The post How to Create a Pep Ventosa Inspired Still Life appeared first on Digital Photography School. It was authored by Charlie Moss.

Pep Ventosa is a Catalan artist who creates incredible images that explore the boundaries of photography. Made from multiple layers of similar photographs, they create an abstract and often surreal effect with a painterly feel. Ventosa usually creates pictures outside, but you can borrow his ideas to create a Pep Ventosa inspired still life. This […]

The post How to Create a Pep Ventosa Inspired Still Life appeared first on Digital Photography School. It was authored by Charlie Moss.

Manchester City fans are fuming with manager Pep Guardiola over his team selection for the FA Cup tie against Sheffield Wednesday, as both Phil Foden and Eric Garcia are left out of his starting line up for the clash at Hillsborough.

Co-op is recalling Co-op Sliced Pepperoni because E. coli O157 has been found in the product.

The plasmas of diabetic or uremic patients and of those receiving peritoneal dialysis treatment have increased levels of the glucose-derived dicarbonyl metabolites like methylglyoxal (MGO), glyoxal (GO), and 3-deoxyglucosone (3-DG). The elevated dicarbonyl levels can contribute to the development of painful neuropathies. Here, we used stimulated immunoreactive Calcitonin Gene–Related Peptide (iCGRP) release as a measure of nociceptor activation, and we found that each dicarbonyl metabolite induces a concentration-, TRPA1-, and Ca2+-dependent iCGRP release. MGO, GO, and 3-DG were about equally potent in the millimolar range. We hypothesized that another dicarbonyl, 3,4-dideoxyglucosone-3-ene (3,4-DGE), which is present in peritoneal dialysis (PD) solutions after heat sterilization, activates nociceptors. We also showed that at body temperatures 3,4-DGE is formed from 3-DG and that concentrations of 3,4-DGE in the micromolar range effectively induced iCGRP release from isolated murine skin. In a novel preparation of the isolated parietal peritoneum PD fluid or 3,4-DGE alone, at concentrations found in PD solutions, stimulated iCGRP release. We also tested whether inflammatory tissue conditions synergize with dicarbonyls to induce iCGRP release from isolated skin. Application of MGO together with bradykinin or prostaglandin E2 resulted in an overadditive effect on iCGRP release, whereas MGO applied at a pH of 5.2 resulted in reduced release, probably due to an MGO-mediated inhibition of transient receptor potential (TRP) V1 receptors. These results indicate that several reactive dicarbonyls activate nociceptors and potentiate inflammatory mediators. Our findings underline the roles of dicarbonyls and TRPA1 receptors in causing pain during diabetes or renal disease.

Neoantimycins are anticancer compounds of 15-membered ring antimycin-type depsipeptides. They are biosynthesized by a hybrid multimodular protein complex of nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS), typically from the starting precursor 3-formamidosalicylate. Examining fermentation extracts of Streptomyces conglobatus, here we discovered four new neoantimycin analogs, unantimycins B–E, in which 3-formamidosalicylates are replaced by an unusual 3-hydroxybenzoate (3-HBA) moiety. Unantimycins B–E exhibited levels of anticancer activities similar to those of the chemotherapeutic drug cisplatin in human lung cancer, colorectal cancer, and melanoma cells. Notably, they mostly displayed no significant toxicity toward noncancerous cells, unlike the serious toxicities generally reported for antimycin-type natural products. Using site-directed mutagenesis and heterologous expression, we found that unantimycin productions are correlated with the activity of a chorismatase homolog, the nat-hyg5 gene, from a type I PKS gene cluster. Biochemical analysis confirmed that the catalytic activity of Nat-hyg5 generates 3-HBA from chorismate. Finally, we achieved selective production of unantimycins B and C by engineering a chassis host. On the basis of these findings, we propose that unantimycin biosynthesis is directed by the neoantimycin-producing NRPS–PKS complex and initiated with the starter unit of 3-HBA. The elucidation of the biosynthetic unantimycin pathway reported here paves the way to improve the yield of these compounds for evaluation in oncotherapeutic applications.

Michal Bassani-Sternberg
Mar 1, 2015; 14:658-673
Research

Ignat V. Shilov
Sep 1, 2007; 6:1638-1655
Technology

Martin R. Larsen
Jul 1, 2005; 4:873-886
Technology

Yasushi Ishihama
Sep 1, 2005; 4:1265-1272
Research

Jürgen Cox
Sep 1, 2014; 13:2513-2526
Technological Innovation and Resources

The plasmas of diabetic or uremic patients and of those receiving peritoneal dialysis treatment have increased levels of the glucose-derived dicarbonyl metabolites like methylglyoxal (MGO), glyoxal (GO), and 3-deoxyglucosone (3-DG). The elevated dicarbonyl levels can contribute to the development of painful neuropathies. Here, we used stimulated immunoreactive Calcitonin Gene–Related Peptide (iCGRP) release as a measure of nociceptor activation, and we found that each dicarbonyl metabolite induces a concentration-, TRPA1-, and Ca2+-dependent iCGRP release. MGO, GO, and 3-DG were about equally potent in the millimolar range. We hypothesized that another dicarbonyl, 3,4-dideoxyglucosone-3-ene (3,4-DGE), which is present in peritoneal dialysis (PD) solutions after heat sterilization, activates nociceptors. We also showed that at body temperatures 3,4-DGE is formed from 3-DG and that concentrations of 3,4-DGE in the micromolar range effectively induced iCGRP release from isolated murine skin. In a novel preparation of the isolated parietal peritoneum PD fluid or 3,4-DGE alone, at concentrations found in PD solutions, stimulated iCGRP release. We also tested whether inflammatory tissue conditions synergize with dicarbonyls to induce iCGRP release from isolated skin. Application of MGO together with bradykinin or prostaglandin E2 resulted in an overadditive effect on iCGRP release, whereas MGO applied at a pH of 5.2 resulted in reduced release, probably due to an MGO-mediated inhibition of transient receptor potential (TRP) V1 receptors. These results indicate that several reactive dicarbonyls activate nociceptors and potentiate inflammatory mediators. Our findings underline the roles of dicarbonyls and TRPA1 receptors in causing pain during diabetes or renal disease.

Human leukocyte antigen (HLA) B*51:01 and endoplasmic reticulum aminopeptidase 1 (ERAP1) are strongly genetically associated with Behcet's disease (BD). Previous studies have defined two subgroups of HLA-B*51 peptidome containing proline (Pro) or alanine (Ala) at position 2 (P2). Little is known about the unconventional non-Pro/Ala2 HLA-B*51-bound peptides. We aimed to study the features of this novel subpeptidome, and investigate its regulation by ERAP1. CRISPR-Cas9 was used to generate an HLA-ABC-triple knockout HeLa cell line (HeLa.ABC-KO), which was subsequently transduced to express HLA-B*51:01 (HeLa.ABC-KO.B51). ERAP1 was silenced using lentiviral shRNA. Peptides bound to HLA-B*51:01 were eluted and analyzed by mass spectrometry. The characteristics of non-Pro/Ala2, Pro2, and Ala2 peptides and their alteration by ERAP1 silencing were investigated. Effects of ERAP1 silencing on cell surface expression of HLA-B*51:01 were studied using flow cytometry. More than 20% of peptides eluted from HLA-B*51:01 lacked Pro or Ala at P2. This unconventional group of HLA-B*51:01-bound peptides was relatively enriched for 8-mers (with relatively fewer 9-mers) compared with the Pro2 and Ala2 subpeptidomes and had similar N-terminal and C-terminal residue usages to Ala2 peptides (with the exception of the less abundant leucine at position ). Knockdown of ERAP1 increased the percentage of non-Pro/Ala2 from 20% to ~40%, increased the percentage of longer (10-mer and 11-mer) peptides eluted from HLA-B*51:01 complexes, and abrogated the predominance of leucine at P1. Interestingly knockdown of ERAP1 altered the length and N-terminal residue usage of non-Ala2&Pro2 and Ala2 but not the Pro2 peptides. Finally, ERAP1 silencing regulated the expression levels of cell surface HLA-B*51 in a cell-type-dependent manner. In conclusion, we have used a novel methodology to identify an unconventional but surprisingly abundant non-Pro/Ala2 HLA-B*51:01 subpeptidome. It is increased by knockdown of ERAP1, a gene affecting the risk of developing BD. This has implications for theories of disease pathogenesis.

Deborah Hinnen
Aug 1, 2017; 30:202-210
Feature Articles

Neoantimycins are anticancer compounds of 15-membered ring antimycin-type depsipeptides. They are biosynthesized by a hybrid multimodular protein complex of nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS), typically from the starting precursor 3-formamidosalicylate. Examining fermentation extracts of Streptomyces conglobatus, here we discovered four new neoantimycin analogs, unantimycins B–E, in which 3-formamidosalicylates are replaced by an unusual 3-hydroxybenzoate (3-HBA) moiety. Unantimycins B–E exhibited levels of anticancer activities similar to those of the chemotherapeutic drug cisplatin in human lung cancer, colorectal cancer, and melanoma cells. Notably, they mostly displayed no significant toxicity toward noncancerous cells, unlike the serious toxicities generally reported for antimycin-type natural products. Using site-directed mutagenesis and heterologous expression, we found that unantimycin productions are correlated with the activity of a chorismatase homolog, the nat-hyg5 gene, from a type I PKS gene cluster. Biochemical analysis confirmed that the catalytic activity of Nat-hyg5 generates 3-HBA from chorismate. Finally, we achieved selective production of unantimycins B and C by engineering a chassis host. On the basis of these findings, we propose that unantimycin biosynthesis is directed by the neoantimycin-producing NRPS–PKS complex and initiated with the starter unit of 3-HBA. The elucidation of the biosynthetic unantimycin pathway reported here paves the way to improve the yield of these compounds for evaluation in oncotherapeutic applications.

Katrina Meyer
Apr 28, 2020; 0:R120.002034v1-mcp.R120.002034
Review

Elena Lorente
Apr 7, 2020; 0:RA120.002014v1-mcp.RA120.002014
Research

James A. Wilkins
Apr 13, 2020; 0:RA120.001944v1-mcp.RA120.001944
Research

Liye Chen
May 1, 2020; 19:871-883
Research

Mona Radwan
Apr 1, 2020; 19:640-654
Research

Qingbo Shu
Apr 1, 2020; 19:672-689
Research

Rationale: The treatment of choice for insulinomas and focal lesions in congenital hyperinsulinism (CHI) is surgery. However, intra-operative detection can be challenging. This could be overcome with intra-operative fluorescence imaging, which provides real-time lesion detection with a high spatial resolution. Here, a novel method for targeted near-infrared (NIR) fluorescence imaging of glucagon-like peptide 1 receptor (GLP-1R) positive lesions, using the GLP-1 agonist exendin-4, labeled with IRDye800CW, was examined in vitro and in vivo. Methods: A competitive binding assay was performed using Chinese hamster lung (CHL) cells transfected with the GLP-1R. Tracer biodistribution was determined in BALB/c nude mice bearing subcutaneous CHL-GLP-1R xenografts. In vivo NIR fluorescence imaging of CHL-GLP-1R xenografts was performed. Localization of the tracer in the pancreatic islets of BALB/c nude mice was examined using fluorescence microscopy. Laparoscopic imaging was performed to detect the fluorescent signal of the tracer in the pancreas of mini pigs. Results: Exendin-4-IRDye800CW binds the GLP-1R with an IC50 value of 3.96 nM. The tracer accumulates in CHL-GLP-1R xenografts. Subcutaneous CHL-GLP-1R xenografts were visualized using in vivo NIR fluorescence imaging. The tracer accumulates specifically in the pancreatic islets of mice and a clear fluorescent signal was detected in the pancreas of mini pigs. Conclusion: These date provide the first in vivo evidence of the feasibility of targeted fluorescence imaging of GLP-1R positive lesions. Intra-operative lesion delineation using exendin-4-IRDye800CW could benefit open as well as laparoscopic surgical procedures for removal of insulinomas and focal lesions in CHI.

Acidosis is a key driver for many diseases, including cancer, sepsis, and stroke. The spatiotemporal dynamics of dysregulated pH across disease remains elusive and current diagnostic strategies do not provide localization of pH alterations. We sought to explore if PET imaging using hydrophobic cyclic peptides that partition into the cellular membrane at low extracellular pH (denoted as "pHLIC") can permit accurate in vivo visualization of acidosis. Methods: Acid-sensitive cyclic peptide c[E4W5C] pHLIC was conjugated to bifunctional maleimide-NO2A and radiolabeled with copper-64 (t1/2 = 12.7 h). C57BL/6J mice were administered LPS (15 mg/kg) or saline (vehicle) and serially imaged with [⁶⁴Cu]Cu-c[E4W5C] over 24 h. Ex vivo autoradiography was performed on resected brain slices and subsequently stained with cresyl violet to enable high-resolution spatial analysis of tracer accumulation. A non- pH-sensitive cell-penetrating control peptide (c[R4W5C]) was used to confirm specificity of [⁶⁴Cu]Cu-c[E4W5C]. CD11b (macrophage/microglia) and TMEM119 (microglia) immunostaining was performed to correlate extent of neuroinflammation with [⁶⁴Cu]Cu-c[E4W5C] PET signal. Results: [⁶⁴Cu]Cu-c[E4W5C] radiochemical yield and purity was >95% and >99% respectively, with molar activity >0.925 MBq/nmol. Significantly increased [⁶⁴Cu]Cu-c[E4W5C] uptake was observed in LPS-treated mice (vs. vehicle) within peripheral tissues including blood, lungs, liver, and small intestines (P < 0.001-0.05). Additionally, there was significantly increased [⁶⁴Cu]Cu-c[E4W5C] uptake in the brains of LPS-treated animals. Autoradiography confirmed increased uptake in the cerebellum, cortex, hippocampus, striatum, and hypothalamus of LPS-treated mice (vs. vehicle). Immunohistochemical (IHC) analysis revealed microglial/macrophage infiltrate, suggesting activation in brain regions containing increased tracer uptake. [⁶⁴Cu]Cu-c[R4W5C] demonstrated significantly reduced uptake in the brain and periphery of LPS mice compared to the acid-mediated [⁶⁴Cu]Cu-c[E4W5C] tracer. Conclusion: Here, we demonstrate that a pH-sensitive PET tracer specifically detects acidosis in regions associated with sepsis-driven pro-inflammatory responses. This study suggests that [⁶⁴Cu]Cu-pHLIC is a valuable tool to noninvasively assess acidosis associated with both central and peripheral innate immune activation.

Peptide receptor radiotherapy using ¹⁷⁷Lu-labeled somatostatin ligand analogs is a well-established treatment for neuroendocrine tumors (NET), with ¹⁷⁷Lu-DOTATATE having acquired marketing authorization in Europe and the USA. The investigation of the pharmacokinetics of those radiopharmaceuticals in vivo in humans is crucial for personalized treatment management and understanding of treatment effects. It requires input data on the in vivo stability of the radiopharmaceuticals in blood and plasma. The work presented here is devoted to the investigation of in vivo stability of ¹⁷⁷Lu-DOTATATE in humans affected by NET. Unexpectedly, fast metabolism of the radiopharmaceutical was observed, with fraction of intact ¹⁷⁷Lu-DOTATATE in plasma decreasing rapidly to 23±5% (mean ± SD) at 24 h and 1.7±0.9% at 96 h after injection.

Objectives: Esthesioneuroblastoma (ENB) is rare with limited therapeutic options when unresectable or metastatic; however, expression of somatostatin receptors qualifies it for peptide receptor radionuclide therapy (PRRT). We report outcomes of PRRT in ENB from two referral centers. Methods: Using PRRT databases at two European Neuroendocrine Tumour Society Centers of Excellence, case finding was undertaken between 2004-2018 for patients who had PRRT with recurrent/metastatic ENB deemed unsuitable for further conventional therapies. Evaluations of response using a composite reference standard and for survival were performed. Results: Of seven patients, four had partial response, two had disease stabilization and one had early progression. Possible side effects include worsening CSF-leaks. Median progression-free survival was 17 months (range, 0-30), and median overall survival was 32 months (range, 4–53). Conclusion: PRRT shows promising efficacy and moderate survival duration in unresectable locally advanced or metastatic ENB warranting larger cohort studies incorporating measures of quality of life.

Targeting tumor-expressed receptors using selective molecules for diagnostic, therapeutic or both diagnostic and therapeutic (theragnostic) purposes is a promising approach in oncological applications. Such approaches have increased significantly over the past decade. Peptides such as gastrin-releasing peptide receptors (GRPR) targeting radiopharmaceuticals are small molecules with fast blood clearance and urinary excretion. They demonstrate good tissue diffusion, low immunogenicity, and highly selective binding to their target cell-surface receptors. They are also easily produced. GRPR, part of the bombesin (BBN) family, are overexpressed in many tumors, including breast and prostate cancer, and therefore represent an attractive target for future development.

The objective of this retrospective study was to determine the role of ¹⁸F-FDG PET/CT in a large cohort of 495 patients with metastatic neuroendocrine neoplasms (NENs) who were treated with peptide receptor radionuclide therapy (PRRT) with a long-term follow-up. Methods: The 495 patients were treated with ¹⁷⁷Lu- and/or ⁹⁰Y- DOTATOC/DOTATATE PRRT between 2/2002 and 7/2018. All subjects received both ⁶⁸Ga-DOTATOC/TATE/NOC and ¹⁸F-FDG PET/CT prior to treatment and were followed 3-189 months. Kaplan-Meier analysis, log-rank test (Mantel-Cox), and Cox regression analysis were performed for overall survival (OS) and progression-free survival (PFS). Results: 199 patients (40.2%) presented with pancreatic NEN, 49 with CUP (cancer of unknown primary), 139 with midgut NEN, whereas the primary tumor was present in the rectum in 20, in the lung in 38, in the stomach in 8 and other locations in 42 patients. FDG-PET/CT was positive in 382 (77.2%) patients and 113 (22.8%) were FDG-negative before PRRT, while 100% were ⁶⁸Ga-DOTATOC/TATE/NOC positive. For all patients, the median PFS and OS, defined from start of PRRT, were 19.6 mo and 58.7 mo, respectively. Positive FDG predicted shorter PFS (18.5 mo vs 24.1 mo; P = 0.0015) and OS (53.2 mo vs 83.1 mo; P < 0.001) than negative FDG. Amongst the pancreatic NEN, the median OS was 52.8 mo in FDG positive and 114.3 mo in FDG negative subjects (P = 0.0006). For all patients with positive ¹⁸F-FDG uptake, and a ratio of the highest SUV_max on ⁶⁸Ga-SSTR PET to the most ¹⁸F-FDG-avid tumor lesions >2, the median OS was 53.0 mo, compared to 43.4 mo in those patients with a ratio <2 (P = 0.030). For patients with no ¹⁸F-FDG uptake (complete "mismatch" imaging pattern), the median OS was 108.3 mo vs 76.9 mo for SUV_max >15.0 and ≤15.0 on ⁶⁸Ga-SSTR PET/CT, respectively. Conclusion: The presence of positive lesions on ¹⁸F-FDG PET is an independent prognostic factor in patients with NEN treated with PRRT. Metabolic imaging with ¹⁸F-FDG PET/CT compliments the molecular imaging aspect of ⁶⁸Ga-SSTR PET/CT for the prognosis of survival after PRRT. High SSTR expression combined with negative ¹⁸F-FDG PET/CT imaging is associated with the most favorable long-term prognosis.

Introduction: Gastrin Releasing peptide receptors (GRPRs) are potential molecular imaging targets in a variety of tumors. Recently, a ⁶⁸Ga-labelled antagonist to GRPRs, NeoBOMB1, was developed for PET. We report on the outcome of a Phase I/IIa clinical trial (EudraCT 2016-002053-38) within the EU-FP7 project Closed-loop Molecular Environment for Minimally Invasive Treatment of Patients with Metastatic Gastrointestinal Stromal Tumours (‘MITIGATE’) (grant agreement number 602306) in patients with oligometastatic gastrointestinal stromal tumors (GIST). Materials and Methods: The main objectives were evaluation of safety, biodistribution, dosimetry and preliminary tumor targeting of ⁶⁸Ga-NeoBOMB1 in patients with advanced TKI-treated GIST using PET/CT. Six patients with histologically confirmed GIST and unresectable primary or metastases undergoing an extended protocol for detailed pharmacokinetic analysis were included. ⁶⁸Ga-NeoBOMB1 was prepared using a kit procedure with a licensed ⁶⁸Ge/⁶⁸Ga generator. 3 MBq/kg body-weight were injected intravenously and safety parameters were assessed. PET/CT included dynamic imaging at 5 min, 11 min and 19 min as well as static imaging at 1, 2 and 3-4 h p.i. for dosimetry calculations. Venous blood samples and urine were collected for pharmacokinetics. Tumor targeting was assessed on a per-lesion and per-patient basis. Results: ⁶⁸Ga-NeoBOMB1 (50 µg) was prepared with high radiochemical purity (yield >97%). Patients received 174 ± 28 MBq of the radiotracer, which was well tolerated in all patients over a follow-up period of 4 weeks. Dosimetry calculations revealed a mean adsorbed effective dose of 0.029 ± 0.06 mSv/MBq with highest organ dose to the pancreas (0.274 ± 0.099 mSv/MBq). Mean plasma half-life was 27.3 min with primarily renal clearance (mean 25.7 ± 5.4% of injected dose 4h p.i.). Plasma metabolite analyses revealed high stability, metabolites were only detected in the urine. In three patients a significant uptake with increasing maximum standard uptake values (SUV_max at 2h p.i.: 4.3 to 25.9) over time was found in tumor lesions. Conclusion: This Phase I/IIa study provides safety data for ⁶⁸Ga-NeoBOMB1, a promising radiopharmaceutical for targeting GRPR-expressing tumors. Safety profiles and pharmacokinetics are suitable for PET imaging and absorbed dose estimates are comparable to other ⁶⁸Ga-labelled radiopharmaceuticals used in clinical routine.

Treatment of hyperinsulinemic hypoglycemia is challenging. Surgical treatment of insulinomas and focal lesions in congenital hyperinsulinism (CHI) is invasive and carries major risks of morbidity. Medication to treat nesidioblastosis and diffuse CHI has varying efficacy and causes significant side effects. Here, we describe a novel method for therapy of hyperinsulinemic hyperglycemia, highly selectively killing beta cells by targeted photodynamic therapy (tPDT) with exendin-4-IRDye700DX, targeting the glucagon-like peptide 1 receptor (GLP-1R). A competitive binding assay was performed using Chinese hamster lung (CHL) cells transfected with the GLP-1R. The efficacy and specificity of tPDT with exendin-4-IRDye700DX was examined in vitro in cells with different levels of GLP-1R expression. Tracer biodistribution was determined in BALB/c nude mice bearing subcutaneous CHL-GLP-1R xenografts. Induction of cellular damage and the effect on tumor growth were analyzed to determine treatment efficacy. Exendin-4-IRDye700DX has a high affinity for the GLP-1R with an IC50 value of 6.3 nM. TPDT caused significant specific phototoxicity in GLP-1R positive cells (2.3 ± 0.8 % and 2.7 ± 0.3 % remaining cell viability in CHL-GLP-1R and INS-1 cells resp.). The tracer accumulates dose-dependently in GLP-1R positive tumors. In vivo tPDT induces cellular damage in tumors, shown by strong expression of cleaved-caspase-3 and leads to a prolonged median survival of the mice (36.5 vs. 22.5 days resp. p<0.05). These data show in vitro as well as in vivo evidence for the potency of tPDT using exendin-4-IRDye700DX. This could in the future provide a new, minimally invasive and highly specific treatment method for hyperinsulinemic hypoglycemia.

The presentation of peptides on class I human leukocyte antigen (HLA-I) molecules plays a central role in immune recognition of infected or malignant cells. In cancer, non-self HLA-I ligands can arise from many different alterations, including non-synonymous mutations, gene fusion, cancer-specific alternative mRNA splicing or aberrant post-translational modifications. Identifying HLA-I ligands remains a challenging task that requires either heavy experimental work for in vivo identification or optimized bioinformatics tools for accurate predictions. To date, no HLA-I ligand predictor includes post-translational modifications. To fill this gap, we curated phosphorylated HLA-I ligands from several immunopeptidomics studies (including six newly measured samples) covering 72 HLA-I alleles and retrieved a total of 2,066 unique phosphorylated peptides. We then expanded our motif deconvolution tool to identify precise binding motifs of phosphorylated HLA-I ligands. Our results reveal a clear enrichment of phosphorylated peptides among HLA-C ligands and demonstrate a prevalent role of both HLA-I motifs and kinase motifs on the presentation of phosphorylated peptides. These data further enabled us to develop and validate the first predictor of interactions between HLA-I molecules and phosphorylated peptides.

Mass spectrometry (MS) and proteomics offer comprehensive characterization and identification of microorganisms and discovery of protein biomarkers that are applicable for diagnostics of infectious diseases. The use of biomarkers for diagnostics is widely applied in the clinic and the use of peptide biomarkers is increasingly being investigated for applications in the clinical laboratory. Respiratory-tract infections are a predominant cause for medical treatment, although, clinical assessments and standard clinical laboratory protocols are time-consuming and often inadequate for reliable diagnoses. Novel methods, preferably applied directly to clinical samples, excluding cultivation steps, are needed to improve diagnostics of infectious diseases, provide adequate treatment and reduce the use of antibiotics and associated development of antibiotic resistance. This study applied nano-liquid chromatography (LC) coupled with tandem MS, with a bioinformatics pipeline and an in-house database of curated high-quality reference genome sequences to identify species-unique peptides as potential biomarkers for four bacterial pathogens commonly found in respiratory tract infections (RTIs): Staphylococcus aureus; Moraxella catarrhalis; Haemophilus influenzae and Streptococcus pneumoniae. The species-unique peptides were initially identified in pure cultures of bacterial reference strains, reflecting the genomic variation in the four species and, furthermore, in clinical respiratory tract samples, without prior cultivation, elucidating proteins expressed in clinical conditions of infection. For each of the four bacterial pathogens, the peptide biomarker candidates most predominantly found in clinical samples, are presented. Data are available via ProteomeXchange with identifier PXD014522. As proof-of-principle, the most promising species-unique peptides were applied in targeted tandem MS-analyses of clinical samples and their relevance for identifications of the pathogens, i.e. proteotyping, was validated, thus demonstrating their potential as peptide biomarker candidates for diagnostics of infectious diseases.

Chronic hyperglycemia is known to disrupt the proteolytic milieu, initiating compensatory and maladaptive pathways in the diabetic kidney. Such changes in intrarenal proteolysis are captured by the urinary peptidome. To elucidate the early kidney response to chronic hyperglycemia, we conducted a peptidomic investigation into urines from otherwise healthy youths with type 1 diabetes and their non-diabetic peers using unbiased and targeted mass spectrometry-based techniques. This cross-sectional study included two separate cohorts for the discovery (n = 30) and internal validation (n = 30) of differential peptide excretion. Peptide bioactivity was predicted using PeptideRanker and subsequently verified in vitro. Proteasix and the Nephroseq database were used to identify putative proteases responsible for peptide generation and examine their expression in diabetic nephropathy. A total of 6550 urinary peptides were identified in the discovery analysis. We further examined the subset of 162 peptides, which were quantified across all thirty samples. Of the 15 differentially excreted peptides (p < 0.05), seven derived from a C-terminal region (⁵⁸⁹SGSVIDQSRVLNLGPITRK⁶⁰⁷) of uromodulin, a kidney-specific protein. Increased excretion of five uromodulin peptides was replicated in the validation cohort using parallel reaction monitoring (p < 0.05). One of the validated peptides (SGSVIDQSRVLNLGPI) activated NFB and AP-1 signaling, stimulated cytokine release, and enhanced neutrophil migration in vitro. In silico analyses highlighted several potential proteases such as hepsin, meprin A, and cathepsin B to be responsible for generating these peptides. In summary, we identified a urinary signature of uromodulin peptides associated with early type 1 diabetes before clinical manifestations of kidney disease and discovered novel bioactivity of uromodulin peptides in vitro. Our present findings lay the groundwork for future studies to validate peptide excretion in larger and broader populations, to investigate the role of bioactive uromodulin peptides in high glucose conditions, and to examine proteases that cleave uromodulin.

For more than two decades naturally presented, human leukocyte antigen (HLA)-restricted peptides (immunopeptidome) have been eluted and sequenced using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Since, identified disease-associated HLA ligands have been characterized and evaluated as potential active substances. Treatments based on HLA-presented peptides have shown promising results in clinical application as personalized T cell-based immunotherapy. Peptide vaccination cocktails are produced as investigational medicinal products under GMP conditions. To support clinical trials based on HLA-presented tumor-associated antigens, in this study the sensitive LC-MS/MS HLA class I antigen identification pipeline was fully validated for our technical equipment according to the current US Food and Drug Administration (FDA) and European Medicines Agency (EMA) guidelines.

The immunopeptidomes of JY cells with or without spiked-in, isotope labeled peptides, of peripheral blood mononuclear cells of healthy volunteers as well as a chronic lymphocytic leukemia and a bladder cancer sample were reliably identified using a data-dependent acquisition method. As the LC-MS/MS pipeline is used for identification purposes, the validation parameters include accuracy, precision, specificity, limit of detection and robustness.

Large-scale identification of N-linked intact glycopeptides by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) in human serum is challenging because of the wide dynamic range of serum protein abundances, the lack of a complete serum N-glycan database and the existence of proteoforms. In this regard, a spectral library search method was presented for the identification of N-linked intact glycopeptides from N-linked glycoproteins in human serum with target-decoy and motif-specific false discovery rate (FDR) control. Serum proteins were firstly separated into low-abundance and high-abundance proteins by acetonitrile (ACN) precipitation. After digestion, the N-linked intact glycopeptides were enriched by hydrophilic interaction liquid chromatography (HILIC) and a portion of the enriched N-linked intact glycopeptides were processed by Peptide-N-Glycosidase F (PNGase F) to generate N-linked deglycopeptides. Both N-linked intact glycopeptides and deglycopeptides were analyzed by LC-MS/MS. From N-linked deglycopeptides data sets, 764 N-linked glycoproteins, 1699 N-linked glycosites and 3328 unique N-linked deglycopeptides were identified. Four types of N-linked glycosylation motifs (NXS/T/C/V, X=P) were used to recognize the N-linked deglycopeptides. The spectra of these N-linked deglycopeptides were utilized for N-linked deglycopeptides library construction and identification of N-linked intact glycopeptides. A database containing 739 N-glycan masses was constructed and utilized during spectral library search for the identification of N-linked intact glycopeptides. In total, 526 N-linked glycoproteins, 1036 N-linked glycosites, 22,677 N-linked intact glycopeptides and 738 N-glycan masses were identified under 1% FDR, representing the most in-depth serum N-glycoproteome identified by LC-MS/MS at N-linked intact glycopeptide level.

C9ORF72-associated Motor Neuron Disease patients feature abnormal expression of 5 dipeptide repeat (DPR) polymers. Here we used quantitative proteomics in a mouse neuronal-like cell line (Neuro2a) to demonstrate that the Arg residues in the most toxic DPRS, PR and GR, leads to a promiscuous binding to the proteome compared with a relative sparse binding of the more inert AP and GA. Notable targets included ribosomal proteins, translation initiation factors and translation elongation factors. PR and GR comprising more than 10 repeats appeared to robustly stall on ribosomes during translation suggesting Arg-rich peptide domains can electrostatically jam the ribosome exit tunnel during synthesis. Poly-GR also recruited arginine methylases, induced hypomethylation of endogenous proteins, and induced a profound destabilization of the actin cytoskeleton. Our findings point to arginine in GR and PR polymers as multivalent toxins to translation as well as arginine methylation that may explain the dysfunction of biological processes including ribosome biogenesis, mRNA splicing and cytoskeleton assembly.

Human leukocyte antigen (HLA) B*51:01 and endoplasmic reticulum aminopeptidase 1 (ERAP1) are strongly genetically associated with Behcet's disease (BD). Previous studies have defined two subgroups of HLA-B*51 peptidome containing proline (Pro) or alanine (Ala) at position 2 (P2). Little is known about the unconventional non-Pro/Ala2 HLA-B*51-bound peptides. We aimed to study the features of this novel subpeptidome, and investigate its regulation by ERAP1. CRISPR-Cas9 was used to generate an HLA-ABC-triple knockout HeLa cell line (HeLa.ABC-KO), which was subsequently transduced to express HLA-B*51:01 (HeLa.ABC-KO.B51). ERAP1 was silenced using lentiviral shRNA. Peptides bound to HLA-B*51:01 were eluted and analyzed by mass spectrometry. The characteristics of non-Pro/Ala2, Pro2, and Ala2 peptides and their alteration by ERAP1 silencing were investigated. Effects of ERAP1 silencing on cell surface expression of HLA-B*51:01 were studied using flow cytometry. More than 20% of peptides eluted from HLA-B*51:01 lacked Pro or Ala at P2. This unconventional group of HLA-B*51:01-bound peptides was relatively enriched for 8-mers (with relatively fewer 9-mers) compared with the Pro2 and Ala2 subpeptidomes and had similar N-terminal and C-terminal residue usages to Ala2 peptides (with the exception of the less abundant leucine at position ). Knockdown of ERAP1 increased the percentage of non-Pro/Ala2 from 20% to ~40%, increased the percentage of longer (10-mer and 11-mer) peptides eluted from HLA-B*51:01 complexes, and abrogated the predominance of leucine at P1. Interestingly knockdown of ERAP1 altered the length and N-terminal residue usage of non-Ala2&Pro2 and Ala2 but not the Pro2 peptides. Finally, ERAP1 silencing regulated the expression levels of cell surface HLA-B*51 in a cell-type-dependent manner. In conclusion, we have used a novel methodology to identify an unconventional but surprisingly abundant non-Pro/Ala2 HLA-B*51:01 subpeptidome. It is increased by knockdown of ERAP1, a gene affecting the risk of developing BD. This has implications for theories of disease pathogenesis.

Neoantimycins are anticancer compounds of 15-membered ring antimycin-type depsipeptides. They are biosynthesized by a hybrid multimodular protein complex of nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS), typically from the starting precursor 3-formamidosalicylate. Examining fermentation extracts of Streptomyces conglobatus, here we discovered four new neoantimycin analogs, unantimycins B–E, in which 3-formamidosalicylates are replaced by an unusual 3-hydroxybenzoate (3-HBA) moiety. Unantimycins B–E exhibited levels of anticancer activities similar to those of the chemotherapeutic drug cisplatin in human lung cancer, colorectal cancer, and melanoma cells. Notably, they mostly displayed no significant toxicity toward noncancerous cells, unlike the serious toxicities generally reported for antimycin-type natural products. Using site-directed mutagenesis and heterologous expression, we found that unantimycin productions are correlated with the activity of a chorismatase homolog, the nat-hyg5 gene, from a type I PKS gene cluster. Biochemical analysis confirmed that the catalytic activity of Nat-hyg5 generates 3-HBA from chorismate. Finally, we achieved selective production of unantimycins B and C by engineering a chassis host. On the basis of these findings, we propose that unantimycin biosynthesis is directed by the neoantimycin-producing NRPS–PKS complex and initiated with the starter unit of 3-HBA. The elucidation of the biosynthetic unantimycin pathway reported here paves the way to improve the yield of these compounds for evaluation in oncotherapeutic applications.

The human leucocyte antigen (HLA)-B*27:05 allele and the endoplasmic reticulum-resident aminopeptidases are strongly associated with ankylosing spondylitis (AS), a chronic inflammatory spondyloarthropathy. This study examined the effect of endoplasmic reticulum aminopeptidase 2 (ERAP2) in the generation of the natural HLA-B*27:05 ligandome in live cells. Complexes of HLA-B*27:05-bound peptide pools were isolated from human ERAP2-edited cell clones and the peptides were identified using high throughput mass spectrometry analyses. The relative abundance of thousand ligands was established by quantitative tandem mass spectrometry and bioinformatics analysis. The residue frequencies at different peptide position, identified in presence or absence of ERAP2, determined structural features of ligands and their interactions with specific pockets of antigen binding site of HLA-B*27:05 molecule. Sequence alignment of ligands identified with species of bacteria associated with HLA-B*27-dependent reactive arthritis was performed. In the absence of ERAP2, peptides with N-terminal basic residues, and minority canonical P2 residues are enriched in the natural ligandome. Further, alterations of residue frequencies and hydrophobicity profile at P3, P7, and P positions were detected. In addition, several ERAP2-dependent cellular peptides were highly similar to protein sequences of arthritogenic bacteria, including one human HLA-B*27:05 ligand fully conserved in a protein from Campylobacter jejuni. These findings highlight the pathogenic role of this aminopeptidase in the triggering of AS autoimmune disease.

Post-translational modifications play a critical and diverse role in regulating cellular activities. Despite their fundamentally important role in cellular function, there has been no report to date of an effective generalized approach to the targeting, extraction and characterization of the critical c-terminal regions of natively prenylated proteins. Various chemical modification and metabolic labelling strategies in cell culture have been reported. However, their applicability is limited to cell culture systems and does not allow for analysis of tissue samples. The chemical characteristics (hydrophobicity, low abundance, highly basic charge) of many of the c-terminal regions of prenylated proteins have impaired the use of standard proteomic workflows. In this context, we sought a direct approach to the problem in order to examine these proteins in tissue without the use of labelling.  Here we demonstrate that prenylated proteins can be captured on chromatographic resins functionalized with mixed disulfide functions. Protease treatment of resin-bound proteins using chymotryptic digestion revealed peptides from many known prenylated proteins. Exposure of the protease-treated resin to reducing agents and hydro organic mixtures released c-terminal peptides with intact prenyl groups along with other enzymatic modifications expected in this protein family. Database and search parameters were selected to allow for c-terminal modifications unique to these molecules such as CAAX box processing and c-terminal methylation. In summary, we present a direct approach to enrich and obtain information at a molecular level of detail about prenylation of proteins from tissue and cell extracts using high performance LCMS without the need for metabolic labeling and derivatization.

Protein-protein interactions are often mediated by short linear motifs (SLiMs) that are located in intrinsically disordered regions (IDRs) of proteins. Interactions mediated by SLiMs are notoriously difficult to study, and many functionally relevant interactions likely remain to be uncovered. Recently, pull-downs with synthetic peptides in combination with quantitative mass spectrometry emerged as a powerful screening approach to study protein-protein interactions mediated by SLiMs. Specifically, arrays of synthetic peptides immobilized on cellulose membranes provide a scalable means to identify the interaction partners of many peptides in parallel. In this minireview we briefly highlight the relevance of SLiMs for protein-protein interactions, outline existing screening technologies, discuss unique advantages of peptide-based interaction screens and provide practical suggestions for setting up such peptide-based screens.

The presentation of post-translationally modified (PTM) peptides by cell surface HLA molecules has the potential to increase the diversity of targets for surveilling T cells. Whilst immunopeptidomics studies routinely identify thousands of HLA-bound peptides from cell lines and tissue samples, in-depth analyses of the proportion and nature of peptides bearing one or more PTMs remains challenging. Here we have analyzed HLA-bound peptides from a variety of allotypes and assessed the distribution of mass spectrometry-detected PTMs, finding deamidation of asparagine or glutamine to be highly prevalent. Given that asparagine deamidation may arise either spontaneously or through enzymatic reaction, we assessed allele-specific and global motifs flanking the modified residues. Notably, we found that the N-linked glycosylation motif NX(S/T) was highly abundant across asparagine-deamidated HLA-bound peptides. This finding, demonstrated previously for a handful of deamidated T cell epitopes, implicates a more global role for the retrograde transport of nascently N-glycosylated polypeptides from the ER and their subsequent degradation within the cytosol to form HLA-ligand precursors. Chemical inhibition of Peptide:N-Glycanase (PNGase), the endoglycosidase responsible for the removal of glycans from misfolded and retrotranslocated glycoproteins, greatly reduced presentation of this subset of deamidated HLA-bound peptides. Importantly, there was no impact of PNGase inhibition on peptides not containing a consensus NX(S/T) motif. This indicates that a large proportion of HLA-I bound asparagine deamidated peptides are generated from formerly glycosylated proteins that have undergone deglycosylation via the ER-associated protein degradation (ERAD) pathway. The information herein will help train deamidation prediction models for HLA-peptide repertoires and aid in the design of novel T cell therapeutic targets derived from glycoprotein antigens.

Xanthophyllomyces dendrorhous is a basidiomycete yeast known as a natural producer of astaxanthin, a carotenoid of commercial interest because of its antioxidant properties. Recent studies indicated that X. dendrorhous has a functional SREBP pathway involved in the regulation of isoprenoid compound biosynthesis, which includes ergosterol and carotenoids. SREBP is a major regulator of sterol metabolism and homeostasis in mammals; characterization in fungi also provides information about its role in the hypoxia adaptation response and virulence. SREBP protease processing is required to activate SREBP pathway functions in fungi. Here, we identified and described the STP1 gene, which encodes a metallopeptidase of the M50 family involved in the proteolytic activation of the transcription factor Sre1 of the SREBP pathway, in X. dendrorhous. We assessed STP1 function in stp1 strains derived from the wild-type and a mutant of ergosterol biosynthesis that overproduces carotenoids and sterols. Bioinformatic analysis of the deduced protein predicted the presence of characteristic features identified in homologs from mammals and fungi. The stp1 mutation decreased yeast growth in the presence of azole drugs and reduced transcript levels of Sre1-dependent genes. This mutation also negatively affected the carotenoid- and sterol-overproducing phenotype. Western blot analysis demonstrated that Sre1 was activated in the yeast ergosterol biosynthesis mutant and that the stp1 mutation introduced in this strain prevented Sre1 proteolytic activation. Overall, our results demonstrate that STP1 encodes a metallopeptidase involved in proteolytic activation of Sre1 in X. dendrorhous, contributing to our understanding of fungal SREBP pathways.

T cells isolated from the pancreatic infiltrates of non-obese diabetic mice have been shown to recognize epitopes formed by the covalent cross-linking of proinsulin and secretory granule peptides. Formation of such hybrid insulin peptides (HIPs) was confirmed through mass spectrometry and responses to HIPs were observed among the islet-infiltrating T cells of pancreatic organ donors and in the peripheral blood of individuals with type 1 diabetes (T1D). However, questions remain about the prevalence of HIP-specific T cells in humans, the sequences they recognize, and their role in disease. We identified six novel HIPs that are recognized in the context of DRB1*04:01, discovered by utilizing a library of theoretical HIP sequences derived from insulin fragments covalently linked to one another or to fragments of secretory granule proteins or other islet-derived proteins. We demonstrate that T cells that recognize these HIPs are detectable in the peripheral blood of subjects with T1D and exhibit an effector memory phenotype. HIP-reactive T cell clones produced Th1-associated cytokines and proliferated in response to human islet preparations. These results support the relevance of HIPs in human disease, further establishing a novel post-translational modification that may contribute to the loss of peripheral tolerance in T1D.

Bio-Synthesis is USA based Custom Peptide Synthesis Company. In organic chemistry, peptide synthesis is the production of peptides. Peptides are chemically synthesized by the condensation reaction of the carboxyl group of amino acid.

PEPSI-COLA JAMAICA, whose success has been built on the unwavering support of Jamaicans, is raising the bar in humanitarian leadership by aiding in the local COVID-19 fight through a donation valued at over $15 million The donation, made to the...

We have had two articles published recently on bmj.com, looking at drug prevention of HIV; PeP - Post-exposure Prophylaxis and PreP - Pre-exposure Prophylaxis, neither prevent the virus from entering the body, but they do prevent the infection from taking hold. There are lots of questions that doctors have about these - what are the risk...

Christophe Bonny
Jan 1, 2001; 50:77-82
Islet Studies

Eve Van Cauter
Mar 1, 1992; 41:368-377
Original Article

Incretin hormone dysregulation contributes to reduced insulin secretion and hyperglycemia in patients with type 2 diabetes mellitus (T2DM). Resistance to glucose-dependent insulinotropic polypeptide (GIP) action may occur through desensitization or downregulation of β-cell GIP receptors (GIP-R). Studies in rodents and cell lines show GIP-R expression can be regulated through peroxisome proliferator–activated receptor (PPAR) response elements (PPREs). Whether this occurs in humans is unknown. To test this, we conducted a randomized, double-blind, placebo-controlled trial of pioglitazone therapy on GIP-mediated insulin secretion and adipocyte GIP-R expression in subjects with well-controlled T2DM. Insulin sensitivity improved, but the insulinotropic effect of infused GIP was unchanged following 12 weeks of pioglitazone treatment. In parallel, we observed increased GIP-R mRNA expression in subcutaneous abdominal adipocytes from subjects treated with pioglitazone. Treatment of cultured human adipocytes with troglitazone increased PPAR binding to GIP-R PPREs. These results show PPAR agonists regulate GIP-R expression through PPREs in human adipocytes, but suggest this mechanism is not important for regulation of the insulinotropic effect of GIP in subjects with T2DM. Because GIP has antilipolytic and lipogenic effects in adipocytes, the increased GIP-R expression may mediate accretion of fat in patients with T2DM treated with PPAR agonists.

The signal peptide of preproinsulin is a major source for HLA class I autoantigen epitopes implicated in CD8 T cell (CTL)–mediated β-cell destruction in type 1 diabetes (T1D). Among them, the 10-mer epitope located at the C-terminal end of the signal peptide was found to be the most prevalent in patients with recent-onset T1D. While the combined action of signal peptide peptidase and endoplasmic reticulum (ER) aminopeptidase 1 (ERAP1) is required for processing of the signal peptide, the mechanisms controlling signal peptide trimming and the contribution of the T1D inflammatory milieu on these mechanisms are unknown. Here, we show in human β-cells that ER stress regulates ERAP1 gene expression at posttranscriptional level via the IRE1α/miR-17-5p axis and demonstrate that inhibition of the IRE1α activity impairs processing of preproinsulin signal peptide antigen and its recognition by specific autoreactive CTLs during inflammation. These results underscore the impact of ER stress in the increased visibility of β-cells to the immune system and position the IRE1α/miR-17 pathway as a central component in β-cell destruction processes and as a potential target for the treatment of autoimmune T1D.