Experimental design methods for bioengineering applications

Experimental design is a form of process analysis in which certain factors are selected to obtain the desired responses of interest. It may also be used for the determination of the effects of various independent factors on a dependent factor. The bioengineering discipline includes many different areas of scientific interest, and each study area is affected and governed by many different factors. Briefly analyzing the important factors and selecting an experimental design for optimization are very effective tools for the design of any bioprocess under question. This review summarizes experimental design methods that can be used to investigate various factors relating to bioengineering processes. The experimental methods generally used in bioengineering are as follows: full factorial design, fractional factorial design, Plackett–Burman design, Taguchi design, Box–Behnken design and central composite design. These design methods are briefly introduced, and then the application of these design methods to study different bioengineering processes is analyzed.     

The matri-cellular proteins 'cysteine-rich, angiogenic-inducer, 61' and 'connective tissue growth factor' are regulated in experimentally-induced sepsis with multiple organ dysfunction

Organ failure is a severe complication in sepsis for which the pathophysiology remains incompletely understood. Recently, the matri-cellular cysteine-rich, angiogenic induced, 61 (Cyr61/CCN1); connective tissue growth factor (Ctgf/CCN2); and nephroblastoma overexpressed gene (Nov/CCN3) (CCN)-protein family have been attributed organ-protective properties. Their expression is sensitive to mediators of sepsis pathophysiology but a potential role in sepsis remains elusive. To provide an initial assessment, 50 rats were subjected to 18?h of cecal-ligation and puncture or sham operation. Hepatic and pulmonary CCN1 mRNA displayed an average 7.4- and 3.3-fold induction, while its cardiac expression was unchanged. The changes coincided with excessive hepatic and pulmonary inflammatory gene activation and a restricted cardiac inflammation. Furthermore, hepatocytes displayed a dosage-dependent CCN1 mRNA response in?vitro, supporting a cytokine-mediated CCN1 regulation in sepsis. CCN2 mRNA was 2.2-fold induced in the liver, while 2.0-fold and 1.4-fold repressed in the heart and lung. Meanwhile, it did not respond to TNF-α exposure in?vitro, which indicates different means of regulation than for CCN1. Taken together, this study provides the first evidence for multi-organ regulation of CCN1 and CCN2 in early stages of sepsis, and implies the eruption of inflammatory mediators as a potential mechanism behind the observed CCN1 regulation.     

Temporal transmission studies of mouse parvovirus 1 in BALB/c and C.B-17/Icr-Prkdc(scid) mice

Fecal shedding and transmission of mouse parvovirus 1 (MPV) to naive sentinels, breeding mates, and progeny were assessed. Neonatal SCID and BALB/c mice inoculated with MPV were evaluated over 24 wk; several mice from each strain were mated once during this period. Fecal MPV loads for each cage were determined weekly by quantitative polymerase chain reaction (PCR) analysis, and all mice were evaluated by quantitative PCR analysis of lymphoid tissues and seroconversion to MPV antigens in immunocompetent mice. Results indicated persistently high fecal shedding of MPV in SCID mice throughout the evaluation period sufficient to allow transmission to sentinels, naive breeding partners, and the progeny of infected male mice and naive partners. Lymphoid tissue viral loads in the progeny of infected female SCID mice were high at weaning but low at 6 wk of age. Infected BALB/c mice shed high levels of MPV in feces for 3 wk postinoculation, with seroconversion only in sentinels exposed during the first 2 wk postinoculation. Thereafter the feces of infected BALB/c mice and the lymphoid tissues of sentinels, naive breeding partners, and progeny intermittently contained extremely low levels of MPV DNA. Although pregnancy and lactation did not increase viral shedding in BALB/c mice, MPV exposure levels were sufficient to induce productive infection in some BALB/c progeny. These data indicate that the adaptive immune response suppresses, but does not eliminate, MPV shedding; this suppression is sufficient to inhibit infection of weanling and adult mice but allows productive infection of some progeny.     

Immune response to bacteria induces dissemination of Ras-activated Drosophila hindgut cells

Although pathogenic bacteria are suspected contributors to colorectal cancer progression, cancer-promoting bacteria and their mode of action remain largely unknown. Here we report that sustained infection with the human intestinal colonizer Pseudomonas aeruginosa synergizes with the Ras1V12 oncogene to induce basal invasion and dissemination of hindgut cells to distant sites. Cross-talk between infection and dissemination requires sustained activation by the bacteria of the Imd-dTab2-dTak1 innate immune pathway, which converges with Ras1V12 signalling on JNK pathway activation, culminating in extracellular matrix degradation. Hindgut, but not midgut, cells are amenable to this cooperative dissemination, which is progressive and genetically and pharmacologically inhibitable. Thus, Drosophila hindgut provides a valuable system for the study of intestinal malignancies.     

FisB relies on homo-oligomerization and lipid binding to catalyze membrane fission in bacteria

Little is known about mechanisms of membrane fission in bacteria despite their requirement for cytokinesis. The only known dedicated membrane fission machinery in bacteria, fission protein B (FisB), is expressed during sporulation in Bacillus subtilis and is required to release the developing spore into the mother cell cytoplasm. Here, we characterized the requirements for FisB-mediated membrane fission. FisB forms mobile clusters of approximately 12 molecules that give way to an immobile cluster at the engulfment pole containing approximately 40 proteins at the time of membrane fission. Analysis of FisB mutants revealed that binding to acidic lipids and homo-oligomerization are both critical for targeting FisB to the engulfment pole and membrane fission. Experiments using artificial membranes and filamentous cells suggest that FisB does not have an intrinsic ability to sense or induce membrane curvature but can bridge membranes. Finally, modeling suggests that homo-oligomerization and trans-interactions with membranes are sufficient to explain FisB accumulation at the membrane neck that connects the engulfment membrane to the rest of the mother cell membrane during late stages of engulfment. Together, our results show that FisB is a robust and unusual membrane fission protein that relies on homo-oligomerization, lipid binding, and the unique membrane topology generated during engulfment for localization and membrane scission, but surprisingly, not on lipid microdomains, negative-curvature lipids, or curvature sensing.

Little is known about how membrane fission occurs in bacteria; this study suggests that the membrane fission protein FisB exploits the unique cellular geometry encountered during sporulation to enable its localization to the fission site through a novel mechanism, where it catalyzes membrane scission.     

Inhibition of RPS6K reveals context-dependent Akt activity in luminal breast cancer cells

Aberrant signaling through insulin (Ins) and insulin-like growth factor I (IGF1) receptors contribute to the risk and advancement of many cancer types by activating cell survival cascades. Similarities between these pathways have thus far prevented the development of pharmacological interventions that specifically target either Ins or IGF1 signaling. To identify differences in early Ins and IGF1 signaling mechanisms, we developed a dual receptor (IGF1R & InsR) computational response model. The model suggested that ribosomal protein S6 kinase (RPS6K) plays a critical role in regulating MAPK and Akt activation levels in response to Ins and IGF1 stimulation. As predicted, perturbing RPS6K kinase activity led to an increased Akt activation with Ins stimulation compared to IGF1 stimulation. Being able to discern differential downstream signaling, we can explore improved anti-IGF1R cancer therapies by eliminating the emergence of compensation mechanisms without disrupting InsR signaling.