Bioengineering of the mammary gland to produce proteins of therapeutic and industrial value is the result of extensive investigation of the physiology of the mammary gland and the ability to generate transgenic animals. Targeting the expression of heterologous proteins to mammary tissue requires a thorough understanding of the biochemical events that coordinate growth and differentiation of the mammary gland and of the hormonal and developmental regulation of expression of milk protein genes. The characterization of mammary-specific promoter regions in milk protein genes and knowledge of the mechanisms that confer integration site-independent expression of transgenes have significantly contributed to modifying the mammary gland to produce heterologous proteins of therapeutic interest. The generation of large transgenic farm animals provides the opportunity for large-scale production of proteins in milk that have a therapeutic value but are naturally present at low concentrations in biological fluids. Transgenic mammary epithelial cells offer a versatile research model in biomedical, environmental health, and neonatal toxicology research.
Secreted parasitism proteins encoded by parasitism genes expressed in esophageal gland cells mediate infection and parasitism of plants by root-knot nematodes (RKN). Parasitism gene 16D10 encodes a conserved RKN secretory peptide that stimulates root growth and functions as a ligand for a putative plant transcription factor. We used in vitro and in vivo RNA interference approaches to silence this parasitism gene in RKN and validate that the parasitism gene has an essential function in RKN parasitism of plants. Ingestion of 16D10 dsRNA in vitro silenced the target parasitism gene in RKN and resulted in reduced nematode infectivity. In vivo expression of 16D10 dsRNA in Arabidopsis resulted in resistance effective against the four major RKN species. Because no known natural resistance gene has this wide effective range of RKN resistance, bioengineering crops expressing dsRNA that silence target RKN parasitism genes to disrupt the parasitic process represents a viable and flexible means of developing novel durable RKN-resistant crops and could provide crops with unprecedented broad resistance to RKN.
Membrane fusion is a vital process of life involved, for example, in cellular secretion via exocytosis, signaling between nerve cells, and virus infection. In both the life sciences and bioengineering, controlled membrane fusion has many possible applications, such as drug delivery, gene transfer, chemical microreactors, or synthesis of nanomaterials. Until now, the fusion dynamics has been elusive because direct observations have been limited to time scales that exceed several milliseconds. Here, the fusion of giant lipid vesicles is induced in a controlled manner and monitored with a temporal resolution of 50 μs. Two different fusion protocols are used that are based on synthetic fusogenic molecules and electroporation. For both protocols, the opening of the fusion necks is very fast, with an average expansion velocity of centimeters per second. This velocity indicates that the initial formation of a single fusion neck can be completed in a few hundred nanoseconds.
Objective: The objective was to examine the efficacy of several treatment regimens in improving wound healing of cutaneous sulfur mustard (HD) injuries. Methods: Wound healing studies were conducted in weanling pigs. Superficial dermal HD injuries were debrided at 48 hours postexposure using an erbium-doped yttrium aluminum garnet (Er:YAG) laser, followed by application of a treatment adjunct. A variety of noninvasive bioengineering methods were conducted during the postsurgical observation period to examine the various cosmetic and functional aspects of the skin. Histopathology was performed at the end of each study (14 or 21 days postsurgery). Results: As noted clinically, reepithelialization was nearly complete by 7 days postsurgery for many of the sites treated with petrolatum and scarlet red dressings. By 21 days, the skin elasticity of the petrolatum-dressed sites was not significantly different from that of sham-exposed skin. Upon dressing removal on postsurgery day 4, the neoepidermis of allograft- and thin film-dressed sites was partially removed, with resultant petechial hemorrhaging. Mean pathology scores for hydrocolloid-dressed sites were significantly lower than those of untreated HD-exposed sites on postsurgery day 14. Conclusions: Care must be taken during bandage changes...
Urochondra setulosa (Trin.) C.E. Hubbard is a coastal halophytic grass thriving on the coastal dunes along the Pakistani seashore. This grass could be useful in coastal sand dune stabilization using seawater irrigation. The purpose of this investigation was to test the hypothesis that Ca2+ (0.0, 2.5, 5.0, 10.0 and 50.0 mmol/L) alleviates the adverse effects of KCl, MgSO4, NaCl and Na2SO4 at 0, 200, 400, 600, 800 and 1000 mmol/L on the germination of Urochondra setulosa. Seed germination was inhibited with increase in salt concentration with few seeds germinated at and above 400 mmol/L concentration. No seed germinated in any of the KCl treatments. Inclusion of CaCl2 substantially alleviated the inhibitory effects of all salts. Germination was higher under photoperiod in comparison to those seeds germinated under complete darkness. Among the CaCl2 concentrations used, 10 mmol/L was most effective in alleviating salinity effects and allowing few seeds to germinate at 1000 mmol/L KCl, MgSO4, NaCl and Na2SO4 solution.
Factorial design and response surface techniques were used to design and optimize increasing P450 BM-3 expression in E. coli. Operational conditions for maximum production were determined with twelve parameters under consideration: the concentration of FeCl3, induction at OD578 (optical density measured at 578 nm), induction time and inoculum concentration. Initially, Plackett-Burman (PB) design was used to evaluate the process variables relevant in relation to P450 BM-3 production. Four statistically significant parameters for response were selected and utilized in order to optimize the process. With the 416C model of hybrid design, response surfaces were generated, and P450 BM-3 production was improved to 57.90×10−3 U/ml by the best combinations of the physicochemical parameters at optimum levels of 0.12 mg/L FeCl3, inoculum concentration of 2.10%, induction at OD578 equal to 1.07, and with 6.05 h of induction.
Forkhead box (Fox) proteins play critical roles in the regulation of differentiation, proliferation, immunity and aging of cells. Most studies on Fox proteins are limited to cultured cells and rodent. The aim of the current study is to detect by immunohistrochemistry whether FoxO1, FoxO3a and FoxO4 proteins are localized in the stomach and intestine of the pig. The results showed that FoxO4 exists in the mucosa in all parts of the stomach and intestine; FoxO3a exists mainly in the lamina propria and muscularis of some parts. However, FoxO1 is not detectable in all parts of the stomach and intestine. Collectively, the results of the present study indicate that there exists a distinct expression pattern of Fox proteins, and that FoxO4 is a primary forkhead transcriptional factor localized in the gastrointestinal tracts of the pig.
The rice water weevil, Lissorhoptrus oryzophilus Kuschel, has two generations in southern Zhejiang, China. To determine oogenesis in first-generation females (summer females) and its relations to temperature, females were collected from a rice field in early and mid-July and reared on young rice plants at 28, 31 and 34 °C in the laboratory. Percentage of females having oocytes, number of oocytes of different stages (stage-I, from early previtellogenesis to middle vitellogenesis; stage-II, late vitellogenesis; and mature-oocyte stage), and length of ovarioles were determined every 10 d of feeding. At each temperature, oogenesis took place in over 40% of females after 20~40 d of feeding, but only 0.0~3.3 stage-I, 0.0~0.8 stage-II and 0.0~1.1 mature oocytes were observed at each observation date. Temperature had significant effect on number of stage-I oocytes but not on number of stage-II and mature oocytes in early July females; temperature had no significant effect on number of oocytes of either stage in mid-July females. Conclusively, in southern Zhejiang, summer L. oryzophilus females have great potential to become reproductive on rice, but their oogenesis activity is very low, with the overall procedures little affected by temperature.
Tissue and organ replacement have quickly outpaced available supply.
Tissue bioengineering holds the promise for additional tissue availability.
Various scaffolds are currently used, whereas polyglycolic acid (PGA), which is
currently used in absorbable sutures and orthopedic pins, provides an excellent
support for tissue development. Unfortunately, PGA can induce a local
inflammatory response following implantation, so we investigated the molecular
mechanism of inflammation in vitro and in vivo. Degraded PGA induced an acute
peritonitis, characterized by neutrophil (PMN) infiltration following
intraperitoneal injection in mice. Similar observations were observed using the
metabolite of PGA, glycolide. Dissolved PGA or glycolide, but not native PGA,
activated the classical complement pathway in human sera, as determined by
classical complement pathway hemolytic assays, C3a and C5a production, C3 and
immunoglobulin deposition. To investigate whether these in vitro observations
translated to in vivo findings...
The quest to determine how precise neural activity patterns mediate computation, behavior, and pathology would be greatly aided by a set of tools for reliably activating and inactivating genetically targeted neurons, in a temporally precise and rapidly reversible fashion. Having earlier adapted a light-activated cation channel, channelrhodopsin-2 (ChR2), for allowing neurons to be stimulated by blue light, we searched for a complementary tool that would enable optical neuronal inhibition, driven by light of a second color. Here we report that targeting the codon-optimized form of the light-driven chloride pump halorhodopsin from the archaebacterium Natronomas pharaonis (hereafter abbreviated Halo) to genetically-specified neurons enables them to be silenced reliably, and reversibly, by millisecond-timescale pulses of yellow light. We show that trains of yellow and blue light pulses can drive high-fidelity sequences of hyperpolarizations and depolarizations in neurons simultaneously expressing yellow light-driven Halo and blue light-driven ChR2, allowing for the first time manipulations of neural synchrony without perturbation of other parameters such as spiking rates. The Halo/ChR2 system thus constitutes a powerful toolbox for multichannel photoinhibition and photostimulation of virally or transgenically targeted neural circuits without need for exogenous chemicals...
Acinetobacter venetianus Rag1 produces an extracellular, polymeric lipoheteropolysaccharide termed apoemulsan. This polymer is putatively produced via a Wzy-dependent pathway. According to this model, the length of the polymer is regulated by polysaccharide-copolymerase (PCP) protein. A highly conserved proline and glycine motif was identified in all members of the PCP family of proteins and is involved in regulation of polymer chain length. In order to control the structure of apoemulsan, defined point mutations in the proline-glycine-rich region of the apoemulsan PCP protein (Wzc) were introduced. Modified wzc variants were introduced into the Rag1 genome via homologous recombination. Stable chromosomal mutants were confirmed by Southern blot analysis. The molecular weight of the polymer was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Five of the eight point mutants produced polymers having molecular weights higher than the molecular weight of the polymer produced by the wild type. Moreover, four of these five polymers had modified biological properties. Replacement of arginine by leucine (R418L) resulted in the most significant change in the molecular weight of the polymer. The R418L mutant was the most hydrophilic mutant...
Keratocytes of the corneal stroma secrete a specialized extracellular matrix essential for vision. These quiescent cells exhibit limited capacity for self-renewal and after cell division become fibroblastic, secreting nontransparent tissue. This study sought to identify progenitor cells for human keratocytes. Near the corneal limbus, stromal cells expressed ABCG2, a protein present in many adult stem cells. The ABCG2-expressing cell population was isolated as a side population (SP) by cell sorting after exposure to Hoechst 33342 dye. The SP cells exhibited clonal growth and continued to express ABCG2 and also PAX6, product of a homeobox gene not expressed in adult keratocytes. Cloned SP cells cultured in medium with fibroblast growth factor-2 lost ABCG2 and PAX6 expression and upregulated several molecular markers of keratocytes, including keratocan, aldehyde dehydrogenase 3A1, and keratan sulfate. Cloned corneal SP cells under chondrogenic conditions produced matrix staining with toluidine blue and expressed cartilage-specific markers: collagen II, cartilage oligomatrix protein, and aggrecan. Exposure of cloned SP cells to neurogenic culture medium upregulated mRNA and protein for glial fibrillary acidic protein, neuro filament protein...
Evolution has used many different strategies to build eyes and lenses. However, the genetic regulation involved seems to be quite conserved. Likewise, the regeneration of eye structures is remarkable, especially in salamanders. This review outlines the basic mechanisms of lens regeneration and its induction and the possibility of creating lenses by transdifferentiation of the pigment epithelial cells, by stem cells or by bioengineering.
Glycosidases play a key role in a number of biological processes and, as such, are of considerable clinical and biotechnological importance. Knowledge of the identifies of catalytically important active site residues is essential for understanding the catalytic mechanism, for enzyme classification, and for targeted bioengineering of glycosidases with altered characteristics. Here we review and discuss traditional strategies and novel approaches based on tandem mass spectrometry for the identification of the key active site residues in glycosidases.
Biomedical informatics includes the application of computers, information networks and systems, and a growing body of scientific understanding to a range of problems. As skill in this field increases and as progress in virtually all modern biomedical science becomes more data intensive, informatics becomes a precious resource. Applications areas include access to knowledge, discovery in genomics, medical records, mathematical modeling, and bioengineering. At the same time, progress in informatics is deeply dependent on resolution of four major public policy issues: digital intellectual property rights, genetic testing protection, medical data privacy, and the role of biomedical data in the context of information warfare and homeland security.