Bioengineering Studies Research Articles

Gravity-induced swirl of nanoparticles in microfluidics

Parallel flows of two fluids in microfluidic devices are used for miniaturized chemistry, physics, biology and bioengineering studies, and the streams are often considered to remain parallel. However, as the two fluids do not always have the same density, interface reorientation induced by density stratification is unavoidable. In this paper, flow characteristics of an aqueous polystyrene nanofluid and a sucrose-densified aqueous solution flowing parallel in microchannels are examined. Nanoparticles 100 nm in diameter are used in the study. The motion of the nanoparticles is simulated using the Lagrangian description and directly observed by a confocal microscope. Matched results are obtained from computational and empirical analysis. Although solution density homogenizes rapidly resulting from a fast diffusion of sucrose in water, the nanofluid is observed to rotate for an extended period. Angular displacement of the nanofluid depends on the ratio of gravitational force to viscous force, Re/Fr2, where Re is the Reynolds number and Fr is the Froude number. In the developing region at the steady state, the angular displacement is related to y/Dh, the ratio between distance from the inlet and the hydraulic diameter of the microfluidic channel. The development of nanofluid flow feature also depends on h/w, the ratio of microfluidic channel's height to width. The quantitative description of the angular displacement of nanofluid will aid rational designs of microfluidic devices utilizing multistream, multiphase flows.

A comparative study on the stability and structure of two different green fluorescent proteins in organic co-solvent systems

Green fluorescent protein (GFP) has been used as a reporter marker in a wide range of biological and bioengineering studies. The expanded use of GFP in the field of biosensors, biochips and bio-conjugations requires the stability of GFP in organic co-solvent systems. This prompted us to examine the kinetic stability of two different GFP sequences, n-GFP and s-GFP, showing different folding robustness and thermodynamic stability, under a range of organic co-solvent systems. n-GFP and s-GFP are variants whose biophysical properties are comparable to wild type and super folder GFPs, respectively. The stability of n-GFP and s-GFP in 50% water-miscible organic solvents showed that s-GFP with higher thermodynamic stability exhibited much higher stability against organic solvents than n-GFP, which has lower thermodynamic stability. s-GFP was quite stable even in 90% organic solvents. Circular dichroism analysis confirmed that s-GFP maintained its native structure in organic co-solvent systems, whereas n-GFP showed structural variations under these conditions. Four highly fluctuating loop regions were identified from molecular dynamic simulations under the organic cosolvent conditions. A structural comparison of n-GFP and s-GFP suggested that the improved kinetic stability of s-GFP was due to its larger number of hydrogen bonds and salt-bridges that were present in four loop regions. This study suggests that thermodynamically stable s-GFP can be a good choice for use under harsh organic co-solvent conditions.

Separation and Purification of ε-Poly-L-lysine from the Culture Broth Based on Precipitation with the Tetraphenylborate Anion

A biopolymer ε-poly-L-lysine (εPL) exists as a fully protonated form (εPLH(n+)) in solutions at pH < 4. Around pH 3.5, at which the tetraphenylborate (TPB(-)) anion is not decomposed immediately, the εPLH(n+) cation associates with the TPB(-) anion to form a precipitate of 1:n stoichiometry, εPLH(TPB)(n). By the addition of NaTPB to a culture broth containing εPL and NH(4)(+) and K(+) ions, not only the polycationic εPLH(n+) but also the monovalent cations would be precipitated with the TPB(-) anion. However, εPLH(TPB)(n) was purified by washing the mixed precipitate with acetone, in which NH(4)TPB and KTPB are soluble. By mixing the εPLH(TPB)(n) precipitate and a high-concentration HCl solution, the TPB(-) anion was decomposed immediately to hydrophobic molecules. By the addition of a much larger volume of acetone to the reaction mixture, the decomposition products dissolved in the solvent. Simultaneously, εPL was precipitated as the hydrochloride salt. Thus, εPL has been separated and purified from the culture broth. Also, the method has been successfully applied to the separation of oligomeric εPL species. The present chemical separation method is rapid, simple, and easy to carry out, and can be utilized in bioengineering studies of such basic peptides or polyamines.

Bacteriophages as Templates for Manufacturing Supramolecular Structures

SS phages are genetically enginnered by replacing 2 N-terminal amino acids of the p8 coat protein of the fd phage. AGE and zeta potential measurements show that SS phages carry at least 1/4 less net negative surface charge than fd phages. Morphology and thickness of phages are studied at different counterion concentrations (10(-3) , 10(-2) and 10(-1) M) by AFM, SEM and immunofluorescence analyses. Bundles induced by CoCl2 and CaCl2 are either metallized by chemical reduction or biomineralized for apatite-like material formation. EDX spectroscopy confirms the presence of Co, P and Ca peaks in mineralized samples. Such bottom-up manufactured phage scaffolds might be applied in bioengineering studies.

Role of the jelly-roll fold in substrate binding by 2-oxoglutarate oxygenases

2-Oxoglutarate (2OG) and ferrous iron dependent oxygenases catalyze two-electron oxidations of a range of small and large molecule substrates, including proteins/peptides/amino acids, nucleic acids/bases, and lipids, as well as natural products including antibiotics and signaling molecules. 2OG oxygenases employ variations of a core double-stranded β-helix (DSBH; a.k.a. jelly-roll, cupin or jumonji C (JmjC)) fold to enable binding of Fe(II) and 2OG in a subfamily conserved manner. The topology of the DSBH limits regions directly involved in substrate binding: commonly the first, second and eighth strands, loops between the second/third and fourth/fifth DSBH strands, and the N-terminal and C-terminal regions are involved in primary substrate, co-substrate and cofactor binding. Insights into substrate recognition by 2OG oxygenases will help to enable selective inhibition and bioengineering studies.

Evaluating the Benefits of Peri-Urban Agriculture.

By uniting literature from farmland preservation, growth management, food systems, economics, bioengineering, and environmental studies, this article provides an overview and valuation of the services that farms provide for urban areas. This article first analyzes the mission statements of 130 nationally accredited land trusts to ascertain the criteria used in preserving farmland. Land trusts present uniform preference for parcels that provide ecosystem services, wildlife habitat, viewsheds, local heritage, and agricultural productivity. The list of benefits provided by land trusts was compared to a literature review drawing from farmland amenity, agritourism, farmland preservation, and ecosystems studies to reveal the range of market values for the various benefits of farmland. The market value of farmland services varies from -$37,541 to 124,000 per acre depending on the method of analysis and location of the farm. This research has strong implications for land-use planning, economic opportunities, and ecosystems infrastructure in peri-urban areas.

Application of Bioengineering Modalities in Vascular Research: Evaluating the Clinical Gain

Using knowledge gained from bioengineering studies, current vascular research focuses on the delineation of the natural history and risk assessment of clinical vascular entities with significant morbidity and mortality, making the development of new, more accurate predictive criteria a great challenge. Additionally, conclusions derived from computational simulation studies have enabled the improvement and modification of many biotechnology products that are used routinely in the treatment of vascular diseases. This review highlights the promising role of the bioengineering applications in the vascular field.

A Review of Bioreactor Protocols for Human Neural Precursor Cell Expansion in Preparation for Clinical Trials

Tissue-specific human neural precursor cells (hNPCs) can be isolated from various regions of the developing or adult central nervous system and may serve as a viable source of cells in cell replacement therapies for the treatment of neurodegenerative disorders. However, in order for cell replacement strategies to become a routine therapeutic option for the treatment of neurodegenerative disorders, hNPCs should be generated under standardized and controlled conditions. Studies over the last two decades have focused on developing cell growth media and cell handling protocols for expansion and differentiation of hNPCs in culture. Key studies have reported the development of serum-free growth media and large-scale computer-controlled suspension bioreactors that can support high cell proliferation rates (doubling times < 3 days), multipotentiality, and potential neurogenic differentiation (more than 60% neurons). Moreover, bioengineering studies have focused on controlling culture conditions in suspension bioreactors including inoculation, hydrodynamics of culture, oxygen and nutrients transfer to the cells, monitoring in situ physiological parameters using process control techniques, and expansion for extended periods of time. In addition, in vitro and in vivo characterization of hNPCs have been performed, providing information on stem/progenitor cell characteristics, cell surface analysis, and appropriate type of cells to use in transplantation studies.

Molecular determinants of azo reduction activity in the strain Pseudomonas putida MET94

Azo dyes are the major group of synthetic colourants used in industry and are serious environmental pollutants. In this study, Pseudomonas putida MET94 was selected from 48 bacterial strains on the basis of its superior ability to degrade a wide range of structurally diverse azo dyes. P. putida is a versatile microorganism with a well-recognised potential for biodegradation or bioremediation applications. P. putida MET94 removes, in 24 h and under anaerobic growing conditions, more than 80% of the majority of the structurally diverse azo dyes tested. Whole cell assays performed under anaerobic conditions revealed up to 90% decolourisation in dye wastewater bath models. The involvement of a FMN dependent NADPH: dye oxidoreductase in the decolourisation process was suggested by enzymatic measurements in cell crude extracts. The gene encoding a putative azoreductase was cloned from P. putida MET94 and expressed in Escherichia coli. The purified P. putida azoreductase is a 40 kDa homodimer with broad substrate specificity for azo dye reduction. The presence of dioxygen leads to the inhibition of the decolourisation activity in agreement with the results of cell cultures. The kinetic mechanism follows a ping-pong bi-bi reaction scheme and aromatic amine products were detected in stoichiometric amounts by high-performance liquid chromatography. Overall, the results indicate that P. putida MET94 is a promising candidate for bioengineering studies aimed at generating more effective dye-reducing strains.

Esophageal injury in fatal rear-impact collisions

Neck injuries resulting from motor vehicle collisions (MVC), often referred to as whiplash trauma and injury, often demonstrate little or no evidence of significant tissue damage. In rare instances, however, serious injury to the anterior neck organ injuries can result from such trauma. The present study describes esophageal injury associated with rear-impact collisions, based on a unique case report, review of the scientific literature and a query in the National Automotive Sampling System (NASS) database of the US National Highway Traffic Safety Administration. The Medline search and present case study totaled five cases of rear-impact collision-related serious esophageal injury (laceration or rupture). In the four published cases all patients survived, whereas in the presented case study, the patient died due to mediastinitis and sepsis. The NASS query revealed an additional three cases out of a total of 55,926 investigated crashes. All three cases were associated with fatalities. Although no anatomical or bioengineering studies have presented data on the behavior of the esophagus during rear-impact whiplash loading, sudden tensile and/or compressive forces is the likely explanation of injury, often in combination with a local fracture of a vertebral body. In these 8 cases significant esophageal injury carried a substantial (50%) risk of mortality. Clinicians should be aware of the potential for significant complications in the whiplash trauma-exposed patient who complains of chest pain, mid-thoracic pain, discomfort in the neck and throat, respiratory distress, or hoarseness. For those forensic specialists involved in whiplash cases these study results highlight the need to consider esophageal injuries as a rare but potential consequence of whiplash trauma.

Application of spontaneously immortalized odontoblast cells in tooth regeneration

Here, we report on the first attempt to bioengineer tooth using a spontaneously immortalized mesenchymal cell line. To assess the odontogenic potential of this cell line, odontoblast-lineage cells (OLC) were re-associated with competent dental epithelium isolated from E14.5 mice. A novel three-dimensional organ germ culture method was applied to nurture the constructs in vitro. Additionally, recombinants were transplanted under the kidney capsule in host animals for 2 weeks. Transplants developed into tooth tissues in one-third of the cases. OLC-derived GFP-positive cells could be identified in mineralizing tooth germs by immunohistochemistry. OLCs were capable of intercellular and cell-matrix communication, thus they eventually differentiated into functional odontoblasts. In summary, we managed to utilize OLCs for dental mesenchyme substitution in tooth regeneration experiments. Therefore, our spontaneously transformed cell line proved its potential for future complex, tooth developmental and bioengineering studies.

Lantibiotics: Mode of Action, Biosynthesis and Bioengineering

Lantibiotics are gene-encoded peptides that contain intramolecular ring structures, introduced through the thioether containing lanthionine and methyllanthionine residues. The overwhelming majority of the lantibiotics shows antibacterial activity. Some lantibiotics, e.g. nisin, are characterized by a dual mode of action. These peptides form a complex with the ultimate cell wall precursor lipid II, thereby inhibiting cell wall biosynthesis. The complexes then aggregate, incorporate further peptides and form a pore in the bacterial membrane. Recent results show that complexing of lipid II is widespread among lantibiotics; however, pore formation depends on the overall length of the peptide and the lipid composition of the test strain membrane. In the two-component system of lacticin 3147, the two functions are performed by the two different peptides. The genetic information for production of lantibiotics is organized in gene clusters which contain a structural gene (lanA) for the lantibiotic prepeptide. The modifications are introduced by one biosynthetic enzyme (LanM) or a combination of a dehydratase (LanB) and a cyclase (LanC). These enzymes have been in the focus of recent bioengineering studies: The structure of NisC has been resolved, the reaction mechanism of LctM was elucidated and the active site residues were characterized by mutagenesis studies. In vitro modification systems have successfully been used to introduce thioether rings into other biologically active peptides. Furthermore, variant lantibiotics with enhanced properties have been engineered and at least one promising new lantibiotic with strong activity against multiresistant pathogens has been described.

Análise dos Aspectos Biomecânicos e Biológicos na Reabilitação Oral com União Dente/Implante

The consequences as well the criteria for the prosthetic oral rehabilitations with tooth/implant connection come in the literature, in most of the cases, with controversial results. On one side, many studies consider the connection viable, while the others studies consider impracticable because of the difference biomechanics and physiologic of the two systems. The purpose of this study was to organize, to synthesize and to select literary studies that can standardize solutions when this rehabilitation type is necessary. The study showed that the clinical follow-up result in a high survival rate in a short period and the bioengineering studies did not accept the tooth/implant connection.

Aerodynamically assisted bio-jets: the development of a novel and direct non-electric field-driven methodology for engineering living organisms

We recently demonstrated the ability to use electrified jets under stable conditions for the generation of cell-bearing droplets to the formation of composite threads which are biologically active. Our studies established that processed cells were viable over several generations post-jetting and -threading. These harmless and successful techniques for jet-based cell handling to deployment for precision deposition have great potential and widespread applications in bioengineering and biotechnology. Nonetheless, our investigations into ‘bio-electrosprays’ and ‘cell electrospinning’ have elucidated these jets having direct applicability in regenerative and therapeutic medicine to studies in developmental biology. For these very reasons, jet methodologies having the capability to safely handle living organisms for drop and placing are increasingly gaining the interests of life scientists. We now demonstrate yet another technique (a non-electric field-driven approach, previously never explored with jetting living cells), possessing the ability to directly handle the processing of primary living organisms by means of the flow of a cell suspension within a needle placed in a pressure chamber in the presence of an applied pressure difference. The technique we introduce here is referred to as ‘aerodynamically assisted bio-jets/-jetting’ which is driven completely by aerodynamic forces applied over an exit orifice by way of a differential pressure. Our investigations present an operational window in which stable jetting conditions are achieved for the formation of a near-monodispersed distribution of cell-bearing droplets and droplet residues. Finally, the aerodynamically bio-jetted living primary organisms are assessed (over both short and long time points) for cellular viability by means of FACScan, a flow cytometry technology which quantifies the percentage of living and dead cells. These advanced biophysical and bioengineering studies elucidate the emergence of a non-electric field-driven bio-jetting technology which now joins the cell jetting race.

Printing technologies for fabrication of bioactive and regular microarrays of streptavidin

In this study, we report and compare two methods for fabricating patterns of streptavidin protein using soft litography microprinting technique (μCP) and laser-based method termed ‘matrix assisted pulsed laser evaporation direct write’ (MAPLE DW). The μCP approach is a parallel deposition technique capable of X depositions per stamper. The technique is limited in more sophisticated multicomponent deposition by the size of patterns that can be produced and the features obtained during the transfer process. The computer-aided design/computer-aided manufacturing (CAD/CAM) ability of MAPLE DW overcomes the limitations of the μCP approach. (i) We establish the science and engineering principles behind the effective transfer of microarrays and (ii) we explore issues regarding the direct immobilization, morphology and function of the deposited protein at the interface with an aqueous environment and in the precision of controlled ligand-receptor reactions. In summary, our objective was to develop simple, robust microfabrication techniques for the construction of model 2D and 3D bioscaffolds to be used in fundamental bioengineering studies.

ChrR, a Soluble Quinone Reductase of Pseudomonas putida That Defends against H2O2

Most bacteria contain soluble quinone-reducing flavoenzymes. However, no biological benefit for this activity has previously been demonstrated. ChrR of Pseudomonas putida is one such enzyme that has also been characterized as a chromate reductase; yet we propose that it is the quinone-reducing activity of ChrR that has the greatest biological significance. ChrR reduces quinones by simultaneous two-electron transfer, avoiding formation of highly reactive semiquinone intermediates and producing quinols that promote tolerance of H(2)O(2). Expression of chrR was induced by H(2)O(2), and levels of chrR expression in overexpressing, wild type, and knock-out mutant strains correlated with the H(2)O(2) tolerance and scavenging ability of each strain. The chrR expression level also correlated with intracellular H(2)O(2) levels as measured by protein carbonylation assays and fluorescence-activated cell scanning analysis with the H(2)O(2)-responsive dye H(2)DCFDA. Thus, enhancing the activity of ChrR in a chromate-remediating bacterial strain may not only increase the rate of chromate transformation, it may also augment the capacity of these cells to withstand the unavoidable production of H(2)O(2) that accompanies chromate reduction.

Reprogramming somatic cell differentiation and the Hayflick Limit: contrasting two modern molecular bioengineering aims and their impact on the future of mankind.

The molecular biology of human cloning and aging research depend on the closely related laboratory techniques supported by a thorough understanding of cell-signaling processes. Unfortunately, the link between these two research fields has received only marginal attention in the lay press. Cloning is possible when somatic cell differentiation is successfully reprogrammed, and clinical control of cellular senescence depends on a proper reconfiguration of the predetermined number of divisions permitted during the cell life-cycle (the so-called "Hayflick Limit"). In this paper, we discuss these two concepts and compare the impact likely to be associated with bioengineering studies that facilitate both human cloning and longevity therapy.

Proper statistical analysis of transepidermal water loss (TEWL) measurements in bioengineering studies.

In irritancy studies, measurement of transepidermal water loss (TEWL) is a widely used technique to assess barrier function. Using inappropriate statistical methods, however, leads to loss of information and misinterpretation of results. In this paper, we discuss some problems and pitfalls when using a suitable statistical technique for most designs in bioengineering studies, analysis of variance (ANOVA): multiple comparisons, choice of sample size and violation of statistical assumptions. For clarification of these points, a practical example will be given. Using the proposed adequate statistical methods correctly will, although accompanied by increased complexity, increase the efficiency of bioengineering studies.

Effect of methanol concentration on the production of human β2-glycoprotein I domain V by a recombinant Pichia pastoris: A simple system for the control of methanol concentration using a semiconductor gas sensor

The methylotrophic yeast Pichia pastoris is one of the best hosts for the production of foreign proteins because of the presence of the strong AOX1 promoter induced by methanol. Methanol feeding during the production phase of the foreign proteins is important because methanol not only induces protein production but also provides energy source for the host cells. Excess methanol inhibits the growth of host cells, while an insufficient amount of energy source and/or methanol starvation lead to poor growth and production. We constructed a simple methanol control system consisting of a semiconductor gas sensor and a relay. Using this system, we studied the effect of methanol concentration on the production of a model foreign protein, human β 2-glycoprotein I domain V. The methanol concentrations were kept constant at 1.5, 10, 17, or 31 g· l −1 (±5%) during the production phase. Although the specific rates of growth and methanol consumption decreased with increase in the methanol concentration, the specific production rates increased, indicating that the energy for the production competed with that for cell growth. Accordingly, we provided glycerol as an extra energy source during the production phase, with the result that the specific production rate increased two times. Our simple and inexpensive system will help bioengineering studies on the production of recombinant proteins in P. pastoris, the growth and production of objective proteins in which are dependent on the methanol concentration.

Establishment of a clinical engineering department in a Venezuelan national reference hospital.

Since 1976, Clinical Engineering (CE) has been studied at the Simón Bolívar University (USB) as part of the Bioengineering Studies program developed at that University. However, it was not until 1996 that Clinical Engineering activities were established in a Venezuelan hospital. This paper describes how the USB, using its own human resources, has achieved the establishment of a Clinical Engineering Department in a national reference hospital for the first time.