An integrated cell-free metabolic platform for protein production and synthetic biology

Cell-free systems offer a unique platform for expanding the capabilities of natural biological systems for useful purposes, i.e. synthetic biology. They reduce complexity, remove structural barriers, and do not require the maintenance of cell viability. Cell-free systems, however, have been limited by their inability to co-activate multiple biochemical networks in a single integrated platform. Here, we report the assessment of biochemical reactions in an Escherichia coli cell-free platform designed to activate natural metabolism, the Cytomim system. We reveal that central catabolism, oxidative phosphorylation, and protein synthesis can be co-activated in a single reaction system. Never before have these complex systems been shown to be simultaneously activated without living cells. The Cytomim system therefore promises to provide the metabolic foundation for diverse ab initio cell-free synthetic biology projects. In addition, we describe an improved Cytomim system with enhanced protein synthesis yields (up to 1200 mg/l in 2 h) and lower costs to facilitate production of protein therapeutics and biochemicals that are difficult to make in vivo because of their toxicity, complexity, or unusual cofactor requirements

Extraction of [18F]fluoride from [18O]water by a fast fibrous anion exchange resin

[18F]Fluoride for nucleophilic radiofluorination was recovered from target water by trapping on a fibrous anion exchange resin in the hydroxide form and subsequent displacement into wet methanolic K2CO3. Extraction into methanol facilitated rapid evaporation and resolubilization of the [18F]fluoride as an ion pair. The resin was first dried in situ and rehydrated with [18O]H2O to avoid isotopic dilution of the target water.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/28900/1/0000737.pd

An integrated cell-free metabolic platform for protein production and synthetic biology

Cell-free systems offer a unique platform for expanding the capabilities of natural biological systems for useful purposes, i.e. synthetic biology. They reduce complexity, remove structural barriers, and do not require the maintenance of cell viability. Cell-free systems, however, have been limited by their inability to co-activate multiple biochemical networks in a single integrated platform. Here, we report the assessment of biochemical reactions in an Escherichia coli cell-free platform designed to activate natural metabolism, the Cytomim system. We reveal that central catabolism, oxidative phosphorylation, and protein synthesis can be co-activated in a single reaction system. Never before have these complex systems been shown to be simultaneously activated without living cells. The Cytomim system therefore promises to provide the metabolic foundation for diverse ab initio cell-free synthetic biology projects. In addition, we describe an improved Cytomim system with enhanced protein synthesis yields (up to 1200 mg/l in 2 h) and lower costs to facilitate production of protein therapeutics and biochemicals that are difficult to make in vivo because of their toxicity, complexity, or unusual cofactor requirements

Routine production of 2-deoxy-2-[18F]fluoro--glucose by direct nucleophilic exchange on a quaternary 4-aminopyridinium resin

Resin-supported [18F]fluoride ion has been prepared and applied to a rapid, convenient synthesis of [18F]FDG. "No-carrier-added" [18F]fluoride ion is collected on a quaternary 4-(N, N-dialkylamino)-pyridinium functionalized polystyrene anion exchange resin directly from a [18O]water target, dried by rinsing with acetonitrile, and then reacted with 1,3,4,6-tetra-O-acetyl-2-O-trifluoromethanesulfonyl-[beta]--mannopyrannose. Acidic hydrolysis yields [18F]FDG in a synthesis time of 40 min with overall yields presently averaging above 50%.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/28859/1/0000694.pd

A captive solvent method for rapid N-[11C]methylation of secondary amides: Application to the benzodiazepine, 4'-chlorodiazepam (RO5-4864)

[11C]4'-Chlorodiazepam (RO5-4864), for PET studies of peripheral benzodiazepine receptors, was synthesized by alkylation of 1-desmethyl-4'-chlorodiazepam, in a small volume of acetone adsorbed on acrylic yarn, with [11C]methyl iodide in the injection loop of a liquid chromatograph. The reaction mixture was introduced directly onto a small, disposable alumina chromatographic column. Elution with pentane:ethanol gave a product of high chemical and radiochemical purity. A simple heating and cooling device for the injection loop is described.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27565/1/0000609.pd

Technical Note: Method to correlate whole‐specimen histopathology of radical prostatectomy with diagnostic MR imaging

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/134778/1/mp1016.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/134778/2/mp1016_am.pd

Multiphase extraction: Rapid phase-transfer of [18F]fluoride ion for nucleophilic radiolabeling reactions

In multiphase extraction [18F]fluoride ion for radiolabeling is recovered from target water by passage through a small column of microporous polymer impregnated with a lipophilic cryptand or quaternary ammonium salt. The 18O enriched water can be recovered for reuse. The [18F]fluoride ion-pair is eluted from the column by a small volume of acetonitrile or other organic solvent. Evaporation of the acetonitrile removes traces of water to yield a reactive ion pair for nucleophilic radiofluorination reactions. A wide range of ion-pairs based on K+ or NH4+ cryptands or quaternary ammonium salts can be employed. The method was applied to the synthesis of [18F]FDG.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27603/1/0000647.pd

A Canadian approach to the regionalization of testis cancer: A review

At the Canadian Testis Cancer Workshop, the rationale and feasibility of regionalization of testis cancer care were discussed. The two-day workshop involved urologists, medical and radiation oncologists, pathologists, radiologists, physician’s assistants, residents and fellows, and nurses, as well as patients and patient advocacy groups. This review summarizes the discussion and recommendations of one of the central topics of the workshop — the centralization of testis cancer in Canada. It was acknowledged that non-guideline-concordant care in testis cancer occurs frequently, in the range of 18–30%. The National Health Service in the U.K. stipulates various testis cancer care modalities be delivered through supra-regional network. All cases are reviewed at a multidisciplinary team meeting and aspects of care can be delivered locally through the network. In Germany, no such network exists, but an insurance-supported online second opinion network was developed that currently achieves expert case review in over 30% of cases. There are clear benefits to regionalization in terms of survival, treatment morbidity, and cost. There was agreement at the workshop that a structured pathway for diagnosis and treatment of testis cancer patients is required. Regionalization may be challenging in Canada because of geography; independent administration of healthcare by each province; physicians fearing loss of autonomy and revenue; patient unwillingness to travel long distances from home; and the inability of the larger centers to handle the ensuing increase in volume. We feel the first step is to identify the key performance indicators and quality metrics to track the quality of care received. After identifying these metrics, implementation of a “networks of excellence” model, similar to that seen in sarcoma care in Ontario, could be effective, coupled with increased use of health technology, such as virtual clinics and telemedicine

Towards a pan-Arctic inventory of the species diversity of the macro- and megabenthic fauna of the Arctic shelf seas

Although knowledge of Arctic seas has increased tremendously in the past decade, benthic diversity was investigated at regional scales only, and no attempt had been made to examine it across the entire Arctic. We present a first pan-Arctic account of the species diversity of the macro- and megabenthic fauna of the Arctic marginal shelf seas. It is based on an analysis of 25 published and unpublished species-level data sets, together encompassing 14 of the 19 marine Arctic shelf ecoregions and comprising a total of 2636 species, including 847 Arthropoda, 668 Annelida, 392 Mollusca, 228 Echinodermata, and 501 species of other phyla. For the four major phyla, we also analyze the differences in faunal composition and diversity among the ecoregions. Furthermore, we compute gross estimates of the expected species numbers of these phyla on a regional scale. Extrapolated to the entire fauna and study area, we arrive at the conservative estimate that 3900 to 4700 macro- and megabenthic species can be expected to occur on the Arctic shelves. These numbers are smaller than analogous estimates for the Antarctic shelf but the difference is on the order of about two and thus less pronounced than previously assumed. On a global scale, the Arctic shelves are characterized by intermediate macro- and megabenthic species numbers. Our preliminary pan-Arctic inventory provides an urgently needed assessment of current diversity patterns that can be used by future investigations for evaluating the effects of climate change and anthropogenic activities in the Arctic

Updated Clinical Guidelines for Diagnosing Fetal Alcohol Spectrum Disorders

The adverse effects of prenatal alcohol exposure constitute a continuum of disabilities (fetal alcohol spectrum disorders [FASD]). In 1996, the Institute of Medicine established diagnostic categories delineating the spectrum but not specifying clinical criteria by which diagnoses could be assigned. In 2005, the authors published practical guidelines operationalizing the Institute of Medicine categories, allowing for standardization of FASD diagnoses in clinical settings. The purpose of the current report is to present updated diagnostic guidelines based on a thorough review of the literature and the authors’ combined expertise based on the evaluation of >10 000 children for potential FASD in clinical settings and in epidemiologic studies in conjunction with National Institute on Alcohol Abuse and Alcoholism–funded studies, the Collaborative Initiative on Fetal Alcohol Spectrum Disorders, and the Collaboration on FASD Prevalence. The guidelines were formulated through conference calls and meetings held at National Institute on Alcohol Abuse and Alcoholism offices in Rockville, MD. Specific areas addressed include the following: precise definition of documented prenatal alcohol exposure; neurobehavioral criteria for diagnosis of fetal alcohol syndrome, partial fetal alcohol syndrome, and alcohol-related neurodevelopmental disorder; revised diagnostic criteria for alcohol-related birth defects; an updated comprehensive research dysmorphology scoring system; and a new lip/philtrum guide for the white population, incorporating a 45-degree view. The guidelines reflect consensus among a large and experienced cadre of FASD investigators in the fields of dysmorphology, epidemiology, neurology, psychology, developmental/behavioral pediatrics, and educational diagnostics. Their improved clarity and specificity will guide clinicians in accurate diagnosis of infants and children prenatally exposed to alcohol