique structure and biochemistry; AlfA nucleates rapidly, polymerizes in the presence of ATP or GTP, and forms highly twisted, ribbon-like, helical filaments with a left-handed pitch and protomer nucleotide binding pockets rotated away from the filament axis. Intriguingly, AlfA filaments spontaneously associate to form uniformly sized, mixed-polarity bundles. Most surprisingly, our biochemical characterization revealed that AlfA does not display dynamic instability and is relatively stable in the presence of diphosphate nucleotides. These results (i) show that there is remarkable structural diversity among bacterial actin filaments and (ii) indicate that AlfA filaments partition DNA by a novel mechanism.

p53-cofactor JMY is a multifunctional actin nucleation factor.

Many cellular structures are assembled from networks of actin filaments, and the architecture of these networks depends on the mechanism by which the filaments are formed. Several classes of proteins are known to assemble new filaments, including the Arp2/3 complex, which creates branched filament networks, and Spire, which creates unbranched filaments. We find that JMY, a vertebrate protein first identified as a transcriptional co-activator of p53, combines these two nucleating activities by both activating Arp2/3 and assembling filaments directly using a Spire-like mechanism. Increased levels of JMY expression enhance motility, whereas loss of JMY slows cell migration. When slowly migrating HL-60 cells are differentiated into highly motile neutrophil-like cells, JMY moves from the nucleus to the cytoplasm and is concentrated at the leading edge. Thus, JMY represents a new class of multifunctional actin assembly factor whose activity is regulated, at least in part, by sequestration in the nucleus.

Macrophages in neovascular age-related macular degeneration: friends or foes?

The events that lead to choroidal neovascularization in eyes with age-related macular degeneration are poorly understood. One possibility that has been explored in a number of studies is that macrophages can promote neovascular changes. In this paper, we summarize the evidence for inflammation in general and macrophages in particular in pathologic neovascularization, and discuss how the diverse functions of these cells may promote or inhibit macular disease. We also discuss some of the conflicting findings regarding the role of macrophages in experimental choroidal neovascularization in mouse models, and suggest areas for future research.