Roles of a Glycolipid MPIase in Sec-Independent Membrane Protein Insertion.

Membrane protein integrase (MPIase), an endogenous glycolipid in Escherichia coli (E. coli) membranes, is essential for membrane protein insertion in E. coli. We have examined Sec-independent membrane protein insertion mechanisms facilitated by MPIase using physicochemical analytical techniques, namely solid-state nuclear magnetic resonance, fluorescence measurements, and surface plasmon resonance. In this review, we outline the physicochemical characteristics of membranes that may affect membrane insertion of proteins. Subsequently, we introduce our results verifying the effects of membrane lipids on insertion and estimate the impact of MPIase. Although MPIase is a minor component of E. coli membranes, it regulates insertion by altering the physicochemical properties of the membrane. In addition, MPIase promotes insertion by interacting with substrate proteins. We propose comprehensive mechanisms for the membrane insertion of proteins involving MPIase, which provide a physicochemical basis for understanding the roles of glycolipids in protein translocation.

Horrifying Basal Cell Carcinoma Presenting as Progressive Pyoderma Gangrenosum: Case Report.

Introduction: Skin ulcers can be challenging to diagnose and manage, particularly with comorbid autoimmune and gastrointestinal diseases. Occam's razor encourages the simplest explanation to guide care, but reconsideration must occur when intervention proves futile. Case Presentation: We report the case of a 70-year-old male, with a 17-year history of expanding pretibial skin ulcer, presumed by prior care providers to be pyoderma gangrenosum related to Crohn's disease. A surgical biopsy performed upon presentation to our institution revealed basal cell carcinoma of the skin, invasive to the proximal tibia with associated deep infection, prompting transfemoral amputation. Conclusion: This report is written as a reminder to reconsider a diagnosis and consider seeking additional expertise when a patient's condition progressively worsens despite intervention. Earlier diagnosis likely would have facilitated therapeutic limb salvage care.

Bacterial Glycolipid Acting on Protein Transport Across Membranes.

The process of protein transport across membranes involves a variety of factors and has been extensively investigated. Traditionally, proteinaceous translocons and chaperones have been recognized as crucial factors in this process. However, recent studies have highlighted the significant roles played by lipids and a glycolipid present in biological membranes in membrane protein transport. Membrane lipids can influence transport efficiency by altering the physicochemical properties of membranes. Notably, our studies have revealed that diacylglycerol (DAG) attenuates mobility in the membrane core region, leading to a dramatic suppression of membrane protein integration. Conversely, a glycolipid in Escherichia coli inner membranes, named membrane protein integrase (MPIase), enhances integration not only through the alteration of membrane properties but also via direct interactions with membrane proteins. This review explores the mechanisms of membrane protein integration mediated by membrane lipids, specifically DAG, and MPIase. Our results, along with the employed physicochemical analysis methods such as fluorescence measurements, nuclear magnetic resonance, surface plasmon resonance, and docking simulation, are presented to elucidate these mechanisms.

Mechanism of rate controllability of water-soluble bifunctional cyclooctadiynes through cation-anion interactions.

Click reactions are used for chemoselective functionalization in many research fields. Despite the utility of small, bioinert azide groups as a counterpart, applications of strain-promoted alkyne-azide cycloaddition (SPAAC) reactions for this purpose are still limited by slow reaction kinetics. Here, we report ion-pair-guided reaction rate enhancement by the use of water-soluble cyclooctadiynes (WS-CODYs) composed of bifunctional strained alkynes and polar side chains. Arrhenius plot analysis revealed that the rate enhancement by WS-CODYs is due to a kinetic salt effect between the polar substituents and the target azide. We demonstrate the utility of these compounds for rapid protein labelling and isoelectric point-dependent labelling.

Staging System Performance and Clinical Outcomes for Cutaneous Squamous Cell Carcinoma of the Ear: A Single-Center Retrospective Study.

BACKGROUND: Cutaneous squamous cell carcinoma (cSCC) of the ear is associated with poor outcomes. No studies have evaluated current staging system performance in this specific location. OBJECTIVE: Describe clinicopathologic characteristics and outcomes of ear cSCC and evaluate the performance of current staging systems. METHODS: Retrospective study including cases diagnosed and treated at a cancer center from January 2000 to December 2014. Demographic, clinical, and pathologic data were collected from clinical records. Biopsy slides were rereviewed and patients were staged according to the American Joint Committee on Cancer (AJCC) seventh, eighth, and Brigham Women's Hospital (BWH) staging. RESULTS: Of 125 patients, the mean age at diagnosis was 71.9 years (SD 12.5), with most men (89.6%, n = 112). Median follow-up was 22.3 months. Local recurrence and survival risk factors were similar to cSCC outside the ear. The Akaike's Information Criterion (AIC) estimates showed that the BWH system better predicted outcomes than the AJCC seventh, and the AJCC eighth, with AIC values of 189.9, 270.5, and 274.1, respectively. Limitations of the study include retrospective design, single center study, and no control group. CONCLUSION: Current staging systems perform well at stratifying risk in ear cSCC.

Structural Requirements of a Glycolipid MPIase for Membrane Protein Integration.

MPIase is a glycolipid involved in membrane protein integration in the inner membrane of Escherichia coli. To overcome the trace amounts and heterogeneity of natural MPIase, we systematically synthesized MPIase analogs. Structure-activity relationship studies revealed the contribution of distinctive functional groups and the effect of the MPIase glycan length on membrane protein integration activity. In addition, both the synergistic effects of these analogs with the membrane chaperone/insertase YidC, and the chaperone-like activity of the phosphorylated glycan were observed. These results verified the translocon-independent membrane integration mechanism in the inner membrane of E. coli, in which MPIase captures the highly hydrophobic nascent proteins via its characteristic functional groups, prevents protein aggregation, attracts the proteins to the membrane surface, and delivers them to YidC in order to regenerate its own integration activity.

A bacterial glycolipid essential for membrane protein integration.

The proper conformation and orientation of membrane protein integration in cells is an important biological event. Interestingly, a new factor named MPIase (membrane protein integrase) was proven essential in this process in Escherichia coli, besides proteinaceous factors, such as Sec translocons and an insertase YidC. A combination of spectroscopic analyses and synthetic work has revealed that MPIase is a glycolipid despite its enzyme-like activity. MPIase has a long glycan chain comprised of repeating trisaccharide units, a pyrophosphate linker, and a diacylglycerol anchor. In order to determine the mechanism of its activity, we synthesized a trisaccharyl pyrophospholipid termed mini-MPIase-3, a minimal unit of MPIase, and its derivatives. A significant activity of mini-MPIase-3 indicated that it involves an essential structure for membrane protein integration. We also analyzed intermolecular interactions of MPIase or its synthetic analogs with a model substrate protein using physicochemical methods. The structure-activity relationship studies demonstrated that the glycan part of MPIase prevents the aggregation of substrate proteins, and the 6-O-acetyl group on glucosamine and the phosphate of MPIase play important roles for interactions with substrate proteins. MPIase serves at an initial step in the Sec-independent integration, whereas YidC, proton motive force, and/or SecYEG cooperatively function(s) with MPIase at the following step in vivo. Furthermore, depletion of the biosynthetic enzyme demonstrated that MPIase is crucial for membrane protein integration and cell growth. Thus, we elucidated new biological functions of glycolipids using a combination of synthetic chemistry, biochemistry, physicochemical measurements, and molecular-biological approaches.

Interaction between glycolipid MPIase and proteinaceous factors during protein integration into the cytoplasmic membrane of E. coli.

Protein integration into biomembranes is an essential biological phenomenon common to all organisms. While various factors involved in protein integration, such as SRP, SecYEG and YidC, are proteinaceous, we identified a glycolipid named MPIase (Membrane Protein Integrase), which is present in the cytoplasmic membrane of E. coli. In vitro experiments using inverted membrane vesicles prepared from MPIase-depleted strains, and liposomes containing MPIase showed that MPIase is required for insertion of a subset of membrane proteins, which has been thought to be SecYEG-independent and YidC-dependent. Also, SecYEG-dependent substrate membrane proteins require MPIase in addition. Furthermore, MPIase is also essential for insertion of proteins with multiple negative charges, which requires both YidC and the proton motive force (PMF). MPIase directly interacts with SecYEG and YidC on the membrane. MPIase not only cooperates with these factors but also has a molecular chaperone-like function specific to the substrate membrane proteins through direct interaction with the glycan chain. Thus, MPIase catalyzes membrane insertion by accepting nascent membrane proteins on the membrane through its chaperone-like function, i.e., direct interaction with the substrate proteins, and then MPIase functionally interacts with SecYEG and YidC for substrate delivery, and acts with PMF to facilitate and complete membrane insertion when necessary. In this review, we will outline the mechanisms underlying membrane insertion catalyzed by MPIase, which cooperates with proteinaceous factors and PMF.

Role of a bacterial glycolipid in Sec-independent membrane protein insertion.

Non-proteinaceous components in membranes regulate membrane protein insertion cooperatively with proteinaceous translocons. An endogenous glycolipid in the Escherichia coli membrane called membrane protein integrase (MPIase) is one such component. Here, we focused on the Sec translocon-independent pathway and examined the mechanisms of MPIase-facilitated protein insertion using physicochemical techniques. We determined the membrane insertion efficiency of a small hydrophobic protein using solid-state nuclear magnetic resonance, which showed good agreement with that determined by the insertion assay using an in vitro translation system. The observed insertion efficiency was strongly correlated with membrane physicochemical properties measured using fluorescence techniques. Diacylglycerol, a trace component of E. coli membrane, reduced the acyl chain mobility in the core region and inhibited the insertion, whereas MPIase restored them. We observed the electrostatic intermolecular interactions between MPIase and the side chain of basic amino acids in the protein, suggesting that the negatively charged pyrophosphate of MPIase attracts the positively charged residues of a protein near the membrane surface, which triggers the insertion. Thus, this study demonstrated the ingenious approach of MPIase to support membrane insertion of proteins by using its unique molecular structure in various ways.

Intermolecular Interactions between a Membrane Protein and a Glycolipid Essential for Membrane Protein Integration.

Inducing newly synthesized proteins to appropriate locations is an indispensable biological function in every organism. Integration of proteins into biomembranes in Escherichia coli is mediated by proteinaceous factors, such as Sec translocons and an insertase YidC. Additionally, a glycolipid named MPIase (membrane protein integrase), composed of a long sugar chain and pyrophospholipid, was proven essential for membrane protein integration. We reported that a synthesized minimal unit of MPIase possessing only one trisaccharide, mini-MPIase-3, involves an essential structure for the integration activity. Here, to elucidate integration mechanisms using MPIase, we analyzed intermolecular interactions of MPIase or its synthetic analogs with a model substrate, the Pf3 coat protein, using physicochemical methods. Surface plasmon resonance (SPR) analyses revealed the importance of a pyrophosphate for affinity to the Pf3 coat protein. Compared with mini-MPIase-3, natural MPIase showed faster association and dissociation due to its long sugar chain despite the slight difference in affinity. To focus on more detailed MPIase substructures, we performed docking simulations and saturation transfer difference-nuclear magnetic resonance. These experiments yielded that the 6-O-acetyl group on glucosamine and the phosphate of MPIase play important roles leading to interactions with the Pf3 coat protein. The high affinity of MPIase to the hydrophobic region and the basic amino acid residues of the protein was suggested by docking simulations and proven experimentally by SPR using protein mutants devoid of target regions. These results demonstrated the direct interactions of MPIase with a substrate protein and revealed detailed mechanisms of membrane protein integration.

Association of interleukin-6 and tumor necrosis factor-α with mortality in hospitalized patients with cancer.

BACKGROUND: Severe cutaneous adverse reactions (SCARs) are associated with high morbidity and mortality in patients with cancer. Early identification and treatment of SCARs may improve outcomes. OBJECTIVE: To identify biomarkers to predict outcomes in hospitalized patients with cancer who developed SCARs. METHODS: Retrospective review of 144 hospitalized patients with cancer with a morbilliform rash, recorded testing for serum cytokines (interleukin [IL]-6, IL-10, and tumor necrosis factor [TNF]-α) or elafin, and a dermatology consultation. Rashes were categorized as simple morbilliform rash without systemic involvement or complex morbilliform rash with systemic involvement. RESULTS: Fifty-four of 144 (37.5%) patients died during follow-up. Elevated levels of IL-6, IL-10, and TNF-α were associated with decreased survival. Overall survivals in patients with elevated levels of IL-6, IL-10, and TNF-α were 53.7%, 56.6%, 53.6%, respectively, compared with 85.7%, 82.5% and 83.6%, respectively, in those with lower levels. Patients with increased levels of both IL-6 and TNF-α had a nearly 6-fold increase in mortality (hazard ratio, 5.82) compared with patients with lower levels. LIMITATIONS: Retrospective design, limited sample size, and high-risk population. CONCLUSIONS: Hospitalized patients with cancer with rash and elevated IL-6 and TNF-α were nearly 6 times more likely to die over the course of follow-up. These biomarkers may serve as prognostic biomarkers and therapeutic targets for this high-risk population.

Author Correction: Identification of a binding protein for sesamin and characterization of its roles in plant growth.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Author Correction: Identification of a binding protein for sesamin and characterization of its roles in plant growth.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Patient Concerns in the Immediate Postoperative Period After Mohs Micrographic Surgery.

BACKGROUND: Postoperative concerns after Mohs micrographic surgery (MMS) are not well characterized. OBJECTIVE: To better define patient concerns and contributing characteristics in the immediate postoperative period after MMS. MATERIALS AND METHODS: A standardized telephone encounter template was implemented to better assess patient concerns in the 24-hour postoperative period. A review was then performed of patients undergoing MMS from October 2016 to July 2017 to assess for the most common patient concerns and association with clinical characteristics. RESULTS: We included 307 patients. Overall, 60.6% of patients reported a concern. Fifty-four percent of patients reported pain. Most pain was characterized as "a little" (67.7%). On univariate analysis, flap repairs, location on the upper extremities, and swelling were associated with higher mean postoperative pain. Tumor type was not associated with increased pain. On multivariate analyses, patients with larger defects and associated edema were statistically significantly associated with higher degrees of pain. CONCLUSION: Over half of patients experience postoperative concerns after MMS, most commonly pain. The immediate postoperative period may be an optimal time to identify patient concerns allowing for reassurance or early intervention when necessary.

Appearance-related psychosocial distress following facial skin cancer surgery using the FACE-Q Skin Cancer.

Over 2 million facial skin cancers occur globally each year. Facial skin cancer surgery can leave scars that may alter appearance and impact psychosocial functioning. The objective of this study is to assess patient-reported appearance-related psychosocial distress following facial skin cancer surgery, and to identify independent predictors of psychosocial impairment. This was a single-center, cross-sectional study at a tertiary care cancer center including patients who underwent dermatologic surgery on the face from March 1, 2016 to March 31, 2018. Patients completed the FACE-Q Skin Cancer Appearance-related Psychosocial Distress scale postoperatively between May 21, 2018 and October 1, 2018. Patient responses were rated on a 4-point Likert scale and converted on a scale from 0 to 100. In total, 359 patients completed the questionnaire (34.2% response rate). Overall, patients reported a low level of psychosocial distress. Patients most frequently reported items of self-consciousness, unhappiness, and insecurity < 3 months following surgery. Though psychosocial distress significantly improved over time, self-consciousness continued to be reported in the long-term postoperative period. Linear regression analysis determined that younger age, history of anxiety and/or depression, surgery on the nose, and repair by flap were independently predictive of psychosocial distress. Marginal predicted values for distress scores based on age demonstrated an indirect relationship. Patient-reported appearance-related psychosocial distress is low following facial skin cancer surgery, and report of distress decreases over time. The identified predictors of distress may be used as indicators for offering psycho-oncologic support and early interventions to improve scar appearance.

Alteration of Membrane Physicochemical Properties by Two Factors for Membrane Protein Integration.

After a nascent chain of a membrane protein emerges from the ribosomal tunnel, the protein is integrated into the cell membrane. This process is controlled by a series of proteinaceous molecular devices, such as signal recognition particles and Sec translocons. In addition to these proteins, we discovered two endogenous components regulating membrane protein integration in the inner membrane of Escherichia coli. The integration is blocked by diacylglycerol (DAG), whereas the blocking is relieved by a glycolipid named membrane protein integrase (MPIase). Here, we investigated the influence of these integration-blocking and integration-promoting factors on the physicochemical properties of membrane lipids via solid-state NMR and fluorescence measurements. These factors did not have destructive effects on membrane morphology because the membrane maintained its lamellar structure and did not fuse in the presence of DAG and/or MPIase at their effective concentrations. We next focused on membrane flexibility. DAG did not affect the mobility of the membrane surface, whereas the sugar chain in MPIase was highly mobile and enhanced the flexibility of membrane lipid headgroups. Comparison with a synthetic MPIase analog revealed the effects of the long sugar chain on membrane properties. The acyl chain order inside the membrane was increased by DAG, whereas the increase was cancelled by the addition of MPIase. MPIase also loosened the membrane lipid packing. Focusing on the transbilayer movement, MPIase reduced the rapid flip-flop motion of DAG. On the other hand, MPIase could not compensate for the diminished lateral diffusion by DAG. These results suggest that by manipulating the membrane lipids dynamics, DAG inhibits the protein from contacting the inner membrane, whereas the flexible long sugar chain of MPIase increases the opportunity for interaction between the membrane and the protein, leading to membrane integration of the newly formed protein.

Activation of Ralfuranone/Ralstonin Production by Plant Sugars Functions in the Virulence of Ralstonia solanacearum.

Plant pathogenic bacteria possess sophisticated mechanisms to detect the presence of host plants by sensing host-derived compounds. Ralstonia solanacearum, the causative agent of bacterial wilt on solanaceous plants, employs quorum sensing to control the production of the secondary metabolite ralfuranones/ralstonins, which have been suggested to be involved in virulence. Here, we report that d-galactose and d-glucose, plant sugars, activate the production of ralfuranones/ralstonins in R. solanacearum. As a result, two new derivatives, ralfuranone M (1) and ralstonin C (2), were found in the culture extracts, and their structures were elucidated by spectroscopic and chemical methods. Ralstonin C (2) is a cyclic lipopeptide containing a unique fatty acid, (2S,3S,Z)-3-amino-2-hydroxyicos-13-enoic acid, whereas ralfuranone M (1) has a common aryl-furanone structure with other ralfuranones. d-Galactose and d-glucose activated the expression of the biosynthetic ralfuranone/ralstonin genes and in part became the biosynthetic source of ralfuranones/ralstonins. Ralfuranones and ralstonins were detected from the xylem fluid of the infected tomato plants, and their production-deficient mutants exhibited reduced virulence on tomato and tobacco plants. Taken together, these results suggest that activation of ralfuranone/ralstonin production by host sugars functions in R. solanacearum virulence.

Identification of a binding protein for sesamin and characterization of its roles in plant growth.

Sesamin is a furofuran-type lignan that is found abundantly in seeds of Sesamum indicum (sesame) and has been widely accepted as a dietary supplement with positive effects on human health. The biological activity of sesamin in human cells and organs has been analysed extensively, although comparatively few studies show biological functions for sesamin in planta. Herein we screened sesamin-binding proteins (SBP) from sesame seedling extracts using sesamin-immobilized nano-beads. In subsequent peptide mass fingerprinting analyses, we identified a SBP, Steroleosin B, which is one of the membrane proteins found in oil bodies. In addition, pull-down assays and saturation transfer difference-nuclear magnetic resonance (STD-NMR) experiments demonstrated that sesamin binds directly to recombinant Steroleosin B in vitro. Finally, ectopic accumulations of sesamin and Steroleosin B in transgenic Arabidopsis thaliana plants induced severe growth defects including suppression of leaf expansion and root elongation. Collectively, these results indicate that sesamin influences tissue development in the presence of Steroleosin B.

Generation of Yellow Flowers of the Japanese Morning Glory by Engineering Its Flavonoid Biosynthetic Pathway toward Aurones.

Wild-type plants of the Japanese morning glory (Ipomoea nil) produce blue flowers that accumulate anthocyanin pigments, whereas its mutant cultivars show wide range flower color such as red, magenta and white. However, I. nil lacks yellow color varieties even though yellow flowers were curiously described in words and woodblocks printed in the 19th century. Such yellow flowers have been regarded as 'phantom morning glories', and their production has not been achieved despite efforts by breeders of I. nil. The chalcone isomerase (CHI) mutants (including line 54Y) bloom very pale yellow or cream-colored flowers conferred by the accumulation of 2', 4', 6', 4-tetrahydoroxychalcone (THC) 2'-O-glucoside. To produce yellow phantom morning glories, we introduced two snapdragon (Antirrhinum majus) genes to the 54Y line by encoding aureusidin synthase (AmAS1) and chalcone 4'-O-glucosyltransferase (Am4'CGT), which are necessary for the accumulation of aureusidin 6-O-glucoside and yellow coloration in A. majus. The transgenic plants expressing both genes exhibit yellow flowers, a character sought for many years. The flower petals of the transgenic plants contained aureusidin 6-O-glucoside, as well as a reduced amount of THC 2'-O-glucoside. In addition, we identified a novel aurone compound, aureusidin 6-O-(6″-O-malonyl)-glucoside, in the yellow petals. A combination of the coexpression of AmAS1 and Am4'CGT and suppression of CHI is an effective strategy for generating yellow varieties in horticultural plants.