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Cell-free protein synthesis has emerged as a promising platform for the production of therapeutic proteins due to its inherently open reaction environment, flexible reaction conditions and rapid protein synthesis capabilities. In recent years, lyophilized cell-free systems have widened the application space of cell-free technology by improving reagent stability outside of cold-chain storage. Current embodiments of the system, however, demonstrate poor stability at elevated temperatures. Lyoprotectants have long been recognized for the ability to preserve the activity of biological molecules during drying processes, but the application of this technology to lyophilized cell-free systems has been limited and has failed to address the negative effects that such lyoprotectants may have on cell-free systems. Here, several lyoprotected, lyophilized cell-free protein synthesis systems are demonstrated using antiplasticized sugar glasses as lyoprotectants, showing significant improvement over standard lyophilized systems or trehalose-preserved systems. Furthermore, we demonstrate for the first time, preservation and therapeutic expression, specifically of FDA-approved crisantaspase, from a truly single-pot lyophilized, endotoxin-free, cell-free protein synthesis system, exemplifying the potential for on-site therapeutic synthesis.

A polymer electrolyte fuel cell has been designed to allow

Here we present our experience applying the principles of optimization and a wholistic student-centered engineering mindset to create an educational experience focused on long-term student success. Specifically, we have applied this approach to redesign a junior-level chemical engineering project that strives to develop student scholarship, citizenship, and leadership given the constraints of time, financial resources, and student/faculty motivation. We propose that using a wholistic optimization mindset can help other educators in designing customized educational experiences.

Functionalization is often needed to harness the power of proteins for beneficial use but can cause losses to stability and/or activity. State of the art methods to limit these deleterious effects accomplish this by substituting an amino acid in the wild-type molecule into an unnatural amino acid, such as

Acute lymphocytic leukemia (ALL) is a common childhood cancer in the United States, with over 6000 new cases diagnosed each year. Administration of bacterial asparaginase (ASNase) has improved survival rates to nearly 80%, however these therapeutics have high incidence of immunological neutralization and serum activity must be monitored for most effective treatment regimens. Here, a 72% improvement in cell-free protein synthesis (CFPS) of FDA approved l-asparaginase (crisantaspase) is demonstrated by employing an aspartate-fed-batch reactor format. A CFPS-based ASNase activity assay as a tool for therapeutic regimentation and production quality control is also presented. This work suggests that shelf-stable and low-cost Escherichia coli-based CFPS reactions may be employed on-demand to 1) synthesize biologics on-site for patient administration, 2) verify biologic activity for dosage calculations, and 3) monitor therapeutic activity in human serum during the treatment regimen. The combination of both therapeutic production and activity assessment introduces a concept of synergistic utility for bacterial cell lysates in modern medical treatment. Indeed, recent work with CFPS biosensors supports a not-too-distant future when shelf-stable E. coli CFPS systems are used to diagnose, treat, and monitor treatment of diseases in the clinical setting.

Solid-state sodium batteries, a relatively safe and potentially cost-effective energy-storage technology, have attracted increasing scientific attention recently for application in stationary grid-scale energy storage. Identifying solid electrolytes with high electrochemical stability and high Na

Although polyethylene glycol (PEG) is commonly used to improve protein stability and therapeutic efficacy, the optimal location for attaching PEG onto proteins is not well understood. Here, we present a cell-free protein synthesis-based screening platform that facilitates site-specific PEGylation and efficient evaluation of PEG attachment efficiency, thermal stability, and activity for different variants of PEGylated T4 lysozyme, including a di-PEGylated variant. We also report developing a computationally efficient coarse-grain simulation model as a potential tool to narrow experimental screening candidates. We use this simulation method as a novel tool to evaluate the locational impact of PEGylation. Using this screen, we also evaluated the predictive impact of PEGylation site solvent accessibility, conjugation site structure, PEG size, and double PEGylation. Our findings indicate that PEGylation efficiency, protein stability, and protein activity varied considerably with PEGylation site, variations that were not well predicted by common PEGylation guidelines. Overall our results suggest current guidelines are insufficiently predictive, highlighting the need for experimental and simulation screening systems such as the one presented here.

Biomanufacturing has emerged as a promising alternative to chemocatalysis for green, renewable, complex synthesis of biofuels, medicines, and fine chemicals. Cell-free chemical biosynthesis offers additional advantages over in vivo production, enabling plug-and-play assembly of separately produced enzymes into an optimal cascade, versatile reaction conditions, and direct access to the reaction environment. In order for these advantages to be realized on the larger scale of industry, strategies are needed to reduce costs of biocatalyst generation, improve biocatalyst stability, and enable economically sustainable continuous cascade operation. Here we overview the advantages and remaining challenges of applying cell-free chemical biosynthesis for commodity production, and discuss recent advances in cascade engineering, enzyme immobilization, and enzyme encapsulation which constitute important steps towards addressing these challenges.

The engineering of and mastery over biological parts has catalyzed the emergence of synthetic biology. This field has grown exponentially in the past decade. As increasingly more applications of synthetic biology are pursued, more challenges are encountered, such as delivering genetic material into cells and optimizing genetic circuits in vivo. An in vitro or cell-free approach to synthetic biology simplifies and avoids many of the pitfalls of in vivo synthetic biology. In this review, we describe some of the innate features that make cell-free systems compelling platforms for synthetic biology and discuss emerging improvements of cell-free technologies. We also select and highlight recent and emerging applications of cell-free synthetic biology.

Recently reported shelf-stable, on-demand protein synthesis platforms are enabling new possibilities in biotherapeutics, biosensing, biocatalysis, and high throughput protein expression. Lyophilized cell-free protein expression systems not only overcome cold-storage limitations, but also enable stockpiling for on-demand synthesis and completely sterilize the protein synthesis platform. Recently reported high-yield synthesis of cytotoxic protein Onconase from lyophilized E. coli extract preparations demonstrates the utility of lyophilized cell-free protein expression and its potential for creating on-demand biotherapeutics, vaccines, biosensors, biocatalysts, and high throughput protein synthesis.

Approximately one third of protein therapeutics are produced in Escherichia coli, targeting a wide variety of diseases. However, due to immune recognition of endotoxin (a lipid component in the E. coli cell membrane), these protein products must be extensively purified before application to avoid adverse reactions such as septic shock. E. coli-based cell-free protein synthesis (CFPS), which has emerged as a promising platform for the development and production of enhanced protein therapeutics, provides a unique opportunity to remove endotoxins prior to protein expression due to its open environment and the absence of live cells. Pre-expression endotoxin removal from CFPS reagents could simplify downstream processing, potentially enabling on-demand production of unique protein therapeutics. Herein, three strategies for removing endotoxins from E. coli cell lysate are evaluated: Triton X-114 two-phase extraction, polylysine affinity chromatography, and extract preparation from genetically engineered, endotoxin-free ClearColi cells. It is demonstrated that current protocols for endotoxin removal treatments insufficiently reduce endotoxin and significantly reduce protein synthesis yields. Further, the first adaptation of ClearColi cells to prepare cell-free extract with high protein synthesis capability is demonstrated. Finally, production of the acute lymphoblastic leukemia therapeutic crisantaspase from reduced-endotoxin extract and endotoxin-free ClearColi extract is demonstrated.

SummaryThe morphology of the sperm head has often been correlated with the outcome ofin vitrofertilization, and has been shown to be the sole parameter in semen of value in predicting the success of intracytoplasmic sperm injection and intracytoplasmic morphologically selected sperm injection.In this paper, we have studied whether digital holographic microscopy (DHM) may be useful to obtain quantitative data on human sperm head structure and compared this technique with high-power digitally enhanced Nomarski optics. The main advantage of digital holography is that high-resolution three-dimensional quantitative sample imaging may be automatically produced by numerical refocusing of a two-dimensional image at different object planes without any mechanical scanning. We show that DHM generates useful information on the dimensions and structure of human sperm, not revealed by conventional phase-contrast microscopy, in particular the volume of vacuoles, and suggest its use as an additional prognostic tool in assisted reproduction technology.

A national seroprevalence study was performed to determine the prevalence of Haemophilus influenzae type b (Hib) antibodies in England and Wales in 2009, when Hib disease incidence was the lowest ever recorded. A total of 2,693 anonymised residual sera from routine diagnostic testing submitted by participating National Health Service hospital laboratories were tested for Hib anti-polyribosyl-ribitol phosphate (PRP) IgG antibodies using a fluorescent bead assay. Median anti-PRP IgG concentrations were highest in toddlers aged 1-4 years (2.65 µg/ml), followed by children aged 5-9 years (1.95 µg/ml). Antibody concentrations were significantly lower after this age, but were still significantly higher among 10-19 year-olds (0.54 µg/ml) compared with adults aged >20 years (0.16 µg/ml; p

Differences between self-diagnosed premenstrual syndrome (PMS) sufferers and non-sufferers (both oral contraceptive users and normally cycling subjects) on performance, state, and reaction to mild stress at premenstrual or intermenstrual stages of the cycle were examined. All subjects reported increases in arousal and stress in the premenstrual phase but there was a significantly greater increase in reported arousal in the premenstrual phase in PMS subjects than in non-PMS subjects. There were no differences between subject groups on performance during the cycle, or on reaction to stress: for all subjects semantic processing on a word matching test improved premenstrually. No differences were found between pill and non-pill subjects. PMS subjects scored significantly higher on the Neuroticism scale of the EPI than non-PMS subjects, and were significantly more ‘A’ typed in personality on a Framingham A/B personality scale. It is suggested that a multivariate model is adopted for menstrual cycle re...

A double-blind, randomized, controlled phase I study to assess the safety, immunogenicity, and antibody persistence of a new group A conjugate vaccine (PsA-TT) in volunteers aged 18 to 35 years was previously performed. Subjects received one dose of either the PsA-TT conjugate vaccine, meningococcal A/C polysaccharide vaccine (PsA/C), or tetanus toxoid vaccine. The conjugate vaccine was shown to be safe and immunogenic as demonstrated by a standardized group A-specific immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) and by a serum bactericidal antibody (SBA) assay using rabbit complement (rSBA). This report details further analysis of the sera using four additional immunologic assays to investigate the relationship between the different immunoassays. The immunoassays used were an SBA assay that used human complement (hSBA), a group A-specific IgG multiplexed bead assay, and two opsonophagocytic antibody (OPA) assays which used two different methodologies. For each vaccine group, geometric mean concentrations or geometric mean titers were determined for all assays before and 4, 24, and 48 weeks after vaccination. Pearson’s correlation coefficients were used to assess the relationship between the six assays using data from all available visits. An excellent correlation was observed between the group A-specific IgG concentrations obtained by ELISA and those obtained by the multiplexed bead assay. hSBA and rSBA titers correlated moderately, although proportions of subjects with putatively protective titers and those demonstrating a >4-fold rise were similar. The two OPA methods correlated weakly and achieved only a low correlation with the other immunoassays. The correlation between hSBA and group A-specific IgG was higher for the PsA-TT group than for the PsA/C group. Within the African meningitis belt, unpredictable epidemics of meningococcal disease continue to occur every 5 to 15 years. To prevent these epidemics, a novel serogroup A conjugate meningococcal vaccine was developed. The Meningitis Vaccine Project, a partnership between the World Health Organization (WHO) and PATH (Seattle, WA) with core funding from the Bill and Melinda Gates Foundation, was created in 2001 with the goal of eliminating meningococcal epidemics in sub-Saharan Africa through the development, testing and licensure, and widespread use of serogroup A meningococcal conjugate vaccines (11) (http://www.meningvax.org). A group A meningococcal conjugate vaccine using tetanus toxoid as a carrier protein (PsA-TT) was developed at the Serum Institute of India Ltd., using a new licensed conjugation technique from the Center for Biologics Evaluation

This chapter examines some of the most important methods in optical microscopy for the identification of fibres, briefly discussing the underlying optical phenomena on which they are based and indicating areas where each might be advantageously applied. In addition, it considers some of the practical issues involved in preparing fibre specimens for microscopy, and straightforward procedures that can greatly simplify the process of making an unambiguous identification. A list of resources and further reading is provided.

Background: Granulosa-cells are able to produce and store leptin, suggesting that this hormone is locally involved in the regulation of follicular growth. In this study, the role of follicular fluid (FF) leptin concentration in predicting oocyte fertilization and embryo quality was evaluated in 35 normogonadotrophic women undergoing controlled ovarian stimulation (COS) for assisted reproductive techniques. Materials and Methods: Leptin concentration was measured in 47 consecutively collected FF in which a mature oocyte had been found during the ovum pick-up. Embryos deriving from fertilized oocytes were submitted to quality scoring systems. Results: Mean leptin concentration was significantly higher in FF whose oocytes showed 2 pronuclei (no. 25) when compared with those with no evidence of fertilization (no. 22) at the 16–18 h check (26.0±6.1 vs 15.3±10.6 ng/ml, respectively, p

We present an architecture for and prototype of a system for quickly detecting software problem recurrences. Re-discovery of the same problem is very common in many large software products and is a major cost component of product support. At run-time, when a problem occurs, the system collects the problem symptoms, including the program call-stack, and compares it against a database of symptoms to find the closest matches. The database is populated off-line using solved cases and indexed to allow for efficient matching. Thus problems that occur repeatedly can be easily and automatically resolved without requiring any human problem-solving expertise. We describe a prototype implementation of the system, including the matching algorithm, and present some experimental results demonstrating the value of automatically detecting re-occurrence of the same problem for a popular sofware product

Carbohydrate additives to modern embryo culture media are based on three basic energy sources, glucose, pyruvate and lactate. Although the use of these substrates is almost universal, debate continues as to the roles of the individual components in the human. This is mainly due to the lack of human embryos for research and the reliance on animal model systems. In the present work, the human embryo was used to study the role of the above simple substrates in the maintenance of the mitochondrial membrane potential and cell division. The mitochondrial membrane potential was measured with fluorescence techniques. Cell division was scored as the number of blastomeres on day 3. Both the mitochondrial membrane potential and cell division were dramatically lost in the absence of energy sources. The mitochondrial membrane potential and cell division were normal in media containing all three energy sources, or in pyruvate-containing media. Both glucose and lactate individually proved poor energy sources for the maintenance of the mitochondrial membrane potential. However, cell division continued in the presence of glucose, suggesting that some energy production can continue. These data suggest that pyruvate is an absolute requirement for mitochondrial respiration and cell cleavage during human preimplantation development. The role of lactate is as yet unclear.

Verification and certification of flight critical software and application-specific integrated circuits (ASICs) is currently a labor-intensive, manual process involving extensive testing, inspections, and process documentation. The complexity of these systems and devices will increase both because increases in cockpit automation and application integration offer important safety benefits and because astonishing improvements in digital computing technology can potentially improve performance and decrease cost. The current approach to verification and certification will be challenged by this increased complexity. In order to reap fully the benefits of these technological advances we must develop new methods for verification and certification of flight critical devices that provide higher degrees of assurance for increasingly complex systems while simultaneously streamlining the verification process. The development of executable formal models may offer higher degrees of assurance, address increased complexity, and streamline certain aspects of the verification process. Increased assurance can be obtained as a result of rigorous, mechanical, mathematically complete checks of consistency and completeness of system requirements as well as proofs of correctness of specific implementations. As vector-based testing becomes increasingly inadequate to assure correctness in the face of exponentially growing state space, formal proofs of correctness can encompass the entire design, demonstrating correctness once and for all.

This study was designed to measure leptin concentrations in the peritoneal fluid (PF) of women with different aspects of pelvic endometriosis. Among 36 consecutive women undergoing laparoscopy, nine were diagnosed as having minimal-mild endometriosis (stage I-II). Among nine other subjects with advanced stage (III-IV) disease, six showed one or more ovarian endometriotic cysts as the only operative finding. The remaining 18 unaffected women constituted the control group. Patients with endometriosis had significantly higher PF leptin concentrations (32.6 +/- 16.2 versus 17.1 +/- 6.6 ng/ml, P = 0.002); this difference remained significant when corrected for body mass index (BMI) (PF leptin/BMI ratio 1.41 +/- 0.67 versus 0.76 +/- 0.28, P = 0.001). Furthermore, the PF leptin/BMI ratio was significantly higher in women with peritoneal implants than in those in whom no implant was found at laparoscopy (1.6 +/- 0.7 versus 0.83 +/- 0.33, P = 0.007). Conversely, patients with one or more ovarian endometriomata as the only finding, had a PF leptin/BMI ratio comparable with that in women where no cyst was found (1.05 +/- 0.4 versus 1.1 +/- 0.65). In women with stage I-II endometriosis, a higher mean PF leptin/BMI ratio was found compared with those affected by stage III-IV (1.78 +/- 0.68 versus 1.05 +/- 0.43, P = 0.01). These results show that during endometriosis the presence of peritoneal disease, and not of ovarian endometriotic cysts, influences leptin concentrations in PF. The data suggest that leptin may play a role in the development of peritoneal endometriosis, and that different biochemical phenomena might be involved in the pathogenesis of the ovarian form of the disease.

A number of power cable diagnostic techniques are discussed. The quality and detail of the information generated by these techniques varies widely, as does the complexity of measurements and equipment required. Study of cable history and examination of failures and cable samples provides a useful insight into the network with little additional work. DC test methods offer the greatest ease of use due to the small physical size, however, their diagnostic capabilities are limited. AC testing provides a greater level of diagnostic flexibility but brings with it increased size and expense. However, the ability to carry out diagnostic testing, particularly partial discharge mapping, does also attract financial benefits such as optimising replacement and reducing service failures. Most of the techniques discussed have been available for some time. The main development in this area has not been in introducing new techniques but in the better use and application of techniques which have existed for some time. Careful choice of these techniques together with informed diagnosis based on the results can provide a cost effective and valuable asset management tool.

The human monoclonal multireactive IgM-antibody CB-03, developed by Jahn et al. (2), was successful produced in Cell-Pharm I hollow fibre bioreactors over several months. The antibody concentration increased 100-fold in comparison to that in tissue culture flasks. After several weeks of fermentation tissue cell density could be reached in the bioreactor cartridges. Some times later total number of cells and viability in the harvests decreased drastically. In this phase of production a decreased glucose consumtion could be shown. In contrast to that, constant amounts of antibodies were produced. Opening the bioreactor cartridge, unexpected trypsin resistant cell clusters were found. All this clusters were surface IgM positive and were able to secrete antibodies. It remains unclear wether this clusters contained functional complete cells or not. Partial cell free antibody synthesis seems to be possible. Reasons for cluster formation are discussed.

Using a broad band testing framework, indices of performance and state were examined in 10 women during one menstrual cycle, over six separate testing sessions. Indices of state were heart rate, the Cruickshank self-report questionnaire and time to basal skin potential. Results showed that self reported arousal was increased premenstrually. There was dissociation between indices of state, supporting the contention that simple models of arousal are inadequate. Performance tests of cognitive processing, tracking skill, reaction time and vigilance, dual task, speed and accuracy, and short term memory capacity showed only one significant result: semantic processing on a word matching task improved premenstrually. There was no significant relationship between state and performance: with high individual differences found between subjects. It was concluded that menstruation was not acting as a stressor for these subjects, and that there was no evidence for a decrement in performance in the menstrual or premenstrual phases of the cycle. Results are discussed in terms of coping with perceived increase in demand premenstrually, resulting in utilisation of compensatory effort and it is suggested that future work should examine womens ability to cope with perceived increase in demand and attribution of arousal during the menstrual cycle. The implications of the findings in terms of the critique of PMS as a valid construct are discussed, and it is concluded that future work should be carried out within an integrated multi-variate framework.

The amino acid composition of the medium in which seedlings of peas had been grown aseptically with and without quartz sand were determined by ion-exchange chromatography. Besides considerable amounts of amino acids, peptide material was also present.

Chase and Posner reported to the Midwestern Psychological Association in 1965 their investigation of the speed with which subjects determined whether a target letter (TS) was present or not in a comparison set (CS) when the letters were drawn from an acoustically similar population (AC), a visually similar population (VIS) or a neutral population (N), and the stimuli were presented visually. TS was presented either simultaneously with CS (visual comparison search), or before CS (visual recognition search) or after CS (memory search). From the evidence that verbal material is coded auditorily in memory1,2 they anticipated that acoustic similarity might affect speed in this task, changing the slope of the function relating reaction time and the number of letters in CS. In fact, only visual similarity had an effect, increased visual similarity increasing the reaction time in the visual comparison search and memory search. Chase and Posner concluded that a visually coded representation of the stimulus was used in these situations.

Up- and downstream processing of human monoclonal IgM is known to bring about problems with respect to clone stability and quantity of antibodies produced. A human B cell hybridoma producing a natural polyreactive IgM antibody (CB03) was adapted to growth in serum-free medium and scaled-up using a hollow fiber bioreactor system. The process of fermentation has been carried out continuously over a period of 4 months. In comparison to stationary culture conditions in the presence of 10% fetal calf serum, antibody concentrations in hollow fiber bioreactor supernatants were found to be significantly increased. Semicontinuously harvested supernatants contained up to 400 mg/liter immunoreactive IgM antibody. During the last weeks of fermentation, a markedly reduced number of viable cells was observed, whereas antibody production seemed to remain stable. Furthermore, we detected formation of cell clusters in the fermentor system. These clusters carried IgM on the surface and secreted immunoreactive IgM antibodies. Clusters were found to represent fusions of hybridoma cells using electron microscopy. Cluster formation was accompanied by decreased glucose consumption and lactate accumulation and was not seen during growth of other human hybridomas. We discuss these results in the content of the polyreactive binding properties of this particular antibody.

Ss shadowed a prose passage delivered to the right ear and were asked to tap to occurrences of a target in a second passage delivered to the left ear. Group 1 was asked to tap to a sound that could also be two different words (e.g., I and eye), Group 2 to one of these words only. Group 2 performed better than Group 1, reversing the result of Wilding & Underwood (1968). The result is attributed to availability of contextual information for Group 2; arguments of Treisman & Geffen (1967) against such availability are rejected.