Using cryo-electron microscopy, we have solved the structure of a novel icosidodecahedral COPII coat involved in cargo export from the endoplasmic reticulum (ER) co-assembled from purified cargo adaptor Sec23-24 and Sec13-31 lattice forming complexes. The coat structure shows a tetrameric assembly of the Sec23-24 adaptor layer that is positioned beneath the vertices and edges of the Sec13-31 lattice. Fitting the known crystal structures of the COPII proteins into the density map reveals a flexible hinge region stemming from interactions between WD40 β-propeller domains present in Sec13 and Sec31 at the vertices. The structure shows that the hinge region can direct geometric cage expansion to accommodate a wide range of bulky cargo including procollagen and chylomicrons that are sensitive to adaptor function in inherited disease. The COPII coat structure leads us to propose a new mechanism by which cargo drives cage assembly and membrane curvature for budding from the ER.

Proteomic analysis of breast cancer tissue has proven difficult due to its inherent histological complexity. This pilot study presents preliminary evidence for the ability to differentiate adenoma and invasive carcinoma by measuring changes in proteomic profile of matched normal and disease tissues. A dual lysis buffer method was used to maximize protein extraction from each biopsy, proteins digested with trypsin, and the resulting peptides iTRAQ labeled. After combining, the peptide mixtures they were separated using preparative IEF followed by RP nanoHPLC. Following MALDI MS/MS and database searching, identified proteins were combined into a nonredundant list of 481 proteins with associated normal/tumor iTRAQ ratios for each patient. Proteins were categorized by location as blood, extracellular, and cellular, and the iTRAQ ratios were normalized to enable comparison between patients. Of those proteins significantly changed (upper or lower quartile) between matched normal and disease tissues, those from two invasive carcinoma patients had >50% in common with each other but <22% in common with an adenoma patient. In invasive carcinoma patients, several cellular and extracellular proteins that were significantly increased (Periostin, Small breast epithelial mucin) or decreased (Kinectin) have previously been associated with breast cancer, thereby supporting this approach for a larger diseasestage characterization effort.

Rab GTPases and SNARE fusion proteins direct cargo trafficking through the exocytic and endocytic pathways of eukaryotic cells. We have used steady state mRNA expression profiling and computational hierarchical clustering methods to generate a global overview of the distribution of Rabs, SNAREs, and coat machinery components, as well as their respective adaptors, effectors, and regulators in 79 human and 61 mouse nonredundant tissues. We now show that this systems biology approach can be used to define building blocks for membrane trafficking based on Rab-centric protein activity hubs. These Rab-regulated hubs provide a framework for an integrated coding system, the membrome network, which regulates the dynamics of the specialized membrane architecture of differentiated cells. The distribution of Rab-regulated hubs illustrates a number of facets that guides the overall organization of subcellular compartments of cells and tissues through the activity of dynamic protein interaction networks. An interactive website for exploring datasets comprising components of the Rab-regulated hubs that define the membrome of different cell and organ systems in both human and mouse is available at http://www.membrome.org/.

Endomembranes of eukaryotic cells are dynamic structures that are in continuous communication through the activity of specialized cellular machineries 1 , such as the coat protein complex II (COPII), which mediates cargo export from the endoplasmic reticulum (ER) 2,3 . COPII consists of the Sar1 GTPase, Sec23 and Sec24 (Sec23/24), where Sec23 is a Sar1-specific GTPase-activating protein and Sec24 functions in cargo selection, and Sec13 and Sec31 (Sec13/31), which has a structural role 3 . Whereas recent results have shown that Sec23/24 and Sec13/31 can self-assemble to form COPII cage-like particles 4 , we now show that Sec13/31 can self-assemble to form minimal cages in the absence of Sec23/24. We present a three-dimensional reconstruction of these Sec13/31 cages at 30 Å resolution using cryo-electron microscopy and single particle analysis. These results reveal a novel cuboctahedron geometry with the potential to form a flexible lattice and to generate a diverse range of containers. Our data are consistent with a model for COPII coat complex assembly in which Sec23/24 has a non-structural role as a multivalent ligand localizing the selfassembly of Sec13/31 to form a cage lattice driving ER cargo export.

The eukaryotic cell is defined by a complex set of sub-cellular compartments that include endomembrane systems making up the exocytic and endocytic trafficking pathways. Current evidence suggests that both the function and communication between these compartments are regulated by distinct families of proteins that direct membrane fission, targeting and fusion. These families include coat protein complexes (CPCs) involved in vesicle formation/fission, Rab GTPases involved in vesicle targeting, and soluble N-ethyl-maleimide-sensitive factor attachment protein receptors (SNAREs) involved in vesicle fusion. The origins of these gene families and their individual contributions to the evolutionary specialization of the membrane architectures of lower and higher eukaryotes are now better understood with the advent of powerful phylogenetic, structural and systems biology tools. Herein, we provide a perspective that suggests that while the core CPC and SNARE machineries have diversified mode...

Ancestry inference for a person using a panel of SNPs depends on the variation of frequencies of those SNPs around the world and the amount of reference data available for calculation/comparison. The Kidd Lab panel of 55 AISNPs has been incorporated in commercial kits by both Life Technologies and Illumina for massively parallel sequencing. Therefore, a larger set of reference populations will be useful for researchers using those kits. We have added reference population allele frequencies for 52 population samples to the 73 previously entered so that there are now allele frequencies publicly available in ALFRED and FROG-kb for a total of 125 population samples.

Here, we report an incest paternity case involving three biological brothers as alleged fathers (AFs), their biological sister and her child that was investigated using the Investigator ESSplex Plus, AmpFLSTR Identifiler Plus/Investigator IDplex Plus and PowerPlex 16 kits. Initial duo paternity investigations using 15-loci autosomal short tandem repeat (STR) analyses failed to exclude any of the AFs. Despite the fact that one of the brothers, AF1, had a mismatch with the child at a single locus (D2S1338), the possibility of a single-step mutation could not be ruled out. When the number of autosomal STR loci analysed was increased to 22 without the inclusion of the mother, AF2 and AF3 still could not be excluded, since both of them again had no mismatches with the child. A breakthrough was possible only upon inclusion of the mother so that trio paternity investigations were carried out. This time AF1 and AF2 could be excluded at two loci (D2S1338 and D1S1656) and six loci (vWa, D1S16...

This Bud's for the Golgi viding a genetic system for dissection of this early step. In contrast, and unlike P. pastoris, the yeast Saccharomyes cerevisiae lacks tER structures. As a consequence, COPII budding occurs throughout the ER. What, then, is the role of the tER in COPII vesicle formation? Selective cargo export from the endoplasmic reticu-As shown by Glick and colleagues, the tER in P. pastoris lum is brought about by the budding of COPII vesicles.

We explored the production in Pichia pastoris of a membrane-acting immunotoxin (IT) based on the Cyt2Aa1 toxin from the bacterium Bacillus thuringiensis subspecies kyushuensis. Initial attempts at the P. pastoris expression of Cyt2Aa1 were not successful due to the high A + T-content of the native bacterial gene, resulting in premature transcription termination. Accordingly, we designed and constructed a synthetic cyt2Aa1 gene (syncyt2Aa1) 2 that was optimised for expression in this eukaryotic host. This was achieved through a recursive PCR strategy where the overall G + C-content of the cyt2Aa1 DNA sequence was systematically increased to $50% compared to $30% in the native bacterial gene and only the P. pastoris preferred codons were used. A synthetic DNA sequence coding for a soluble and flexible serine/glycine linker was then used to genetically fuse syncyt2Aa1 with the human single-chain antibody fragment (scFv) C6.5 targeting p185 HERÀ2 , a cell-surface glycoprotein overexpressed in 30% of human breast and ovarian cancers. Subsequent expression of the resulting IT construct ½scFvC6:5-syncyt2Aa1ðmychis 6 Þ 2 led to high-level accumulation of the recombinant protein in yeast membranes. Although the solubilisation of scFvC6.5-syncyt2Aa1(mychis 6 ) from P. pastoris membranes necessitated the use of guanidine hydrochloride, the use of subsequent in vitro refolding and immobilised metal affinity chromatography (IMAC) steps allowed purification of the recombinant product at yields as high as $ 10 mg l À1 culture. Despite being core N-linked glycosylated and retaining part of the yeast secretion signal, the P. pastoris produced scFvC6.5-syncyt2Aa1(mychis 6 ) exhibited significant specific activity for p185 HERÀ2 -overexpressing SK-BR-3 cells but not p185 HERÀ2 -negative Swiss 3T3 cells or human erythrocytes. 2 The sequence data corresponding to the syncyt2Aa1 and scFvC6.5-syncyt2Aa1(mychis 6 ) open reading frames were deposited with the EMBL/ GenBank Database under Accession Nos. AF398463 and AF516873, respectively.

The methylotrophic yeast Pichia pastoris is a popular heterologous expression host for the recombinant production of a variety of prokaryotic and eukaryotic proteins. The rapid emergence of P. pastoris as a robust heterologous expression host was facilitated by the ease with which it can be manipulated and propagated, which is comparable to that of Escherichia coli and Saccharomyces cerevisiae. P. pastoris offers further advantages such as the tightly-regulated alcohol oxidase promoter that is particularly suitable for heterologous expression of foreign genes. While recombinant production of bacterial toxins and their derivatives is highly desirable, attempts at their heterologous expression using the traditional E. coli expression system can be problematic due to the formation of inclusion bodies that often severely limit the final yields of biologically active products. However, recent literature now suggests that P. pastoris may be an attractive alternative host for the heterologous production of bacterial toxins, such as those from the genera Bacillus, Clostridium, and Corynebacterium, as well as their more complex derivatives. Here, we review the recombinant production of bacterial toxins and their derivatives in P. pastoris with special emphasis on their potential clinical applications. Considering that de novo design and construction of synthetic toxin genes have often been necessary to achieve optimal heterologous expression in P. pastoris, we also present general guidelines to this end based on our experience with the P. pastoris expression of the Bacillus thuringiensis Cyt2Aa1 toxin.

This technical note presents the methods and techniques developed by the Bi-communal Forensic Team (BCFT) of the Committee
on Missing Persons in Cyprus (CMP) used to excavate and exhume the remains of missing persons, many of whom were buried in deep
wells at sites across the island of Cyprus during the conflict period of the 1960s and 1970s. A total of 493 Turkish Cypriots and 1508 Greek
Cypriots were officially reported missing by the two communities as a result of the conflict. Since the team’s formation, in 2005, the BCFT
has excavated 114 wells, resulting in the recovery of 195 missing individuals from 35 of these well excavations. The standard excavation
approach used by the BCFT, especially for deep well recovery, consists of “ramp,” “pocket,” and “pool” components. These excavation features
enable CMP archaeologists to excavate deep wells safely and efficiently while simultaneously permitting time for thorough documentation
and unimpeded recovery of human remains. The team uses three variants of this approach to cope with the variety of geological, physical, and
hydrological contexts faced in Cyprus’ wells, including hard and soft landforms, the presence or absence of water, and limitations imposed by
surrounding infrastructure. The “terracing”, “double-ramp”, and “single-ramp” variations are detailed with respect to the environmental contexts
which prescribe their use. The BCFT’s general procedures for human remains recovery and standard well safety protocols conclude the article.

İnsan iskeletinden yaş tahmini, biyolojik profil oluşturmada önemli bir
basamaktır. Adli antropologların amacı, ölmüş kişilerin kimliklendirilmesinde, tıbbi ve
hukuki yetkililere muhtemel yaş tahmini sunarak yardım etmektir. Bu tahmin
yetişkinlerde, genellikle yaşla birlikte tahmin edilebilir bir şekilde dejenere olmuş
çeşitli iskelet özelliklerinin incelenmesiyle yapılır. Adli antropologlar bu özelliklerin
karakteristik bulgularını kullanarak mümkün olan en dar yaş aralığını tespit etmeye
çalışırlar. Fakat göreceğiniz üzere, insandan insana değişen dejeneratif özellikler ve
yaşlanma hızı süreci, daha geniş yaş aralıkları tahminine neden olmaktadır.

Adli Bilimler tarihinde, olay yeri incelemeleri ve çalışmalarında arkeolog ve antropologlara ihtiyaç duyulması 1970'lere dayanır. Günümüzde dünyanın birçok ülkesinde adli arkeologlar ve antropologlar polis birimlerinde, profesyonel adli ekiplerde görev almakta ve birçok adli vakada şahitlik yaparak olayların çözülmesinde önemli rol oynamaktadırlar. Kuzey Kıbrıs ve Türkiye Cumhuriyeti'nde yürütülen bu çalışmalarda görev alacak olay yeri inceleme uzmanlarının, temel seviyede iskelet incelemeleri konusunda bilgi sahibi olmaları ve iskelet buluntularını içeren vakalarda, OYİ uzmanlarının yanında adli antropologların da olay yerinde görev almaları gerekmektedir. Bu makalede, adli arkeolog ve adli antropologların olay yeri incelemeleri ve çalışmalarındaki rolleri, önemi ve gerekliliği üzerinde durulacaktır.

In the history of Forensic Sciences, the need for archaeologists and anthropologists assist in crime scene investigations arise around 1970's. Since then forensic archaeologists and anthropologists began to work in police departments or in professional forensic teams in many countries, and played an important role in the courts by providing answers and conclusions to law enforcement agencies. The crime scene investigation experts in North Cyprus and Turkey must have basic knowledge about human bones, and forensic anthropologists should assist CSI experts in the cases which human remains are located. This paper focuses on the role of forensic archaeologists and forensic anthropologists in crime scene investigations and examinations and the need for their expertise in death investigations.