Efficient high‐throughput biological process characterization: Definitive screening design with the Ambr250 bioreactor system

The burgeoning pipeline for new biologic drugs has increased the need for high‐throughput process characterization to efficiently use process development resources. Breakthroughs in highly automated and parallelized upstream process development have led to technologies such as the 250‐mL automated mini bioreactor (ambr250?) system. Furthermore, developments in modern design of experiments (DoE) have promoted the use of definitive screening design (DSD) as an efficient method to combine factor screening and characterization. Here we utilize the 24‐bioreactor ambr250? system with 10‐factor DSD to demonstrate a systematic experimental workflow to efficiently characterize an Escherichia coli (E. coli) fermentation process for recombinant protein production. The generated process model is further validated by laboratory‐scale experiments and shows how the strategy is useful for quality by design (QbD) approaches to control strategies for late‐stage characterization. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1388–1395, 2015     

Survival strategies of a sterol auxotroph

The high sterol concentration in eukaryotic cell membranes is thought to influence membrane properties such as permeability, fluidity and microdomain formation. Drosophila cannot synthesize sterols, but do require them for development. Does this simply reflect a requirement for sterols in steroid hormone biosynthesis, or is bulk membrane sterol also essential in Drosophila? If the latter is true, how do they survive fluctuations in sterol availability and maintain membrane homeostasis? Here, we show that Drosophila require both bulk membrane sterol and steroid hormones in order to complete adult development. When sterol availability is restricted, Drosophila larvae modulate their growth to maintain membrane sterol levels within tight limits. When dietary sterol drops below a minimal threshold, larvae arrest growth and development in a reversible manner. Strikingly, membrane sterol levels in arrested larvae are dramatically reduced (dropping sixfold on average) in most tissues except the nervous system. Thus, sterols are dispensable for maintaining the basic membrane biophysical properties required for cell viability; these functions can be performed by non-sterol lipids when sterols are unavailable. However, bulk membrane sterol is likely to have essential functions in specific tissues during development. In tissues in which sterol levels drop, the overall level of sphingolipids increases and the proportion of different sphingolipid variants is altered. These changes allow survival, but not growth, when membrane sterol levels are low. This relationship between sterols and sphingolipids could be an ancient and conserved principle of membrane homeostasis.     

Surface Plasmon Resonance and Enhanced Fluorescence Application of Single-step Synthesized Elliptical Nano Gold-embedded Antimony Glass Dichroic Nanocomposites

A novel series of elliptical gold (Au⁰) nanoparticles (18–40 nm) embedded antimony glass (K₂O-B₂O₃-Sb₂O₃) dichroic nanocomposites have been synthesized by a single-step melt-quench in-situ thermochemical reduction technique. X-ray and selected area electron diffractions manifest growth of Au⁰ nanoparticles along the (111) and (200) crystallographic planes. The transmission electron microscopic image reveals elliptical Au⁰ nanoparticles having an aspect ratio varying in the range 1.2–2.1. The dichroic behavior of the nanocomposites arises due to elliptical shape of the Au⁰ nanoparticles. These nanocomposites show strong surface plasmon resonance (SPR) band of Au nanoparticles in the range 610–681 nm and it exhibit red-shifts with increasing Au concentration. They, when co-doped with Sm₂O₃ and excited at 949 nm, exhibit about sevenfold enhancement of the upconverted red emission transition of ⁴G_5/2→⁶H_9/2 at 636 nm due to local electric field enhancement effect of Au⁰ nanoparticles induced by its SPR. These nanocomposites are the promising materials for laser, display, and various nanophotonic applications.     

Biological systems modeling and analysis: a biomolecular technique of the twenty-first century.

It is proposed that computational systems biology should be considered a biomolecular technique of the twenty-first century, because it complements experimental biology and bioinformatics in unique ways that will eventually lead to insights and a depth of understanding not achievable without systems approaches. This article begins with a summary of traditional and novel modeling techniques. In the second part, it proposes concept map modeling as a useful link between experimental biology and biological systems modeling and analysis. Concept map modeling requires the collaboration between biologist and modeler. The biologist designs a regulated connectivity diagram of processes comprising a biological system and also provides semi-quantitative information on stimuli and measured or expected responses of the system. The modeler converts this information through methods of forward and inverse modeling into a mathematical construct that can be used for simulations and to generate and test new hypotheses. The biologist and the modeler collaboratively interpret the results and devise improved concept maps. The third part of the article describes software, BST-Box, supporting the various modeling activities.     

Simvastatin potentiates TNF-alpha-induced apoptosis through the down-regulation of NF-kappaB-dependent antiapoptotic gene products: role of IkappaBalpha kinase and TGF-beta-activated kinase-1

Numerous recent reports suggest that statins (hydroxy-3-methylglutaryl-CoA reductase inhibitors) exhibit potential to suppress tumorigenesis through a mechanism that is not fully understood. Therefore, in this article, we investigated the effects of simvastatin on TNF-alpha-induced cell signaling. We found that simvastatin potentiated the apoptosis induced by TNF-alpha as indicated by intracellular esterase activity, caspase activation, TUNEL, and annexin V staining. This effect of simvastatin correlated with down-regulation of various gene products that mediate cell proliferation (cyclin D1 and cyclooxygenase-2), cell survival (Bcl-2, Bcl-x(L), cellular FLIP, inhibitor of apoptosis protein 1, inhibitor of apoptosis protein 2, and survivin), invasion (matrix mellatoproteinase-9 and ICAM-1), and angiogenesis (vascular endothelial growth factor); all known to be regulated by the NF-kappaB. We found that simvastatin inhibited TNF-alpha-induced NF-kappaB activation, and l-mevalonate reversed the suppressive effect, indicating the role of hydroxy-3-methylglutaryl-CoA reductase. Simvastatin suppressed not only the inducible but also the constitutive NF-kappaB activation. Simvastatin inhibited TNF-alpha-induced IkappaBalpha kinase activation, which led to inhibition of IkappaBalpha phosphorylation and degradation, suppression of p65 phosphorylation, and translocation to the nucleus. NF-kappaB-dependent reporter gene expression induced by TNF-alpha, TNFR1, TNFR-associated death domain protein, TNFR-associated factor 2, TGF-beta-activated kinase 1, receptor-interacting protein, NF-kappaB-inducing kinase, and IkappaB kinase beta was abolished by simvastatin. Overall, our results provide novel insight into the role of simvastatin in potentially preventing and treating cancer through modulation of IkappaB kinase and NF-kappaB-regulated gene products.     

Internode structure and cell wall composition in maturing tillers of switchgrass (Panicum virgatum. L)

This work examined cell composition gradients in maturing tillers of switchgrass (Panicum virgatum L.) with the aim of developing baseline information on this important forage and biomass crop. Flowering tillers were collected from plants raised from seeds in a greenhouse and field, harvested at soil level and separated into internodes beginning with the node subtending the peduncle. Internodes were analyzed using microscopy, by fiber digestion, high-performance liquid chromatography and by gas chromatography-mass spectrometry to obtain anatomical and compositional data. Microscopy demonstrated the development and maturation of cortical fibers which eventually became confluent with the fiber sheath surrounding vascular bundles in the lower internodes. Detergent fiber analysis indicated increasing cellulose and lignin contents and decreases in cell solubles and hemicelluloses with increasing distance of the internodes from the top of the plant. Soluble phenolics were greatest in amounts and complexity in top internodes. The lower internodes contained greater levels of wall-bound phenolic acids, principally as 4-coumarate and ferulate.     

Amino acid-based enantiomerically pure 3-substituted 1,4-benzodiazepin-2-ones: a new class of anti-ischemic agents

A series of 3-substituted 1,4-benzodiazepin-2-ones derived from S and R amino acids were evaluated for their anti-ischemic activity in vitro. Treatment with compounds 7h, 16, 9d, and 17 decreased the apoptotic neuronal number, however increased the neuronal viability. The compounds decreasing apoptosis could protect neurons from the ischemic injury. The difference in the activities of 1,4-benzodiazepin-2-ones derived from S- and R-amino acids is discussed and explained on the basis of molecular modeling studies.     

Isolation and characterization of embryonic stem cell-like cells from in vitro-produced buffalo (Bubalus bubalis) embryos

This study was carried out to isolate and characterize buffalo embryonic stem (ES) cell-like cells from in vitro-produced embryos. Inner cell mass (ICM) cells were isolated either mechanically or by enzymatic digestion from 120 blastocysts whereas 28 morulae were used for the isolation of blastomeres mechanically. The ICM cells/ blastomeres were cultured on mitomycin-C-treated feeder layer. Primary cell colony formation was higher (P < 0.05) for hatched blastocysts (73.1%, 30/41) than that for early/expanded blastocysts (25.3%, 20/79). However, no primary cell colonies were formed when blastomeres obtained from morulae were cultured. Primary colonies were formed in 14.1% (12/85) of intact blastocyst culture, which was significantly lower (P < 0.05) than that of 41.6% for ICM culture. These colonies were separated by enzymatic or mechanical disaggregation. Using mechanical disaggregation method, the cells remained undifferentiated and two buffalo ES cell-like cell lines (bES1, bES2) continued to grow in culture up to eight passages. However, disassociation through enzymatic method resulted in differentiation. Undifferentiated cells exhibited stem cell morphological features, normal chromosomal morphology, and expressed specific markers such as alkaline phosphatase (AP) and Oct-4. Cells formed embryoid bodies (EBs) in suspension culture; extended culture of EBs resulted in formation of cystic EBs. Following prolonged in vitro culture, these cells differentiated into several types of cells including neuron-like and epithelium-like cells. Furthermore, the vitrified-thawed ES cell-like cells also exhibited typical stem cell characteristics. In conclusion, buffalo ES cell-like cells could be isolated from in vitro-produced blastocysts and maintained in vitro for prolonged periods of time.