Expression of active human sialyltransferase ST6GalNAcI in Escherichia coli

Georgios Skretas, Sean Carroll, and George Georgiou are with the Department of Chemical Engineering, University of Texas at Austin, Austin, TX 78712, USA -- George Georgiou is with the Department of Biomedical Engineering, University of Texas at Austin and the Section of Microbiology and Molecular Genetics, University of Texas at Austin, Austin, TX 78712, USA -- Georgios Skretas and George Georgiou are with the Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX 78712, USA -- Shawn DeFrees, Karl F. Johnson, and Marc F. Schwartz are with Neose Technologies Inc, 102 Rock Road, Horsham, PA, 19044, USABackground: The presence of terminal, surface-exposed sialic acid moieties can greatly enhance the in vivo half-life of glycosylated biopharmaceuticals and improve their therapeutic efficacy. Complete and homogeneous sialylation of glycoproteins can be efficiently performed enzymically in vitro but this process requires large amounts of catalytically active sialyltransferases. Furthermore, standard microbial hosts used for large-scale production of recombinant enzymes can only produce small quantities of glycosyltransferases of animal origin, which lack catalytic activity. Results and conclusion: In this work, we have expressed the human sialyltransferase ST6GalNAc I (ST6), an enzyme that sialylates O-linked glycoproteins, in Escherichia coli cells. We observed that wild-type bacterial cells are able to produce only very small amounts of soluble ST6 enzyme. We have found, however, that engineered bacterial strains which possess certain types of oxidative cytoplasm or which co-express the molecular chaperones/co-chaperones trigger factor, DnaK/DnaJ, GroEL/GroES, and Skp, can produce greatly enhanced amounts of soluble ST6. Furthermore, we have developed a novel high-throughput assay for the detection of sialyltransferase activity and used it to demonstrate that the bacterially expressed ST6 enzyme is active and able to transfer sialic acid onto a desialylated O-glycoprotein, bovine submaxillary mucin. To the best of our knowledge, this is the first example of expression of active human sialyltransferase in bacteria. This system may be used as a starting point for the evolution of sialyltransferases with better expression characteristics or altered donor/acceptor specificities.Chemical EngineeringBiomedical EngineeringInstitute for Cellular and Molecular [email protected]

Code wars: steganography, signals intelligence, and terrorism

This paper describes and discusses the process of secret communication known as steganography. The argument advanced here is that terrorists are unlikely to be employing digital steganography to facilitate secret intra-group communication as has been claimed. This is because terrorist use of digital steganography is both technically and operationally implausible. The position adopted in this paper is that terrorists are likely to employ low-tech steganography such as semagrams and null ciphers instead

Species-Specific Scaling to Define and Conserve the Northern Great Plains Region

Prairie ecosystems are in a continuous state of flux, shifting by processes that include variable weather patterns and climatic conditions, disturbance regimes, and more recently, human-induced modification. Similarly, wildlife resources fluctuate across the landscape as a result of these ever-changing conditions; however, human alterations have increased, removed, and manipulated the ecological processes of the prairie. Specifically, the spatial scales at which humans manage and interact with the landscape are often inconsistent or incompatible with the scales required for the persistence of wildlife populations. Our synthesis demonstrates how the spatial scales at which wildlife in the Northern Great Plains of North America operate have been constrained by human intervention. This process of anthropogenic scaling has affected the decline of many native wildlife populations and in some cases has resulted in the complete extirpation of species from the landscape. We use historical observations and recent quantitative data to describe the primary cause of spatial scale alteration for prairie focal species (i.e. plains bison, pronghorn, grassland birds, Greater Sage-grouse, black-tailed prairie dogs, swift fox, prairie rattlesnakes) using migration, home range, distribution, and dispersal distances as metrics. We then describe the role that spatial scale plays in wildlife management of the prairie landscape from the non-profit, state, and federal perspective and how these entities are managing at the scales of each focal species

Stress Granules and RNA Processing Bodies are Novel Autoantibody Targets in Systemic Sclerosis

Autoantibody profiles represent important patient stratification markers in systemic sclerosis (SSc). Here, we performed serum-immunoprecipitations with patient antibodies followed by mass spectrometry (LC-MS/MS) to obtain an unbiased view of all possible autoantibody targets and their associated molecular complexes recognized by SSc

Appendectomy versus non-operative treatment for acute uncomplicated appendicitis in children: Study protocol for a multicentre, open-label, non-inferiority, randomised controlled trial

Background Appendectomy is considered the gold standard treatment for acute appendicitis. Recently the need for surgery has been challenged in both adults and children. In children there is growing clinician, patient and parental interest in non-operative treatment of acute appendicitis with antibiotics as opposed to surgery. To date no multicentre randomised controlled trials that are appropriately powered to determine efficacy of nonoperative treatment (antibiotics) for acute appendicitis in children compared with surgery (appendectomy) have been performed. Methods Multicentre, international, randomised controlled trial with a non-inferiority design. Children (age 5–16 years) with a clinical and/or radiological diagnosis of acute uncomplicated appendicitis will be randomised (1:1 ratio) to receive either laparoscopic appendectomy or treatment with intravenous (minimum 12 hours) followed by oral antibiotics (total course 10 days). Allocation to groups will be stratified by gender, duration of symptoms (≫ or \u3c48 hours) and centre. Children in both treatment groups will follow a standardised treatment pathway. Primary outcome is treatment failure defined as additional intervention related to appendicitis requiring general anaesthesia within 1 year of randomisation (including recurrent appendicitis) or negative appendectomy. Important secondary outcomes will be reported and a cost-effectiveness analysis will be performed. The primary outcome will be analysed on a non-inferiority basis using a 20% non-inferiority margin. Planned sample size is 978 children. Discussion The APPY trial will be the first multicentre randomised trial comparing non-operative treatment with appendectomy for acute uncomplicated appendicitis in children. The results of this trial have the potential to revolutionise the treatment of this common gastrointestinal emergency. The randomised design will limit the effect of bias on outcomes seen in other studies. Trial registration number clinicaltrials.gov:NCT02687464. Registered on Jan 13th 2016

A Real Time Metridia Luciferase Based Non-Invasive Reporter Assay of Mammalian Cell Viability and Cytotoxicity via the β-actin Promoter and Enhancer

Secreted reporter molecules offer a means to evaluate biological processes in real time without the need to sacrifice samples at pre-determined endpoints. Here we have adapted the secreted bioluminescent reporter gene, Metridia luciferase, for use in a real-time viability assay for mammalian cells. The coding region of the marine copepod gene has been codon optimized for expression in human cells (hMLuc) and placed under the control of the human β-actin promoter and enhancer. Metridia luciferase activity of stably transfected cell models corresponded linearly with cell number over a 4-log dynamic range, detecting as few as 40 cells. When compared to standard endpoint viability assays, which measure the mitochondrial dehydrogenase reduction of tetrazolium salts, the hMLuc viability assay had a broader linear range of detection, was applicable to large tissue culture vessels, and allowed the same sample to be repeatedly measured over several days. Additional studies confirmed that MLuc activity was inhibited by serum, but demonstrated that assay activity remained linear and was measurable in the serum of mice bearing subcutaneous hMLuc-expressing tumors. In summary, these comparative studies demonstrate the value of humanized Metridia luciferase as an inexpensive and non-invasive method for analyzing viable cell number, growth, tumor volume, and therapeutic response in real time

A proposal for a coordinated effort for the determination of brainwide neuroanatomical connectivity in model organisms at a mesoscopic scale

In this era of complete genomes, our knowledge of neuroanatomical circuitry remains surprisingly sparse. Such knowledge is however critical both for basic and clinical research into brain function. Here we advocate for a concerted effort to fill this gap, through systematic, experimental mapping of neural circuits at a mesoscopic scale of resolution suitable for comprehensive, brain-wide coverage, using injections of tracers or viral vectors. We detail the scientific and medical rationale and briefly review existing knowledge and experimental techniques. We define a set of desiderata, including brain-wide coverage; validated and extensible experimental techniques suitable for standardization and automation; centralized, open access data repository; compatibility with existing resources, and tractability with current informatics technology. We discuss a hypothetical but tractable plan for mouse, additional efforts for the macaque, and technique development for human. We estimate that the mouse connectivity project could be completed within five years with a comparatively modest budget.Comment: 41 page

Load carriage changes tibiofemoral arthrokinematics during ambulatory tasks in recruit-aged women

The introduction of women into U.S. military ground close combat roles requires research into sex-specific effects of military training and operational activities. Knee osteoarthritis is prevalent among military service members; its progression has been linked to occupational tasks such as load carriage. Analyzing tibiofemoral arthrokinematics during load carriage is important to understand potentially injurious motion and osteoarthritis progression. The study purpose was to identify effects of load carriage on knee arthrokinematics during walking and running in recruit-aged women. Twelve healthy recruit-aged women walked and ran while unloaded (bodyweight [BW]) and carrying additional + 25%BW and + 45%BW. Using dynamic biplane radiography and subject-specific bone models, tibiofemoral arthrokinematics, subchondral joint space and center of closest contact location between subchondral bone surfaces were analyzed over 0–30% stance (separate one-way repeated measures analysis of variance, load by locomotion). While walking, medial compartment contact location was 5% (~ 1.6 mm) more medial for BW than + 45%BW at foot strike (p = 0.03). While running, medial compartment contact location was 4% (~ 1.3 mm) more lateral during BW than + 25%BW at 30% stance (p = 0.04). Internal rotation was greater at + 45%BW compared to + 25%BW (p < 0.01) at 30% stance. Carried load affects tibiofemoral arthrokinematics in recruit-aged women. Prolonged load carriage could increase the risk of degenerative joint injury in physically active women