An innovative hybrid modeling approach for simultaneous prediction of cell culture process dynamics and product quality.

The use of hybrid models is extensively described in the literature to predict the process evolution in cell cultures. These models combine mechanistic and machine learning methods, allowing the prediction of complex process behavior, in the presence of many process variables, without the need to collect a large amount of data. Hybrid models cannot be directly used to predict final product critical quality attributes, or CQAs, because they are usually measured only at the end of the process, and more mechanistic knowledge is needed for many classes of CQAs. The historical models can instead predict the CQAs better; however, they cannot directly relate manipulated process parameters to final CQAs, as they require knowledge of the process evolution. In this work, we propose an innovative modeling approach based on combining a hybrid propagation model with a historical data-driven model, that is, the combined hybrid model, for simultaneous prediction of full process dynamics and CQAs. The performance of the combined hybrid model was evaluated on an industrial dataset and compared to classical black-box models, which directly relate manipulated process parameters to CQAs. The proposed combined hybrid model outperforms the black-box model by 33% on average in predicting the CQAs while requiring only around half of the data for model training to match performance. Thus, in terms of model accuracy and experimental costs, the combined hybrid model in this study provides a promising platform for process optimization applications.

Yeast knockout library allows for efficient testing of genomic mutations for cell-free protein synthesis.

Cell-free protein synthesis (CFPS) systems from crude lysates have benefitted from modifications to their enzyme composition. For example, functionally deleting enzymes in the source strain that are deleterious to CFPS can improve protein synthesis yields. However, making such modifications can take substantial time. As a proof-of-concept to accelerate prototyping capabilities, we assessed the feasibility of using the yeast knockout collection to identify negative effectors in a Saccharomyces cerevisiae CFPS platform. We analyzed extracts made from six deletion strains that targeted the single deletion of potentially negative effectors (e.g., nucleases). We found a statistically significant increase in luciferase yields upon loss of function of GCN3, PEP4, PPT1, NGL3, and XRN1 with a maximum increase of over 6-fold as compared to the wild type. Our work has implications for yeast CFPS and for rapidly prototyping strains to enable cell-free synthetic biology applications.

Energizing eukaryotic cell-free protein synthesis with glucose metabolism.

Eukaryotic cell-free protein synthesis (CFPS) is limited by the dependence on costly high-energy phosphate compounds and exogenous enzymes to power protein synthesis (e.g., creatine phosphate and creatine kinase, CrP/CrK). Here, we report the ability to use glucose as a secondary energy substrate to regenerate ATP in a Saccharomyces cerevisiae crude extract CFPS platform. We observed synthesis of 3.64±0.35 µg mL(-1) active luciferase in batch reactions with 16 mM glucose and 25 mM phosphate, resulting in a 16% increase in relative protein yield (µg protein/$ reagents) compared to the CrP/CrK system. Our demonstration provides the foundation for development of cost-effective eukaryotic CFPS platforms.

Characterizing IGR IRES-mediated translation initiation for use in yeast cell-free protein synthesis.

Eukaryotic cell-free protein synthesis (CFPS) systems are limited, in part, by inefficient translation initiation. Here, we report three internal ribosome entry site (IRES) sequences from the Dicistroviridae family that are highly active in yeast CFPS. These include the intergenic region (IGR) IRES from cricket paralysis virus (CrPV), plautia stali intestine virus (PSIV) and Solenopsis invicta virus 1 (SINV1). Optimization of combined transcription and translation (Tx/Tl) CFPS reactions primed with linear DNA containing the CrPV IGR IRES resulted in batch synthesis yields of 0.92 ± 0.17 µg/mL luciferase. Further template engineering, such as including the first 12 nt of native CrPV gene, increased yields to 2.33 ± 0.11 µg/mL. We next observed that the inclusion of a 50 nt poly(A) to the 3' end of the IGR IRES-mediated message increased yields an additional 81% to 4.33 ± 0.37 µg/mL, without any effect on mRNA stability or copy number. This was surprising because the CrPV IGR IRES requires no known translation initiation factors. Lastly, we investigated a method to inhibit background expression through competitive inhibition by supplying the reaction with 5' cap structure analog. This study highlights the crucial role translation initiation plays in yeast CFPS and offers a simple platform to study IRES sequences.

Substrate replenishment and byproduct removal improve yeast cell-free protein synthesis.

Cell-free protein synthesis (CFPS) platforms are now considered a powerful tool for synthesizing a variety of proteins at scales from pL to 100 L with accelerated process development pipelines. We previously reported the advancement of a novel yeast-based CFPS platform. Here, we studied factors that cause termination of yeast CFPS batch reactions. Specifically, we characterized the substrate and byproduct concentrations in batch, fed-batch, and semi-continuous reaction formats through high-performance liquid chromatography (HPLC) and chemical assays. We discovered that creatine phosphate, the secondary energy substrate, and nucleoside triphosphates were rapidly degraded during batch CFPS, causing a significant drop in the reaction's energy charge (E.C.) and eventual termination of protein synthesis. As a consequence of consuming creatine phosphate, inorganic phosphate accumulated as a toxic byproduct. Additionally, we measured amino acid concentrations and found that aspartic acid was rapidly consumed. By adopting a semi-continuous reaction format, where passive diffusion enables substrate replenishment and byproduct removal, we achieved over a 70% increase in active superfolder green fluorescent protein (sfGFP) as compared with the batch system. This study identifies targets for the future improvement of the batch yeast CFPS reaction. Moreover, it outlines a detailed, generalized method to characterize and improve other CFPS platforms.

Optimized extract preparation methods and reaction conditions for improved yeast cell-free protein synthesis.

Cell-free protein synthesis (CFPS) has emerged as a powerful platform technology to help satisfy the growing demand for simple, affordable, and efficient protein production. In this article, we describe a novel CFPS platform derived from the popular bio-manufacturing organism Saccharomyces cerevisiae. By developing a streamlined crude extract preparation protocol and optimizing the CFPS reaction conditions we were able to achieve active firefly luciferase synthesis yields of 7.7 ± 0.5 µg mL(-1) with batch reactions lasting up to 2 h. This duration of synthesis is the longest ever reported for a yeast CFPS batch reaction. Furthermore, by removing extraneous processing steps and eliminating expensive reagents from the cell-free reaction, we have increased relative product yield (µg protein synthesized per $ reagent cost) over an alternative commonly used method up to 2000-fold from ∼2 × 10(-4) to ∼4 × 10(-1) µg $(-1) , which now puts the yeast CPFS platform on par with other eukaryotic CFPS platforms commercially available. Our results set the stage for developing a yeast CFPS platform that provides for high-yielding and cost-effective expression of a variety of protein therapeutics and protein libraries.

Cell-free synthetic biology: thinking outside the cell.

Cell-free synthetic biology is emerging as a powerful approach aimed to understand, harness, and expand the capabilities of natural biological systems without using intact cells. Cell-free systems bypass cell walls and remove genetic regulation to enable direct access to the inner workings of the cell. The unprecedented level of control and freedom of design, relative to in vivo systems, has inspired the rapid development of engineering foundations for cell-free systems in recent years. These efforts have led to programmed circuits, spatially organized pathways, co-activated catalytic ensembles, rational optimization of synthetic multi-enzyme pathways, and linear scalability from the micro-liter to the 100-liter scale. It is now clear that cell-free systems offer a versatile test-bed for understanding why nature's designs work the way they do and also for enabling biosynthetic routes to novel chemicals, sustainable fuels, and new classes of tunable materials. While challenges remain, the emergence of cell-free systems is poised to open the way to novel products that until now have been impractical, if not impossible, to produce by other means.

Cell-free protein synthesis: applications come of age.

Cell-free protein synthesis has emerged as a powerful technology platform to help satisfy the growing demand for simple and efficient protein production. While used for decades as a foundational research tool for understanding transcription and translation, recent advances have made possible cost-effective microscale to manufacturing scale synthesis of complex proteins. Protein yields exceed grams protein produced per liter reaction volume, batch reactions last for multiple hours, costs have been reduced orders of magnitude, and reaction scale has reached the 100-liter milestone. These advances have inspired new applications in the synthesis of protein libraries for functional genomics and structural biology, the production of personalized medicines, and the expression of virus-like particles, among others. In the coming years, cell-free protein synthesis promises new industrial processes where short protein production timelines are crucial as well as innovative approaches to a wide range of applications.

INTERACT: an object oriented protein-protein interaction database.

MOTIVATION: Protein-protein interactions provide vital information concerning the function of proteins, complexes and networks. Currently there is no widely accepted repository of this interaction information. Our aim is to provide a single database with the necessary architecture to fully store, query and analyse interaction data. RESULTS: An object oriented database has been created which provides scientists with a resource for examining existing protein-protein interactions and inferring possible interactions from the data stored. It also provides a basis for examining networks of interacting proteins, via analysis of the data stored. The database contains over a thousand interactions. CONTACT: k.eilbeck@stud.man.ac.uk

Computed tomography of the chest in blunt thoracic trauma: results of a prospective study.

BACKGROUND: Computed tomography of the chest (CTC) is more sensitive than conventional roentgenography at detecting blunt thoracic injuries. Its effect on subsequent therapy remains incompletely characterized. METHODS: Nine criteria believed to represent the presence of, or the potential for, significant thoracic injuries were defined, and patients were followed prospectively. Forty consecutive patients had CTC after initial evaluation. Physiologic and anatomic findings were compared, and the effect of CTC on therapy was analyzed. RESULTS: CTC detected 76 injuries not found on plain roentgenograms, and plain roentgenograms detected 25 injuries not visible on CTC scans. Six patients had therapy changes based on CTC findings, five of which involved chest tube modification. The percentage of pulmonary contusion did not predict the need for mechanical ventilation but did correlate with physiologic contusion. CONCLUSIONS: Blunt thoracic injuries detected by CTC infrequently require immediate therapy. If immediate therapy is needed, findings will be visible on plain roentgenograms or on clinical exam. Routine CTC in blunt trauma is not recommended but may be helpful in selected cases.

Identification of the homologous beige and Chediak-Higashi syndrome genes.

Vesicular transport to and from the lysosome and late endosome is defective in patients with Chediak-Higashi syndrome (CHS) and in mutant beige (bg) mice. CHS and bg cells have giant, perinuclear vesicles with characteristics of late endosomes and lysosomes that arise from dysregulated homotypic fusion. CHS and bg lysosomes also exhibit compartmental missorting of proteins, such as elastase, glucuronidase and cathepsin G. Lyst, a candidate gene for bg, was identified by direct complementary DNA selection from a yeast artificial chromosome (YAC) clone containing a 650-kilobase segment of the bg-critical region on mouse chromosome 13. Lyst is disrupted by a 5-kilobase deletion in bg mice, and Lyst messenger RNA is markedly reduced in bg homozygotes. The homologous human gene, LYST, is highly conserved with mouse Lyst, and contains a frame-shift mutation at nucleotides 117-118 of the coding domain in a CHS patient. Thus bg mice and human CHS patients have homologous disorders associated with Lyst mutations. Lyst encodes a protein with a carboxy-terminal prenylation motif and multiple potential phosphorylation sites. Lyst protein is predicted to form extended helical domains, and has a region of sequence similar to stathmin, a coiled-coil phosphoprotein thought to act as a relay integrating cellular signal response coupling.

The prevalence of abnormal magnetic resonance imaging findings in asymptomatic knees. With correlation of magnetic resonance imaging to arthroscopic findings in symptomatic knees.

The purpose of this study was to prospectively evaluate the prevalence of abnormal magnetic resonance imaging scans of the knees of asymptomatic subjects. A prospective analysis of magnetic resonance imaging to arthroscopic findings in symptomatic knees was also performed. The prevalence of meniscal tears found in asymptomatic knees was 5.6% (medial meniscus, 1.9%; lateral meniscus, 3.7%). Other abnormal findings included a prevalence of 1.9% for degenerative changes of the medial femoral condyle and 3.7% both for ganglion cysts and patellofemoral joint articular cartilage degenerative changes. There was also a prevalence of 24.1% of Grade II signal changes of the posterior horn of the medial meniscus. Statistical comparison of our results to previous studies revealed that the magnetic resonance imaging scan readings on the asymptomatic knees in this study were accurate and lesions were correctly identified. We recommend that clinicians match clinical signs and symptoms with magnetic resonance imaging findings before instituting surgical treatment because of a 5.6% prevalence of meniscal tears in the asymptomatic population. The significance of the high percentage of posterior horn medial meniscal Grade II signal changes is unknown.

Isolated fracture-dislocation of the fourth carpometacarpal joint. A report of two cases.

Dislocations and fracture-dislocations of isolated and multiple carpometacarpal (CMC) joints are rare injuries that usually result from the impact of a clenched hand on an immobile object. Various authors have presented combinations of dislocations of any or all of the CMC joints. Isolated fracture-dislocation of the fourth CMC joint, however, is either exceedingly rare or highly underreported. Two cases of isolated fracture-dislocation of the fourth CMC joint are presented here: in both, the appearance of the hand suggested a fracture-dislocation of the CMC joint, and the isolated lesion was confirmed at the time of surgery. Both patients were treated with open reduction and internal fixation and had good results at 2 years.

Adolescent depression and suicide: rising problems.

In adolescents, as in adults, most depressive persons are not suicidal, and many suicidal persons are not depressed. However, accurate diagnosis and treatment of depression in adolescent patients is essential to suicide prevention. Unfortunately, depression and suicidality remain widely undiagnosed and untreated in the adolescent population.