Criminal Procedure - Match-Game 1990\u27s: The Admissibility of DNA Profiling - State v. Pennington

This Note discusses the nature, history, and effect of DNA profiling and supports the Pennington court\u27s holding as the correct approach. Although the court adopted the majority view, and most likely the correct view, this area of law still remains in a state of confusion

A novel strategy for the targeted analysis of protein and peptide metabolites

The detection and quantitation of exogenously administered biological macromolecules (e.g. vaccines, peptide and protein therapeutics) and their metabolites is frequently complicated by the presence of a complex endogenous mixture of closely related compounds. We describe a method that incorporates stable isotope labeling of the compound of interest allowing the selective screening of the intact molecule and all metabolites using a modified precursor ion scan. This method involves monitoring the low molecular weight fragment ions produced during MS/MS that distinguish isotopically labelled material from related endogenous compounds. All isotopically labelled substances can be selected using this scanning technique for further analysis whilst other unlabelled and irrelevant substances are ignored. The potential for this technique to be used in metabolism and pharmacokinetic experiments is discussed with specific examples looking at the metabolism of α-synuclein in serum and the brain

HLA Class II Specificity Assessed by High-Density Peptide Microarray Interactions

The ability to predict and/or identify MHC binding peptides is an essential component of T cell epitope discovery, something that ultimately should benefit the development of vaccines and immunotherapies. In particular, MHC class I prediction tools have matured to a point where accurate selection of optimal peptide epitopes is possible for virtually all MHC class I allotypes; in comparison, current MHC class II (MHC-II) predictors are less mature. Because MHC-II restricted CD4+ T cells control and orchestrated most immune responses, this shortcoming severely hampers the development of effective immunotherapies. The ability to generate large panels of peptides and subsequently large bodies of peptide-MHC-II interaction data are key to the solution of this problem, a solution that also will support the improvement of bioinformatics predictors, which critically relies on the availability of large amounts of accurate, diverse, and representative data. In this study, we have used rHLA-DRB1*01:01 and HLA-DRB1*03:01 molecules to interrogate high-density peptide arrays, in casu containing 70,000 random peptides in triplicates. We demonstrate that the binding data acquired contains systematic and interpretable information reflecting the specificity of the HLA-DR molecules investigated, suitable of training predictors able to predict T cell epitopes and peptides eluted from human EBV-transformed B cells. Collectively, with a cost per peptide reduced to a few cents, combined with the flexibility of rHLA technology, this poses an attractive strategy to generate vast bodies of MHC-II binding data at an unprecedented speed and for the benefit of generating peptide-MHC-II binding data as well as improving MHC-II prediction tools.Fil: Osterbye, Thomas. Universidad de Copenhagen; DinamarcaFil: Nielsen, Morten. Technical University of Denmark; Dinamarca. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Dudek, Nadine L.. Monash University; AustraliaFil: Ramarathinam, Sri H.. Monash University; AustraliaFil: Purcell, Anthony W.. Monash University; AustraliaFil: Schafer-Nielsen, Claus. No especifíca;Fil: Buus, Soren. University Of Copenhagen, Faculty Of Health Sciences

Protein secretion and outer membrane assembly in Alphaproteobacteria

The assembly of β-barrel proteins into membranes is a fundamental process that is essential in Gram-negative bacteria, mitochondria and plastids. Our understanding of the mechanism of β-barrel assembly is progressing from studies carried out in Escherichia coli and Neisseria meningitidis. Comparative sequence analysis suggests that while many components mediating β-barrel protein assembly are conserved in all groups of bacteria with outer membranes, some components are notably absent. The Alphaproteobacteria in particular seem prone to gene loss and show the presence or absence of specific components mediating the assembly of β-barrels: some components of the pathway appear to be missing from whole groups of bacteria (e.g. Skp, YfgL and NlpB), other proteins are conserved but are missing characteristic domains (e.g. SurA). This comparative analysis is also revealing important structural signatures that are vague unless multiple members from a protein family are considered as a group (e.g. tetratricopeptide repeat (TPR) motifs in YfiO, β-propeller signatures in YfgL). Given that the process of the β-barrel assembly is conserved, analysis of outer membrane biogenesis in Alphaproteobacteria, the bacterial group that gave rise to mitochondria, also promises insight into the assembly of β-barrel proteins in eukaryotes

The interplay between citrullination and HLA-DRB1 polymorphism in shaping peptide binding hierarchies in rheumatoid arthritis

The HLA-DRB1 locus is strongly associated with rheumatoid arthritis (RA) susceptibility, whereupon citrullinated self-peptides bind to HLA-DR molecules bearing the shared epitope (SE) amino acid motif. However, the differing propensity for citrullinated/non-citrullinated self-peptides to bind given HLA-DR allomorphs remains unclear. Here, we used a fluorescence polarization assay to determine a hierarchy of binding affinities of 34 self-peptides implicated in RA against three HLA-DRB1 allomorphs (HLA-DRB1*04:01/*04:04/*04:05) each possessing the SE motif. For all three HLA-DRB1 allomorphs, we observed a strong correlation between binding affinity and citrullination at P4 of the bound peptide ligand. A differing hierarchy of peptide-binding affinities across the three HLA-DRB1 allomorphs was attributable to the β-chain polymorphisms that resided outside the SE motif and were consistent with sequences of naturally presented peptide ligands. Structural determination of eight HLA–DR4–self-epitope complexes revealed strict conformational convergence of the P4-Cit and surrounding HLA β-chain residues. Polymorphic residues that form part of the P1 and P9 pockets of the HLA-DR molecules provided a structural basis for the preferential binding of the citrullinated self-peptides to the HLA-DR4 allomorphs. Collectively, we provide a molecular basis for the interplay between citrullination of self-antigens and HLA polymorphisms that shape peptide–HLA-DR4 binding affinities in RA

Kinetics of antigen expression and epitope presentation during virus infection

Current knowledge about the dynamics of antigen presentation to T cells during viral infection is very poor despite being of fundamental importance to our understanding of anti-viral immunity. Here we use an advanced mass spectrometry method to simultaneously quantify the presentation of eight vaccinia virus peptide-MHC complexes (epitopes) on infected cells and the amounts of their source antigens at multiple times after infection. The results show a startling 1000-fold range in abundance as well as strikingly different kinetics across the epitopes monitored. The tight correlation between onset of protein expression and epitope display for most antigens provides the strongest support to date that antigen presentation is largely linked to translation and not later degradation of antigens. Finally, we show a complete disconnect between the epitope abundance and immunodominance hierarchy of these eight epitopes. This study highlights the complexity of viral antigen presentation by the host and demonstrates the weakness of simple models that assume total protein levels are directly linked to epitope presentation and immunogenicity.NHMRC (National Health and Medical Research Council of Australia

Multi-omic analysis of two common p53 mutations: Proteins regulated by mutated p53 as potential targets for immunotherapy

The p53 protein is mutated in more than 50% of human cancers. Mutated p53 proteins not only lose their normal function but often acquire novel oncogenic functions, a phenomenon termed mutant p53 gain-of-function. Mutant p53 has been shown to affect the transcription of a range of genes, as well as protein–protein interactions with transcription factors and other effectors; however, no one has intensively investigated and identified these proteins, or their MHC presented epitopes, from the viewpoint of their ability to act as targets for immunotherapeutic interventions. We investigated the molecular changes that occurred after the TP53 null osteosarcoma cells, SaOS-2, were transfected with one of two conformational p53-mutants, either R175H or R273H. We then examined the phenotypic and functional changes using macroscopic observations, proliferation, gene expression and proteomics alongside immunopeptidome profiling of peptide antigen presentation in the context of major histocompatibility complex (MHC) class I molecules. We identified several candidate proteins in both TP53 mutant cell lines with differential expression when compared to the TP53 null vector control, SaOS-V. Quantitative SWATH proteomics combined with immune-peptidome analysis of the class-I eluted peptides identified several epitopes presented on pMHC and in silico analysis shortlisted which antigens were expressed in a range of cancerous but not adjacent healthy tissues. Out of all the candidates, KLC1 and TOP2A showed high levels of expression in every tumor type examined. From these proteins, three A2 and four pan HLA-A epitopes were identified in both R175H and R273H from TOP2A. We have now provided a short list of future immunotherapy targets for the treatment of cancers harboring mutated TP53

Die Ballon-Okklusionsangiographie der Pulmonalarterien bei chronischer pulmonaler Hypertonie

Die chronische pulmonale Hypertonie ist eine seltene Erkrankung, die heute noch als unheilbar gilt. Die therapeutischen Möglichkeiten haben sich in den letzten Jahren rasant weiterentwickelt und orientieren sich an der Pathogenese und an den Befunden aus der bildgebenden Diagnostik. Diese muss in ihren Möglichkeiten der differentialdiagnostischen Eingrenzung zugrundeliegender Ursachen der pulmonalen Hypertonie Schritt halten. Insbesondere die Ergebnisse der intraarteriellen Pulmonalisangiographie als Referenzmethode in der Bildgebung der Lungen-strombahn haben meist unmittelbare therapeutische Konsequenz, z.B. ob ein Patient für eine pulmonale Thrombendarteriektomie gegeignet ist oder nicht. Wir haben überprüft, ob die selektive pulmonale Ballon-Okklusionsangiographie als Erweiterung der standardisierten Übersichtsangiographie die Differentialdiagnose z.B. der embolischen (CTEPH) gegenüber der nichtembolischen (NoCTEPH) Erkrankung verbessert. Zu diesem Zweck wurden Untersuchungen von 50 Patienten bestehend aus jeweils einer konventionellen Übersichtsangiographie und der ergänzenden Ballon-Okklusionsangiographie retrospektiv nach einem standardisierten Studienprotokoll ausgewertet. Zunächst wurde eine digitale Subtraktionsangiographie der Pulmonalarterien angefertigt. Anschließend führten wir einen weichen Latex-Ballonkatheter in Segment- oder Subsegmentarterien ein. Dann entfalteten wir den Ballon, um die sondierte Arterie zu verschließen und injizierten jeweils 10 bis 15 ml Kontrastmittel, um die kleinen peripheren Gefäße sichtbar zu machen. 13 Patienten litten an einer nichtthrombembolischen Form der chronischen pulmonalen Hypertonie. Bei 36 von 37 Patienten mit CTEPH fanden wir organisiertes embolisches Material als irreguläre Stenosen, Verschlüsse oder Strickleitersysteme (Webs und Bands). In der Darstellung dieser pathologischen Befunde war die Ballon-Okklusionsangiographie der Übersichtsangiographie sowohl qualitativ als auch quantitativ überlegen. Nach unseren Daten entdeckt die Ballon-Okklusionsangiographie in etwa bei jedem fünften Patienten mit negativer Übersichtsangiographie wenigstens ein thrombembolisches Residuum, sie steigert somit als Verfeinerung der Methode die Sensitivität der Pulmonalisangiographie. Generell stellte sie 2,7 bis 3,6 Aufteilungsgenerationen der peripheren Gefäße mehr dar als die konventionelle selektive DSA. Außerdem fanden wir bei 17 Patienten Kollateralgefäße zu den peripheren Segmenten von zentral verschlossenen Pulmonal-arterien. Dieses Phänomen war nur in der Ballon-Okklusionsangiographie zu beobachten und erwies sich als spezifisch für Patienten mit thrombembolischer pulmonaler Hypertonie. Der Befund ist insofern erstaunlich, als dass Pulmonalarterien eigentlich als funktionelle Endarterien ohne Anastomosen zu Nachbararterien beschrieben werden. Bei 11 Patienten fanden sich Anastomosen zu subpleuralen Bronchialarterien. Dieses bereits bekannte Phänomen steht nach unseren Daten in keinem Zusammenhang mit einer bestimmten Erkrankung und ist somit als unspezifisches Merkmal der chronischen pulmonalen Hypertonie zu deuten. In drei Fällen konnten histologisch postkapilläre Formen der pulmonalen Hypertonie (zweimal pulmonale veno-okklusive Erkrankung (PVOD), einmal primäre kapilläre Hämangiomatose) gesichert werden. Bei diesen Patienten zeigte die Ballon-Okklusionsangiographie eine Füllung der Lungenvenen ohne angiographisch sichtbare Anfärbung des Kapillarbettes (fehlende Parenchymanfärbung). Die geschilderten Erkenntnisse aus unserer Studie lassen sich für die Praxis wie folgt zusammenfassen: 1. Die Ballon-Okklusionsangiographie verbessert die Visualisierung der peripheren Pulmonalarterien. 2. Sie erleichtert die Detektion und Lokalisation thrombembolischer Residuen 3. Sie hilft bei der Differentialdiagnose zwischen thrombembolischer und nicht-thrombembolischer chronischer pulmonaler Hypertonie. 4. Vorher unsichtbare Anastomosen und Kollateralgefäße werden sichtbar. 5. Venöse Füllung ohne Parenchymanfärbung ist offensichtlich ein Zeichen der Parenchymerkrankung; diese Konstellation ist bei Patienten mit chronischer pulmonaler Hypertonie möglicherweise ein Hinweis auf das Vorliegen der pulmonalen venookklusiven Erkrankung (PVOD) oder der primären kapillären Hämangiomatose (PCH). 6. Die selektive Ballon-Okklusionsangiographie segmentaler Pulmonalarterien verbessert in Zusammenschau mit der Computertomographie die Zuverlässigkeit in der Selektion von Kandidaten für eine pulmonale Thrombendarteriektomie oder eine Prostazyklintherapie.Purpose: Test the ability of selective balloon occlusion angiography of pulmonary segmental arteries in the differential diagnosis of chronic pulmonary hypertension: embolic vs. non-embolic disease, pulmonary capillary hemangiomatosis, and venoocclusive disease. Methods and Materials: In 50 patients with pulmonary hypertension, digital subtraction angiography (DSA) of pulmonary arteries were used to assist in the selection of candidates appropriate for thrombo-endarterectomy. In addition to these standard methods, we introduced a soft latex balloon catheter into segmental arteries, inflated the balloon to occlude the artery, and injected 10 to 15 ml contrast medium to visualize small peripheral vessels as completely as possible. Results: 13 patients suffered from non-embolic pulmonary hypertension. In 36 of 37 patients with embolic pulmonary hypertension organizing embolic material was depicted as irregular narrowing or occlusion of pulmonary arteries, and weblike strictures. In all of these patients occlusion technique revealed more tiny webs or organized micro emboli in small peripheral arteries. According to our data balloon occlusion angiography discovers in every fifth patient showing a negative conventional pulmonary angiography at least one thromboembolic residuum and thus increases as a sophisticated method the sensitivity of the pulmonary angiography. Generally, occlusion technique revealed additional 2,7 to 3,6 ramifications of peripheral vessels in comparison to conventional selective DSA. Unexpectedly, we found in 11 patients anastomoses to bronchial arteries and in 17 patients collateral vessels to the peripheral segments of centrally occluded pulmonary arteries. These findings are astonishing, because pulmonary arteries are believed ramifying dichotomically without anastomoses. Obviously, there are alterations of pulmonary perfusion, which overcome normal anatomy. 3 patients with characteristic CT signs of interstitial disease (poorly defined nodular opacities and septal lines) underwent lung biopsy: 2 cases of venoocclusive disease, 1 case of pulmonary capillary hemangiomatosis. Occlusion angiography in these 3 patients revealed filling of veins without opacification of capillaries (failing parenchymal phase). Conclusion: Balloon occlusion technique improves the visualization of peripheral pulmonary arteries. Differential diagnosis of embolic and non-embolic pulmonary hypertension is facilitated. Previously invisible anastomoses and collateral vessels become visible. Venous filling without capillary opacification is apparently a sign of parenchymal disease; in patients with chronic pulmonary hypertension it might be a hint at venoocclusive disease or pulmonary capillary hemangiomatosi

The Shaping of T Cell Receptor Recognition by Self-Tolerance

SummaryDuring selection of the T cell repertoire, the immune system navigates the subtle distinction between self-restriction and self-tolerance, yet how this is achieved is unclear. Here we describe how self-tolerance toward a trans-HLA (human leukocyte antigen) allotype shapes T cell receptor (TCR) recognition of an Epstein-Barr virus (EBV) determinant (FLRGRAYGL). The recognition of HLA-B8-FLRGRAYGL by two archetypal TCRs was compared. One was a publicly selected TCR, LC13, that is alloreactive with HLA-B44; the other, CF34, lacks HLA-B44 reactivity because it arises when HLA-B44 is coinherited in trans with HLA-B8. Whereas the alloreactive LC13 TCR docked at the C terminus of HLA-B8-FLRGRAYGL, the CF34 TCR docked at the N terminus of HLA-B8-FLRGRAYGL, which coincided with a polymorphic region between HLA-B8 and HLA-B44. The markedly contrasting footprints of the LC13 and CF34 TCRs provided a portrait of how self-tolerance shapes the specificity of TCRs selected into the immune repertoire

Assembly of the type II secretion system such as found in Vibrio cholerae depends on the novel Pilotin AspS

The Type II Secretion System (T2SS) is a molecular machine that drives the secretion of fully-folded protein substrates across the bacterial outer membrane. A key element in the machinery is the secretin: an integral, multimeric outer membrane protein that forms the secretion pore. We show that three distinct forms of T2SSs can be distinguished based on the sequence characteristics of their secretin pores. Detailed comparative analysis of two of these, the Klebsiella-type and Vibrio-type, showed them to be further distinguished by the pilotin that mediates their transport and assembly into the outer membrane. We have determined the crystal structure of the novel pilotin AspS from Vibrio cholerae, demonstrating convergent evolution wherein AspS is functionally equivalent and yet structurally unrelated to the pilotins found in Klebsiella and other bacteria. AspS binds to a specific targeting sequence in the Vibrio-type secretins, enhances the kinetics of secretin assembly, and homologs of AspS are found in all species of Vibrio as well those few strains of Escherichia and Shigella that have acquired a Vibrio-type T2SS