An evolutionarily conserved mechanism that amplifies the effect of deleterious mutations in osteosarcoma.

Illustrating the molecular consequence of deleterious mutations is essential for bridging the gap between genotype and phenotype. In the cancer field, differential expression of the two alleles on heterozygous sites could directly reflect the effect of a mutation under certain trans environment. We retrieved transcriptomes of osteosarcoma and normal tissues in human and mouse. We defined tumor-specific heterozygous mutations with stringent criteria by considering sequencing depth and ancestral state. We calculated the relative expression of mutated alleles and normal alleles on the missense mutation sites in osteosarcoma. There is a conserved pattern that the mutated alleles have globally higher expression levels than the normal alleles in tumors. In the shared genes with missense mutations in both human and mouse, the relative expression of mutated alleles is highly correlated. Moreover, shared genes are functionally more important than unshared genes, and are enriched in oncogenes. The oncogenic role of mutations in oncogene KMT2A is experimentally verified. We systematically illustrate the deleterious effects of missense mutations by showing the over-expression of mutated alleles. We partially bridge the gap between genotype and phenotype by surmising that the over-expression of the mutated alleles might break the cellular equilibrium and lead to tumorigenesis.

Cost-Efficiency Optimization Serves as a Conserved Mechanism that Promotes Osteosarcoma in Mammals.

Mutations that reduce the biosynthetic cost of ATP production or increase the gene translation efficiency (tAI) are favorable for rapid cell growth and proliferation and therefore likely to be observed in tumors. Whether the mutations in tumors optimize the trade-off between the ATP biosynthesis cost and gene translation efficiency by increasing the tAI/ATP ratio is currently unknown. We retrieved transcriptome data of normal and osteosarcoma tissue samples from humans and mice and identified tumor-specific mutations in each species by using stringent cutoffs and outgroup information. We compared the tAI/ATP values of genes before and after mutation. The tAI/ATP profile was found to be highly conserved in humans and mice, and also correlated with the essentiality of genes. Tumor-specific rather than shared mutations were found to lead to increased tAI/ATP values in both species. Thus, tumor-specific mutations were found to optimize the cost-efficiency trade-off by increasing the tAI/ATP ratio of genes in osteosarcoma. This may indicate an evolutionarily conserved mechanism that promotes tumorigenesis by facilitating rapid cell growth and proliferation.

Production of highly efficient cellulase mixtures by genetically exploiting the potentials of Trichoderma reesei endogenous cellulases for hydrolysis of corncob residues.

BACKGROUND: Trichoderma reesei is one of the most important fungi utilized for cellulase production. However, its cellulase system has been proven to be present in suboptimal ratio for deconstruction of lignocellulosic substrates. Although previous enzymatic optimization studies have acquired different types of in vitro synthetic mixtures for efficient lignocellulose hydrolysis, production of in vivo optimized cellulase mixtures by industrial strains remains one of the obstacles to reduce enzyme cost in the biofuels production from lignocellulosic biomass. RESULTS: In this study, we used a systematic genetic strategy based on the pyrG marker to overexpress the major cellulase components in a hypercellulolytic T. reesei strain and produce the highly efficient cellulase mixture for saccharification of corncob residues. We found that overexpression of CBH2 exhibited a 32-fold increase in the transcription level and a comparable protein level to CBH1, the most abundant secreted protein in T. reesei, but did not contribute much to the cellulolytic ability. However, when EG2 was overexpressed with a 46-fold increase in the transcription level and a comparable protein level to CBH2, the engineered strain QPE36 showed a 1.5-fold enhancement in the total cellulase activity (up to 5.8 U/mL FPA) and a significant promotion of saccharification efficiency towards differently pretreated corncob residues. To assist the following genetic manipulations, the marker pyrG was successfully excised by homologous recombination based on resistance to 5-FOA. Furthermore, BGL1 was overexpressed in the EG2 overexpression strain QE51 (pyrG-excised) and a 11.6-fold increase in BGL activity was obtained. The EG2-BGL1 double overexpression strain QEB4 displayed a remarkable enhancement of cellulolytic ability on pretreated corncob residues. Especially, a nearly complete cellulose conversion (94.2%) was found for the delignified corncob residues after 48 h enzymatic saccharification. CONCLUSIONS: These results demonstrate that genetically exploiting the potentials of T. reesei endogenous cellulases to produce highly efficient cellulase mixtures is a powerful strategy to promote the saccharification efficiency, which will eventually facilitate cost reduction for lignocellulose-based biofuels.

A molasses habitat-derived fungus Aspergillus tubingensis XG21 with high ß-fructofuranosidase activity and its potential use for fructooligosaccharides production.

The industrial microorganisms used for fructooligosaccharides (FOS) synthesis are generally fermented with sucrose as carbon source to induce the production of ß-fructofuranosidase (FFase) having transfructosylation activity. Consequently, isolation of novel FFase producers from a sucrose-enriched biotope would help improve FOS productivity and reduce the process cost. Here, three fungi isolated from a unique sugarcane molasses habitat were found to possess FFase activity and one of them, XG21, exhibited a high capacity to synthesize FOS. Analysis of its morphological properties and ribosomal internal transcribed spacer (ITS) sequence allowed the taxonomic position to be assigned and it was thus identified as Aspergillus tubingensis XG21. It could utilize various potential carbon sources for vigorous growth, but only produced high-level FFase activity on sucrose. Furthermore, the transfructosylation ability and FOS synthesis were analyzed by TLC and HPLC. During the transfructosylation reaction, an increase in sucrose concentration led to the remarkable enhancement in FOS formation with the maximum content of up to 56.9% within 8 h. Finally, the sugarcane molasses was used to cultivate A. tubingensis XG21 and the optimal FFase activity reached up to 558.3 U/g, which was 88.9% higher than that with sucrose as carbon source. These results indicate that A. tubingensis XG21 can be considered as a new genetic resource adapted to cheaply available carbon sources for FOS production.

Drug-to-antibody determination for an antibody-drug-conjugate utilizing cathepsin B digestion coupled with reversed-phase high-pressure liquid chromatography analysis.

Antibody drug conjugates or ADCs are currently being evaluated for their effectiveness as targeted chemotherapeutic agents across the pharmaceutical industry. Due to the complexity arising from the choice of antibody, drug and linker; analytical methods for release and stability testing are required to provide a detailed understanding of both the antibody and the drug during manufacturing and storage. The ADC analyzed in this work consists of a tubulysin drug analogue that is randomly conjugated to lysine residues in a human IgG1 antibody. The drug is attached to the lysine residue through a peptidic, hydrolytically stable, cathepsin B cleavable linker. The random lysine conjugation produces a heterogeneous mixture of conjugated species with a variable drug-to-antibody ratio (DAR), therefore, the average amount of drug attached to the antibody is a critical parameter that needs to be monitored. In this work we have developed a universal method for determining DAR in ADCs that employ a cathepsin B cleavable linker. The ADC is first cleaved at the hinge region and then mildly reduced prior to treatment with the cathepsin B enzyme to release the drug from the antibody fragments. This pre-treatment allows the cathepsin B enzyme unrestricted access to the cleavage sites and ensures optimal conditions for the cathepsin B to cleave all the drug from the ADC molecule. The cleaved drug is then separated from the protein components by reversed phase high performance liquid chromatography (RP-HPLC) and quantitated using UV absorbance. This method affords superior cleavage efficiency to other methods that only employ a cathepsin digestion step as confirmed by mass spectrometry analysis. This method was shown to be accurate and precise for the quantitation of the DAR for two different random lysine conjugated ADC molecules.

Tear-mediated delivery of nanoparticles through transcytosis of the lacrimal gland.

Rapid clearance from the tears presents a formidable obstacle to the delivery of peptide drugs to the eye surface. This impedes therapies for ocular infections, wound healing, and dry-eye disease that affect the vision of millions worldwide. To overcome this challenge, this manuscript explores a novel strategy to reach the ocular surface via receptor-mediated transcytosis across the lacrimal gland (LG), which produces the bulk of human tears. The LG abundantly expresses the coxsackievirus and adenovirus receptor (CAR); furthermore, we recently reported a peptide-based nanoparticle (KSI) that targets CAR on liver cells. This manuscript reports the unexpected finding that KSI both targets and transcytoses into the LG acinar lumen, which drains to tear ducts. When followed using ex vivo live cell imaging KSI rapidly accumulates in lumen formed by LG acinar cells. LG transduction with a myosin Vb tail, which is dominant negative towards transcytosis, inhibits lumenal accumulation. Transcytosis of KSI was confirmed in vivo by confocal and TEM imaging of LG tissue following administration of KSI nanoparticles. These findings suggest that it is possible to target nanomaterials to the tears by targeting certain receptors on the LG. This design strategy represents a new opportunity to overcome barriers to ocular delivery.

Biodegradation of elastin-like polypeptide nanoparticles.

Protein polymers are repetitive polypeptides produced by ribosomal biosynthetic pathways; furthermore, they offer emerging opportunities in drug and biopharmaceutical delivery. As for any polymer, biodegradation is one of the most important determinants affecting how a protein polymer can be utilized in the body. This study was designed to characterize the proteolytic biodegradation for a library of protein polymers derived from the human tropoelastin, the Elastin-like polypeptides (ELPs). ELPs are of particular interest for controlled drug delivery because they reversibly transition from soluble to insoluble above an inverse phase transition temperature (T(t)). More recently, ELP block copolymers have been developed that can assemble into micelles; however, it remains unclear if proteases can act on these ELP nanoparticles. For the first time, we demonstrate that ELP nanoparticles can be degraded by two model proteases and that comparable proteolysis occurs after cell uptake into a transformed culture of murine hepatocytes. Both elastase and collagenase endopeptidases can proteolytically degrade soluble ELPs. To our surprise, the ELP phase transition was protective against collagenase but not to elastase activity. These findings enhance our ability to predict how ELPs will biodegrade in different physiological microenvironments and are essential to develop protein polymers into biopharmaceuticals.

A Rab11a-enriched subapical membrane compartment regulates a cytoskeleton-dependent transcytotic pathway in secretory epithelial cells of the lacrimal gland.

Despite observations that the lacrimal gland has been identified as the principal source of dimeric immunoglobulin A (dIgA) in tears, the mechanism used by lacrimal gland acinar cells (LGACs) to transcytose dIgA produced by interstitial plasma cells is not well-characterized. This study identifies a transcytotic pathway in LGACs regulated by Rab11a for polymeric immunoglobulin receptor (pIgR) and dIgA. EGFP-tagged Rab11a expressed in primary LGACs labeled a unique membrane compartment of comparable localization to endogenous Rab11a beneath the apical plasma membrane. This compartment was enriched in pIgR and clearly distinct from the regulated secretory pathway. Comparison of dIgA uptake in LGACs expressing wild type and dominant negative EGFP-Rab11a showed that the rapid exocytosis of dIgA was inhibited in acini expressing the dominant-negative protein, which additionally redistributed subapical pIgR. The trafficking of EGFP-Rab11a-enriched vesicles was regulated by microtubule-based and myosin Vb motors at distinct steps. Our data suggest that Rab11a is a crucial regulator of dIgA trafficking in primary acinar secretory epithelial cells and further support a role for microtubules, cytoplasmic dynein, actin filaments and myosin Vb in the maintenance of the Rab11a compartment in this primary secretory epithelial cell.

Design and cellular internalization of genetically engineered polypeptide nanoparticles displaying adenovirus knob domain.

Hepatocytes and acinar cells exhibit high-efficiency, fiber-dependent internalization of adenovirus; however, viral capsids have unpredictable immunological effects and are challenging to develop into targeted drug carriers. To exploit this internalization pathway and minimize the use of viral proteins, we developed a simple gene product that self assembles nanoparticles decorated with the knob domain of adenovirus serotype 5 fiber protein. The most significant advantages of this platform include: (i) compatibility with genetic engineering; (ii) no bioconjugate chemistry is required to link fusion proteins to the nanoparticle surface; and (iii) it can direct the reversible assembly of large nanoparticles, which are monodisperse, multivalent, and biodegradable. These particles are predominantly composed from diblock copolymers of elastin-like polypeptide (ELP). ELPs have unique phase transition behavior, whereby they self-assemble above a transition temperature that is simple to control. The diblock ELP described contains two motifs with distinct transition temperatures, which assemble nanoparticles at physiological temperatures. Analysis by non-denaturing-PAGE demonstrated that the purified knob-ELP formed trimers or dimers, which is a property of the native knob/fiber protein. Dynamic light scattering indicated that the diblock copolymer, with or without knob, is able to self assemble into nanoparticles ~40 nm in diameter. To examine the functionality of knob-ELP, their uptake was assessed in a hepatocyte cell-line that expresses the receptor for adenovirus serotype 5 fiber and knob, the coxsackievirus and adenovirus receptor (CAR). Both plain ELP and knob-ELP were bound to the outside of hepatocytes; however, the knob-ELP fusion protein exhibits more internalization and localization to lysosomes of hepatocytes. These findings suggest that functional fusion proteins may only minimally influence the assembly temperature and diameter of ELP nanoparticles. These results are a proof-of-principal that large fusion proteins (>10 kDa) can be assembled by diblock ELPs without the need for bioconjugate chemistry, which greatly simplifies the design and evaluation of targeted drug carriers.

Efficient Lewis acid ionic liquid-catalyzed synthesis of the key intermediate of coenzyme Q10 under microwave irradiation.

An efficient synthesis of a valuable intermediate of coenzyme Q(10) by microwave-assisted Lewis acidic ionic liquid (IL)-catalyzed Friedel-Crafts alkylation is reported. The acidity of six [Etpy]BF(4)-based ionic liquids was characterized by means of the FT-IR technique using acetonitrile as a molecular probe. The catalytic activities of these ionic liquids were correlated with their Lewis acidity. With increasing Lewis acid strength of the ionic liquids, their catalytic activity in the Friedel-Crafts reaction increased, except for [Etpy]BF(4)-AlCl(3). The effects of the reaction system, the molar fraction of Lewis acid in the Lewis acid ILs and heating techniques were also investigated. Among the six Lewis acid ionic liquids tested [Etpy]BF(4)-ZnCl(2) showed the best catalytic activity, with a yield of 89% after a very short reaction time (150 seconds). This procedure has the advantages of higher efficiency, better reusability of ILs, energy conservation and eco-friendliness. The method has practical value for preparation of CoQ(10) on an industrial scale.

Synthesis, biophysical properties and pharmacokinetics of ultrahigh molecular weight tense and relaxed state polymerized bovine hemoglobins.

Hemoglobin-based oxygen carriers (HBOC) are currently being developed as red blood cell (RBC) substitutes for use in transfusion medicine. Despite significant commercial development, late stage clinical results of polymerized hemoglobin (PolyHb) solutions hamper development. We synthesized two types of PolyHbs with ultrahigh molecular weights: tense (T) state PolyHb (M(W)=16.59 MDa and P(50)=41 mmHg) and relaxed (R) state PolyHb (M(W)=26.33 MDa and P(50)=0.66 mmHg). By maintaining Hb in either the T- or R-state during the polymerization reaction, we were able to synthesize ultrahigh molecular weight PolyHbs in distinct quaternary states with no tetrameric Hb, high viscosity, low colloid osmotic pressure and the ability to maintain O(2) dissociation, CO association and NO dioxygenation reactions. The PolyHbs elicited some in vitro RBC aggregation that was less than 6% dextran (500 kDa) but more than 5% human serum albumin. In vitro, T-state PolybHb autoxidized faster than R-state PolybHb as expected from previously reported studies, conversely, when administered to guinea pigs as a 20% exchange transfusion, R-state PolybHb oxidized faster and to a greater extent than T-state PolybHb, suggesting a more complex oxidative processes in vivo. Our findings also demonstrate that T-state PolybHb exhibited a longer circulating half-life, slower clearance and longer systemic exposure time compared to R-state PolybHb.

The quaternary structure of tetrameric hemoglobin regulates the oxygen affinity of polymerized hemoglobin.

This study focuses on the effect of the initial quaternary structure of bovine hemoglobin (Hb) on the physical properties of glutaraldehyde polymerized Hb (PolyHb) solutions. Tense (T) state PolyHb was synthesized by maintaining the pO(2) of Hb before and after polymerization at 0 mm Hg. In contrast, relaxed (R) state PolyHb was generated by maintaining the pO(2) of Hb before and after polymerization to >749 mm Hg. PolyHb solutions were characterized by measuring the pO(2), methemoglobin (metHb) level, molecular weight distribution, O(2) affinity and cooperativity coefficient. The metHb level of all PolyHb solutions was low (<2%). Analysis of the molecular weight distribution of PolyHb solutions indicates that in general, the molecular weight of PolyHb solutions increased with increasing cross-link density. T-state PolyHb solutions exhibited lower O(2) affinity compared to unmodified Hb, whereas R-state PolyHb solutions exhibited higher O(2) affinity compared to unmodified Hb. In addition, the polymerization reaction resulted in a significant decrease in cooperativity that was more pronounced at higher cross-link densities. All of these results were explained in terms of the quaternary structure of Hb. Taken together, our results yield more insight into the importance Hb's quaternary structure plays in defining the physical properties of glutaraldehyde PolyHb solutions. This information will be useful in designing optimized glutaraldehyde PolyHb oxygen carriers for various applications in transfusion medicine.

Effects of the molecular mass of tense-state polymerized bovine hemoglobin on blood pressure and vasoconstriction.

Despite recent advances in the design of hemoglobin (Hb)-based oxygen carriers (HBOCs), vasoconstriction, presumably caused by nitric oxide (NO) scavenging, vessel wall hyperoxygenation, and/or extravasation, has been identified as the principal road block hampering commercial development of HBOCs. This study was designed to analyze systemic and microvascular responses to the molecular mass and plasma concentration of tense (T)-state polymerized bovine Hb (PolybHb) solutions. Experiments were performed using the hamster window chamber model subjected to successive hypervolemic infusions of T-state PolybHb solutions. PolybHb plasma concentrations were evaluated, namely, 0.5, 1.0 and 1.5 g/dl, respectively. Infusion of PolybHb solutions with molecular mass >500 kDa elicited hypertension and vasoconstriction proportional to the plasma concentration and inversely proportional to the PolybHb cross-link density. However, two high-molecular mass PolybHb solutions, PolybHb(40:1)(high) PolybHb(50:1)(high), did not elicit vasoconstriction at all concentrations studied, whereas PolybHb(50:1)(high) only elicited moderate hypertension at the highest concentration studied. In contrast, infusion of PolybHb solutions with molecular mass <500 kDa elicited significant hypertension and vasoconstriction compared with PolybHb solutions with molecular mass >500 kDa that was proportional to the plasma concentration and inversely proportional to the PolybHb cross-link density. We present promising results for highly cross-linked T-state PolybHb solutions with molecular mass >500 kDa [PolybHb(40:1)(high) PolybHb(50:1)(high)], which supports the concept that HBOC size/molecular mass influences its proximity to the vascular endothelium and molecular diffusivity. The hemodynamics of HBOC within the plasma layer surrounding the abluminal side endothelium regulates NO production and consumption, vessel oxygen flux, and extravasation. Although mechanistically attractive, neither of these hypotheses can be directly tested in vivo and will require further investigation.

Purification of hemoglobin by tangential flow filtration with diafiltration.

A recent study by Palmer, Sun, and Harris (Biotechnol. Prog., 25:189-199, 2009) demonstrated that tangential flow filtration (TFF) can be used to produce HPLC-grade bovine and human hemoglobin (Hb). In this current study, we assessed the quality of bovine Hb (bHb) purified by introducing a 10 L batch-mode diafiltration step to the previously mentioned TFF Hb purification process. The bHb was purified from bovine red blood cells (RBCs) by filtering clarified RBC lysate through 50 nm (stage I) and 500 kDa (stage II) hollow fiber (HF) membranes. The filtrate was then passed through a 100 kDa (stage III) HF membrane with or without an additional 10 L diafiltration step to potentially remove additional small molecular weight impurities. Protein assays, SDS-PAGE, and LC-MS of the purified bHb (stage III retentate) reveal that addition of a diafiltration step has no effect on bHb purity or yield; however, it does increase the methemoglobin level and oxygen affinity of purified bHb. Therefore, we conclude that no additional benefit is gained from diafiltration at stage III and a three stage TFF process is sufficient to produce HPLC-grade bHb.

Tangential flow filtration of hemoglobin.

Bovine and human hemoglobin (bHb and hHb, respectively) was purified from bovine and human red blood cells via tangential flow filtration (TFF) in four successive stages. TFF is a fast and simple method to purify Hb from RBCs using filtration through hollow fiber (HF) membranes. Most of the Hb was retained in stage III (100 kDa HF membrane) and displayed methemoglobin levels less than 1%, yielding final concentrations of 318 and 300 mg/mL for bHb and hHb, respectively. Purified Hb exhibited much lower endotoxin levels than their respective RBCs. The purity of Hb was initially assessed via SDS-PAGE, and showed tiny impurity bands for the stage III retentate. The oxygen affinity (P(50)) and cooperativity coefficient (n) were regressed from the measured oxygen-RBC/Hb equilibrium curves of RBCs and purified Hb. These results suggest that TFF yielded oxygen affinities of bHb and hHb that are comparable to values in the literature. LC-MS was used to measure the molecular weight of the alpha (alpha) and beta (beta) globin chains of purified Hb. No impurity peaks were present in the HPLC chromatograms of purified Hb. The mass of the molecular ions corresponding to the alpha and beta globin chains agreed well with the calculated theoretical mass of the alpha- and beta- globin chains. Taken together, our results demonstrate that HPLC-grade Hb can be generated via TFF. In general, this method can be more broadly applied to purify Hb from any source of RBCs. This work is significant, since it outlines a simple method for generating Hb for synthesis and/or formulation of Hb-based oxygen carriers.

Site-selective glycosylation of hemoglobin on Cys beta93.

In this work, we describe the synthesis and characterization of a novel glycosylated hemoglobin (Hb) with high oxygen affinity as a potential Hb-based oxygen carrier. Site-selective glycosylation of bovine Hb was achieved by conjugating a lactose derivative to Cys 93 on the beta subunit of Hb. LC-MS analysis indicates that the reaction was quantitative, with no unmodified Hb present in the reaction product. The glycosylation site was identified by chymotrypsin digestion of the glycosylated bovine Hb followed with LC-MS/MS and from the X-ray crystal structure of the glycosylated Hb. The chemical conjugation of the lactose derivative at Cys beta93 yields an oxygen carrier with a high oxygen affinity (P(50) of 4.94 mmHg) and low cooperativity coefficient (n) of 1.20. Asymmetric flow field-flow fractionation (AFFFF) coupled with multiangle static light scattering (MASLS) was used to measure the absolute molecular weight of the glycosylated Hb. AFFFF-MASLS analysis indicates that glycosylation of Hb significantly altered the alpha(2)beta(2)-alphabeta equilibrium compared to native Hb. Subsequent X-ray analysis of the glycosylated Hb crystal showed that the covalently linked lactose derivative is sandwiched between the beta(1) and alpha(2) (and hence by symmetry the beta(2) and alpha(1)) subunits of the tetramer, and the interaction between the saccharide and amino acid residues located at the interface is apparently stabilized by hydrogen bonding interactions. The resultant structural analysis of the glycosylated Hb helps to explain the shift in the alpha(2)beta(2)-alphabeta equilibrium in terms of the hydrogen bonding interactions at the beta(1)alpha(2)/beta(2)alpha(1) interface. Taken together, all of these results indicate that it is feasible to site-specifically glycosylate Hb. This work has great potential in developing an oxygen carrier with defined chemistry that can target oxygen delivery to low pO(2) tissues and organs.

Identical-dual-bandpass sampled fiber Bragg grating and its application to ultranarrow filters.

We have theoretically proposed and experimentally demonstrated a new kind of ultranarrow identical-dual-bandpass sampled fiber Bragg gratings (SFBGs) with a pi phase shift technique. The spacing of two bandpasses of the proposed grating can be flexibly adjusted by changing the sampled period, and any desired spacing can be achieved in principle. An experimental example shows that the transmission peaks of two narrow transmission-band are near 1549.1 and 1550.1 nm. Based on the proposed SFBG, an ultranarrow identical-dual-channel filter is designed. Two channels of the proposed filter have an equal bandwidth, an even strength, and the same group delay. The bandwidth of each channel of our filter is as small as 1 pm and up to 10(-3) pm (corresponding to approximately 0.1 MHz), which is less than the bandwidth of the conventional SFBG filters by a factor of 10(2)-10(4). The proposed grating and filter can find potential applications with slow light and dual-wavelength single-longitudinal-mode fiber lasers.

Tunable multiwavelength fiber laser using a comb filter based on erbium-ytterbium co-doped polarization maintaining fiber loop mirror.

A tunable comb filter based on a fiber loop mirror setup, which incorporates a piece of pumped erbium-ytterbium co-doped polarization maintaining fiber, is newly presented. It is accomplished by controlling the pump power or adjusting a polarization controller in the loop mirror, which results from the fact that the effective birefringence of the erbium-ytterbium co-doped polarization maintaining fiber depends on the pump power and polarization state of the traversing signal. By using the proposed comb filter, a continuously tunable multiwavelength fiber ring laser in the L-band is successfully demonstrated.

Preparation of ultrapure bovine and human hemoglobin by anion exchange chromatography.

Bovine and human hemoglobin (Hb) form the basis for many different types of Hb-based O(2) carriers (HBOCs) ranging from chemically modified Hbs to particle encapsulated Hbs. Hence, the development of a facile purification method for preparing ultrapure Hb is essential for the reliable synthesis and formulation of HBOCs. In this work, we describe a simple process for purifying ultrapure solutions of bovine and human Hb. Bovine and human red blood cells (RBCs) were lyzed, and Hb was purified from the cell lysate by anion exchange chromatography. The initial purity of Hb fractions was analyzed by SDS-PAGE. Pure Hb fractions (corresponding to a single band on the SDS-PAGE gel) were pooled together and the overall purity and identity assessed by LC-MS. LC-MS analysis yielded two peaks corresponding to the calculated theoretical molecular weight of the alpha and beta chains of Hb. The activity of HPLC pure Hb was assessed by measuring its oxygen affinity, cooperativity and methemoglobin level. These measures of activity were comparable to values in the literature. Taken together, our results demonstrate that ultrapure Hb (electrophoresis and HPLC pure) can be easily prepared via anion exchange chromatography. In general, this method can be more broadly applied to purify hemoglobin from any source of RBC. This work is significant, since it outlines a simple method for generating ultrapure Hb for synthesis and/or formulation of HBOCs.

The temperature sensitivity of Sagnac loop interferometer based on polarization maintaining side-hole fiber.

The temperature sensitivity of modal birefringence (dB/dT) of different types of polarization-maintaining fibers with side holes was measured using a Sagnac loop interferometer. The thermal expansion coefficient can be varied by controlling the amount of germanium doped in the core region. Using this method, dB/dT could be made higher (~10(-7)/ degrees C) than that of standard PMFs (~10(-8)/ degrees C) or comparable to that of standard PMFs (~10(-9)/ degrees C).