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A combination of gas chromatography and mass spectrometry (GC-MS) was applied to separate and identify the relative substances in 2-chloroethyl phenyl sulfide (CEPS), an industrial intermediate formed in the synthesis of sulfinpyrazone. Six impurities, including 1,2-dichloroethane, thiophenol, diethylmalonate, tributylamine, diphenyl disulfide, and 1,2-dithiophenylethane, were verified. According to the production technique, the sources of all of these related impurities were analyzed, and some practical suggestions are put forward for their removal from or avoidance in CEPS. Furthermore, a technique involving high-performance liquid chromatography with UV detection (HPLC-UV) was developed to separate and determine thiophenol, the key impurity in CEPS. A linear standard curve was obtained for thiophenol in concentrations between 2.65 × 10−4 and 0.106 mg mL−1, and the detection limit of thiophenol was 1.32 × 10−4 mg mL−1. Excellent precision and accuracy were shown from the method validation, with relative...

Antibody-drug conjugates enhance the antitumor effects of antibodies and reduce adverse systemic effects of potent cytotoxic drugs. However, conventional drug conjugation strategies yield heterogenous conjugates with relatively narrow therapeutic index (maximum tolerated dose/curative dose). Using leads from our previously described phage display-based method to predict suitable conjugation sites, we engineered cysteine substitutions at positions on light and heavy chains that provide reactive thiol groups and do not perturb immunoglobulin folding and assembly, or alter antigen binding. When conjugated to monomethyl auristatin E, an antibody against the ovarian cancer antigen MUC16 is as efficacious as a conventional conjugate in mouse xenograft models. Moreover, it is tolerated at higher doses in rats and cynomolgus monkeys than the same conjugate prepared by conventional approaches. The favorable in vivo properties of the near-homogenous composition of this conjugate suggest that our strategy offers a general approach to retaining the antitumor efficacy of antibody-drug conjugates, while minimizing their systemic toxicity.

Virus-like particles (VLPs) represent a promising vaccine against severe acute respiratory syndrome coronavirus (SARS CoV). In this study, recombinant baculovirus vAcS and vAcME were constructed to express the S protein and the M and E proteins of SARS CoV, respectively. Electron microscope analysis demonstrated the formation of VLPs in vAcME and vAcS coinfected insect cells. Mice immunized four times with VLPs developed high antibody titres against SARS CoV. In addition, VLPs elicited cell-mediated immunity as demonstrated by enhanced interferon-gamma and interleukin-4 production. VLPs also conferred protective immunity against the infection of Spike protein pseudotyped murine leukaemia virus. Our findings demonstrate that SARS CoV VLPs are immunogenic and can elicit strong SARS CoV-specific humoral and cellular immune responses in mice. This is the first study describing the immunogenicity of SARS CoV VLPs, providing valuable data for developing a protective vaccine against SARS CoV infection.

The CW domain is a zinc binding domain, composed of approximately 50- 60 amino acid residues with four conserved cysteine (C) and two to four conserved tryptophan (W) residues. The members of the superfamily of CW domain containing proteins, comprised of 12 different eukaryotic nuclear protein families, are extensively expressed in vertebrates, vertebrate-infecting parasites and higher plants, where they are often involved in chromatin remodeling, methylation recognition, epigenetic regulation and early embryonic development. Since the first CW domain structure was determined 5 years ago, structures of five CW domains have been solved so far. In this review, we will discuss these recent advances in understanding the identification, definition, structure, and functions of the CW domain containing proteins.

Purification processes for therapeutic antibodies typically exploit multiple and orthogonal chromatography steps in order to remove impurities, such as host-cell proteins. While the majority of host-cell proteins are cleared through purification processes, individual host-cell proteins such as Phospholipase B-like 2 (PLBL2) are more challenging to remove and can persist into the final purification pool even after multiple chromatography steps. With packed-bed chromatography runs using host-cell protein ELISAs and mass spectrometry analysis, we demonstrated that different therapeutic antibodies interact to varying degrees with host-cell proteins in general, and PLBL2 specifically. We then used a high-throughput Protein A chromatography method to further examine the interaction between our antibodies and PLBL2. Our results showed that the co-elution of PLBL2 during Protein A chromatography is highly dependent on the individual antibody and PLBL2 concentration in the chromatographic load. Process parameters such as antibody resin load density and pre-elution wash conditions also influence the levels of PLBL2 in the Protein A eluate. Furthermore, using surface plasmon resonance, we demonstrated that there is a preference for PLBL2 to interact with IgG4 subclass antibodies compared to IgG1 antibodies.

Elucidating action mechanisms of Chinese medicines has remained a challenging task due to the chemical and biological complexity that needs to be resolved. In this study we applied a gene expression data and connectivity map (CMAP) based approach to study action mechanisms of a Chinese medicine Xuesaitong injection (XST) on preventing cerebral ischemia–reperfusion injury. XST is a standardized patent Chinese medicine of Panax notoginseng roots and it has long been used for the effective prevention and treatment of stroke in China. However, more research is needed to understand the mechanisms underlying its effects against ischemic stroke. We first evaluated the effect of XST against ischemic stroke in an ischemia–reperfusion rat animal model and dissected its mechanisms based on gene expression data of injured brain. The results showed that treatment with XST significantly attenuated infarct area and histological damage. Based upon pathway analysis and the CMAP query of microarray data, anti-inflammatory response and anti-platelet coagulation were found as the major mechanisms of XST against stroke, which were further validated in vitro and with pharmacological assays of serum. We demonstrated the feasibility of applying the combination of the microarray with the CMAP in identifying mechanisms of Chinese medicine.

RNA modification, involving in a wide variety of cellular processes, has been identified over 100 types since 1950s. N(6)-methyladenosine (m6A), as one of the most abundant RNA modifications, is found in several RNA species and predominantly located in the stop codons, long internal exons as well as 3'UTR. It was reported that m6A modification preferentially appears after G in the conserved motif RRm6ACH (R = A/G and H = A/C/U). There are two families of enzymes responsible for maintaining the balance of m6A modification: m6A methyltransferases and demethylases, which add and remove methyl marks for adenosine of RNA, respectively. METTL3 complex, the m6A methyltransferases, and two kinds of demethylases including Fat mass and obesity-associated protein (FTO) and alkylation protein AlkB homolog 5 (ALKBH5) are characterized thus far. Besides the "writers" and "erasers", m6A specific recognizing proteins, such as the YTH (YT521-B homology) domain family proteins, also have attracted significant attention. Herein, we focus on the recent progress in understanding the biological/biochemical functions and structures of proteins responsible for the m6A modification and recognition. Detailed analyses of these important proteins are essential for the further study of their biological function and will also guide us in designing more potent and specific small-molecule chemical inhibitors for these targets.

In order to shorten the revisit period, and improve the efficiency of imaging, currently, all the in orbit high resolution remote sensing satellites adopt side-view imaging technology. When satellites imaging with a side perspective, it would inevitably cause the degradation of image quality, like spatial resolution reduction, image deformation incensement, and the reduction of positioning accuracy. In addition, with the increase of the side perspective, the error of DEM data will be amplified in the process of ortho-rectification, which, thus, will bring bigger error for image mapping accuracy. To address this problem, embarked from the impact of side view and DEM precision on image point error, this paper used Quick bird image data of the same area but different side perspectives, and simulate the impacts of different DEM elevation error on different side view image. At last we put forward a method to determine the maximum side perspective with map precision constraints, and set up a conversion relationship among satellite side view, DEM error and image point error. Finally, the paper combines the errors of 1:50000 DEM, ASTERGDEM and the requirement of ortho-image drawing, discusses and gives the values of maximum side perspective in the mountains and plains.

Object: To determine the morbidity of diabetes mellitus in cynomolgus monkey by performing the Oral Glucose Tolerance Test (OGTT). Method: OGTTs were performed in 600 male cynomolgus monkeys, aging 4-18 years. The values of plasma glucose (PG) and insulin (Ins) were detected. Result: Significant differences were found in the values of PG and Ins among groups with different ages. The values of PG of 4 pathological courses of DM were: Normal glucose tolerance (NGT) group: Fasting plasma glucose (FPG) < 6.10 mmol/L and 2hPG < 7.8 mmol/L; Impaired fasting glucose (IFG) group: 6.10 mmol/L ≤ FPG < 7.0 mmol/L and 2hPG < 7.8 mmol/L; Impaired glucose tolerance (IGT) group: FPG < 7.0 mmol/L and 7.8 mmol/L ≤ 2hPG < 11.1 mmol/L; diabetes mellitus (DM) group: FPG ≥ 7.0 mmol/L or 2hPG ≥ 11.1 mmol/L. The morbidity of diabetes mellitus in the tested cynomolgus monkeys was 11.5%. Conclusion: Our findings pave the ways for a wide range of screening of DM in cynomolgus monkeys. Meanwhile, we also provide the reference to preclinically test new antidiabetic drugs in cynomolgus monkeys.

Summary: PRO70769 is a humanized IgG1 monoclonal antibody against the CD20 molecule that is present on normal and malignant B cells. PRO70769 is being evaluated for treatment of B-cell-mediated diseases and is in a phase 1 trial for rheumatoid arthritis. As part of the preclinical toxicology evaluation, B-cell depletion profiles and safety of PRO70769 were assessed in cynomolgus monkeys. Animals were administered drug (IV) on days 1 and 15 with 10, 50, or 100 mg/kg PRO70769 and killed 2 weeks after the second dose and after a 3-month recovery period. In a parallel study, animals were not necropsied but instead were retreated with a second cycle of PRO70769 administered under an identical regimen. PRO70769 suppressed B cells in the blood to undetectable levels and significantly reduced B cells in lymphoid tissues. Splenic B cells were depleted to a greater extent compared with lymph node B cells. A second cycle of treatment resulted in a greater extent of depletion in lymphoid tissues compared with the depletion observed after one cycle of treatment; however, residual B cells in lymphoid tissues were still detectable, even at the highest dose. The rate of B-cell recovery in peripheral blood appeared similar between one and two cycles of treatment. Upon depletion there was a change in the profile of lymph node B-cell subsets. After recovery, B-cell subsets were reconstituted to normal levels. Depletion of CD20-expressing cells and lymphoid follicular atrophy were the only treatment-related effects.

Severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) is the etiological agent of SARS. It is believed that SARS-CoV originates from wild animals. We have developed a multitarget real-time Taqman reverse transcription-PCR (RT-PCR) assay for the quantitative detection of SARS-CoV. The sequences of the Taqman probes with a minor groove binder and the corresponding primers were based on the sequences of the N gene, open reading frame (ORF) 3, and ORF 8. The overall linear range of this assay was from at least 10 1 to 10 6 copies per reaction, and the detection limit could reach less than 10 copies per reaction. The quantification results for SARS-CoV from cell culture correlated well with those of the RT-PCR by using any two of the three sets of primer and probe used in this assay. However, the results of quantification of SARS-CoV obtained by using a few available throat swab specimens from SARS patients and the N gene as the target were almost 10 times higher than those obtained by using ORF 3 and ORF 8. Using this assay, we also detected an apparently SARS-CoV-related coronavirus in the throat swab specimens from masked palm civets in the west part of Hubei Province, People's Republic of China.

Adenovirus (Ad) assembly has been studied by using pulse-chase kinetic analyses, through the characterization of temperature-sensitive virus mutants blocked at different stages of assembly at the restrictive temperature, by studies of virus mutants defective in the expression of proteins involved in the packaging process, and by studies of mutants defective in the packaging sequences (2-4, 6-8, 12, 25, 31, 32). The results suggest that adenovirus assembly follows an ordered series of assembly and processing events analogous to those found in the assembly of bacteriophages. Whether the viral genome is involved in nucleating the initiating steps of capsid morphogenesis or whether viral DNA is inserted into an empty, preformed capsid has not been resolved. It is clear, however, that the viral genome contains cis-acting sequences that mediate the packaging process (reviewed in reference 20). Adenovirus DNA packaging occurs in a polar manner from the left end of the genome (1, 28). The genomes of adenovirus type 3 (Ad3), Ad5, and Ad16, representatives of adenovirus subgroups B and C, harbor conserved packaging signals (6, 10, 19, 22). These conserved cis-acting packaging domains suggest similar mechanisms of selective and polar DNA packaging for all adenovirus subgroups. Ad5 DNA encapsidation is dependent on cis-acting sequences located between nucleotides (nt) 230 and 380 (Fig. ​(Fig.1A)1A) (6, 7, 13, 25). Seven repeated sequences (termed A-repeats due to their AT-rich content) that contribute to viral DNA packaging are located within this domain. Although A-repeats are functionally redundant, they follow a hierarchy of importance: A1, A2, A5, and A6 are the most important repeats for packaging activity (6, 7, 25). A-repeats contain a bipartite consensus motif (5′-TTTG N8 CG-3′ [Fig. ​[Fig.1A]).1A]). Both the first and the second half-site of the consensus motif, as well as the 8-bp spacing between the half-sites, are critical for viral DNA packaging (25). Several lines of evidence suggest that a limiting trans-acting factor(s), which binds to the viral packaging sequences, plays a role in the packaging process (7, 25, 26). First, in vivo coinfection experiments show that viruses with a greater number of packaging repeats package more efficiently than viruses with fewer packaging repeats. Second, an isolated packaging domain on a multicopy plasmid represses packaging of a wild-type virus in vivo. Both results suggest competition for a limiting packaging factor(s) in vivo. Multimerized individual packaging repeats, termed minimal packaging domains, have been used to study Ad DNA packaging (5, 26). Minimal packaging sequences were shown to support packaging in vivo to various degrees when built into viruses lacking the authentic packaging domain. When minimal packaging sequences were used in binding assays in vitro as probes, three cellular transcription factors that bind to these sites were identified: chicken ovalbumin upstream promoter transcription factor (COUP-TF), OCT-1, and CCAAT displacement protein (CDP) (5, 26). It is very unlikely that COUP-TF and OCT-1 are required for Ad packaging, since viruses with mutated or synthetic packaging domains were identified that functioned efficiently in packaging in vivo but did not bind these transcription factors in vitro. However, a role for CDP in Ad packaging was suggested in earlier studies (5, 26). FIG. 1. Ad5 packaging sequences. (A) Schematic representation of the left end of the Ad5 genome. Nucleotide coordinates, relative to the left terminus, are indicated, and the inverted terminal repeat (ITR) is represented by a gray box. A-repeats 1 to 7 are represented ... Two viral proteins, L1 52/55K and IVa2, have been found to play important roles in Ad packaging and virus assembly (8, 12, 31, 32), although their exact roles in this process remain unclear. A null mutant and a temperature-sensitive mutant of the L1 52/55K protein, respectively, form empty capsids or capsids that contain only the left end of the viral genome (8, 12). The Ad L1 52/55K protein is found within immature virus particles (8, 12) and forms a physical complex with the Ad IVa2 protein (9). In turn, the Ad IVa2 protein is essential for virus assembly and the formation of empty viral capsids. Ad IVa2 is found in both empty and mature virus particles (31, 32). The IVa2 protein binds to packaging A-repeats 1 and 2 as well as to A-repeats 4 and 5 in vitro (30). Collectively, these data demonstrate that the Ad L1 52/55K and IVa2 proteins play a key role(s) in the very early stages of the virus assembly process. IVa2 appears to be a multifunctional protein during Ad infection. In addition to its role in viral assembly, IVa2 has been implicated in the transcriptional control of the major late promoter (MLP) (29) by binding to the DE elements within the MLP (17). In this study, we have examined the binding of the Ad IVa2 and L1 52/55K proteins to wild-type and mutant packaging sequences in vitro and in vivo in comparison to the growth properties of corresponding mutant viruses. Our results demonstrate that the binding of IVa2 to the packaging sequences in vitro correlates directly with the packaging efficiency of the virus in vivo. Furthermore, it appears that the higher-order IVa2-containing complexes that form on adjacent packaging A-repeats in vitro are the complexes required for the packaging activity of these sites in vivo. Further, both the IVa2 and L1 52/55K proteins associate with the packaging domain in vivo in direct correlation with packaging function. Finally, our results indicate that the cellular protein CDP does not play a role in packaging. These data are consistent with the known role of the IVa2 and L1 52/55K proteins in the Ad assembly process, and they provide strong evidence for a causal link between the binding of these proteins to packaging sequences and the assembly of virus particles.

The TiO2 photoassisted degradation of pesticide pyridaben has been examined in acetonitrile/water suspensions under UV light irradiation mainly at wavelength longer than 360 nm. To distinguish from the accompanying photolytic phenomenon, the kinetics of direct photolytic and photocatalytic degradation of pyridaben in TiO2 suspensions were studied by two different UV wavelength irradiations (λ ≥ 300 nm and λ ≥ 360 nm). The results manifested that the photolysis or photocatalysis of pyridaben followed pseudo-first-order kinetics, the direct photolysis was dominant at λ ≥ 300 nm, and the higher photocatalytic efficiency was obtained at λ ≥ 360 nm. The photocatalytic degradation kinetics were studied under different conditions such as water content, pH, catalyst concentration as well as radiant flux, and the degradation rates were found to be strongly influenced by these parameters. A qualitative study of the degradation products generated during the process was performed by GC–MS. Up to fifteen compounds were detected as degradation intermediates, many of which were identical with those detected previously at λ ≥ 300 nm. We also analyzed the evolution of the degradation products semiquantitatively by plotting areas of the corresponding GC peaks as functions of irradiation time. Moreover, a complementary study using molecular model calculation was performed to forecast pyridaben's adsorption point on TiO2 particles and its weak position of molecular cleavage. On the basis of the analytical and kinetic results, a degradation mechanism was proposed. This work is significant to understand the photochemistry of pesticide pyridaben in the environmental treatment.

A human group B rotavirus strain WH-1 was detected in an adult sporadic case of diarrhea in Wuhan, China in 2002. In this study, the gene sequences of WH-1 were determined in order to examine the phylogenetic relatedness to other human group B rotaviruses found previously in China (ADRV, in 1982), India (CAL-1, in 1998), and Bangladesh (Bang373, in 2000), as well as animal viruses, and to estimate the mutation rate of group B rotavirus. VP7 (major outer capsid protein) gene of WH-1 showed extremely high sequence identity (98.6%) to ADRV and showed relatively high sequence identities to CAL-1 (92.5%) and Bang373 (92.4%). In contrast, identities to animal (bovine and murine) group B rotaviruses were considerably lower (61-64%). Other gene segments of WH-1 encoding VP2, VP4, VP6, NSP1-NSP3, and NSP5 also showed high sequence identities to ADRV genes (98-99%), which were generally higher than those to CAL-1 genes and Bang373 genes (90-95%). However, amino acid sequence identities between WH-1 and ADRV were almost the same (VP2, VP6, and NSP3), or lower (NSP2) than those between WH-1 and CAL-1 (or Bang373). Since rates of synonymous substitution and transition between WH-1 and ADRV were similar for all the segments analyzed, genetic evolution was considered to have occurred neutrally and at a similar speed in most of the RNA segments. Based on the sequence divergence between WH-1 and ADRV, the mutation rate in natural condition of human group B rotavirus was estimated as 7.9 x 10(-4) substitution/site per year. The frequency of synonymous substitution between ADRV and Bang373 was 5.7 times higher than that between ADRV and WH-1, suggesting that the group B rotaviruses of Indian-Bangladeshi lineage diverged from that of Chinese lineage several decades ago.

Tissue factor (TF) binds the zymogen (VII) and activated (VIIa) forms of coagulation factor VII with high affinity. The structure determined for the sTF-VIIa complex [Banner, D. W., et al. (1996) Nature 380, 41-46] shows that all four domains of VIIa (Gla, EGF-1, EGF-2, and protease) are in contact with TF. Although a structure is not available for the TF-VII complex, the structure determined for free VII [Eigenbrot, C., et al. (2001) Structure 9, 675-682] suggests a significant conformational change for the zymogen to enzyme transition. In particular, the region of the protease domain that must contact TF has a conformation that is altered from that of VIIa, suggesting that the VII protease domain interacts with TF in a manner different from that of VIIa. To test this hypothesis, a panel of 12 single-site sTF mutants, having substitutions of residues observed to contact the proteolytic domain of VIIa, have been evaluated for binding to both zymogen VII and VIIa. Affinities were determined by surface plasmon resonance measurements using a noninterfering anti-TF monoclonal antibody to capture TF on the sensor chip surface. Dissociation constants (K(D)) measured for binding to wild-type sTF are 7.5 +/- 2.4 nM for VII and 5.1 +/- 2.3 nM for VIIa. All of the sTF mutants except S39A and E95A exhibited a significant decrease (2-fold) in affinity for VIIa. The changes in affinity measured for VII or VIIa binding with substitution in sTF were comparable in magnitude. We conclude that the proteolytic domain of both VII and VIIa interacts with this region of sTF in a nearly identical fashion. Therefore, zymogen VII can readily adopt a VIIa-like conformation required for binding to TF.

Adenovirus (Ad) type 5 DNA packaging is initiated in a polar fashion from the left end of the genome. The packaging process is dependent upon the cis -acting packaging domain located between nucleotides 194 and 380. Seven A/T-rich repeats have been identified within this domain that direct packaging. A1, A2, A5, and A6 are the most important repeats functionally and share a bipartite sequence motif. Several lines of evidence suggest that there is a limiting trans -acting factor(s) that plays a role in packaging. Two cellular activities that bind to minimal packaging domains in vitro have been previously identified. These binding activities are P complex, an uncharacterized protein(s), and chicken ovalbumin upstream promoter transcription factor (COUP-TF). In this work, we report that a third cellular protein, octamer-1 protein (Oct-1), binds to minimal packaging domains. In vitro binding analyses and in vivo packaging assays were used to examine the relevance of these DNA binding activities to Ad DNA packaging. The results of these experiments reveal that COUP-TF and Oct-1 binding does not play a functional role in Ad packaging, whereas P-complex binding directly correlates with packaging function. We demonstrate that P complex contains the cellular protein CCAAT displacement protein (CDP) and that full-length CDP is found in purified virus particles. In addition to cellular factors, previous evidence indicates that viral factors play a role in the initiation of viral DNA packaging. We propose that CDP, in conjunction with one or more viral proteins, binds to the packaging sequences of Ad to initiate the encapsidation process.

The transition from the so-called matrix veins to persistent slip bands (PSBs) in a fatigued copper single crystal was observed by electron channeling contrast (ECC) technique in a scanning electron microscope (SEM). In the four stages of PSBs formation, the different dislocation patterns can be obtained by the experiment, namely, the veins, persistent slip lines (PSLs), embryos and well-developed PSBs dislocation structures. In the consecutive four stages the typical dislocation structures were simulated and the internal stresses were calculated by the three-dimensional discrete dislocation method, correspondingly, the external stresses were calculated by the finite element method (FEM). The simulation shows that in the dislocation dense regions (veins and walls) the stress distributions are relatively more concentrated than that in the dislocation poor regions (channels and within a PSL). At the tips of the PSLs and embryos, the internal stresses were not substantially changed; however, at the tips of the PSLs and embryos the external stresses were much higher than that at the other regions. This will drive the PSLs and embryos to grow.

The effect of a caspase-3 inhibitor on retinal degeneration in C3H mice carrying the rd gene, a mutation of a rod-specific phosphodiesterase, was investigated.A quantity of 2 mg/kg of Ac-DEVD-CHO, as inhibitor, was injected intraperitoneally every other day from 8 days of age, and retinal damage was compared with that in saline-treated C3H mice at 13 days (1 day after the third treatment) and 17 days of age (1 day after the fifth treatment). Retina of ICR mice not carrying rd gene was also evaluated under the same protocol. The efficacy of Ac-DEVD-CHO was evaluated based on total retinal thickness and outer retinal thickness (thickness of outer nuclear layer and photoreceptor layer). An apoptotic index and a cell proliferation index for the photoreceptor cells, at 13 days of age, were calculated based on terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-digoxigenin nick-end labeling (TUNEL) and proliferating cell nuclear antigen (PCNA) labeling, respectively.At 13 days of age, total and outer retinal thickness in saline-treated C3H mice were 140.3 microm and 37.5 microm, compared with 160.4 microm and 49.5 microm, respectively, in Ac-DEVD-CHO-treated C3H mice ( P0.01, respectively). In ICR mice, total and outer retinal thickness were 182.1 microm and 90.9 microm, respectively, in saline-treated mice and 183.8 microm and 89.6 microm in Ac-DEVD-CHO-treated mice (not significant). At this time, the TUNEL index was 23.52 cells/10(4) microm (2) of outer nuclear layer in saline-treated C3H mice; Ac-DEVD-CHO treatment significantly reduced this value to 18.73 cells/10(4) microm(2) ( P0.05). The TUNEL index in saline- and Ac-DEVD-CHO-treated ICR mice was 0.59 cells/10(4) microm(2) and 0.80 cells/10(4) microm(2), respectively (not significant); Ac-DEVD-CHO treatment had no influence on normally developing retina. The PCNA index was not affected by Ac-DEVD-CHO-treatment. However, at 17 days of age, Ac-DEVD-CHO treatment did not ameliorate retinal degeneration.The caspase-3 inhibitor was transiently effective in delaying retinal degeneration through inhibition of the apoptosis of photoreceptor cells in rd gene-carrying mice. The use of caspase-3 inhibitors may have therapeutic applications in the treatment of human retinal degeneration.

SummaryComplexation of factor VIIa (FVIIa) and tissue factor (TF) initiates the extrinsic pathway of blood coagulation. Inappropriate triggering of this pathway has been linked to thrombotic disorders. We have previously shown that a mutant form of soluble tissue factor (sTF, residues 1-219) having Lys165 and Lys166 replaced with alanine residues (hTFAA) gave an antithrombotic effect in a rabbit model of arterial thrombosis. Here we have improved the potency of hTFAA by incorporating amino acid substitutions that increase the affinity of sTF for FVIIa. Phage display has been used to select consensus sequences at two FVIIa-contact regions on sTF, segments 44–50 and 130–140, that individually showed modestly increased (∼2-fold) FVIIa-affinity. These consensus sequences have been combined with other previously selected mutations to generate a series of variants (hTFAA-2, hTFAA-3, and hTFAA-4) having 4 to 10-fold increased FVIIa-binding affinity. Surface plasmon resonance measurements indicate that the increased affinity results mostly from an increase in the association rate constant. All of these variants displayed increased inhibitor potency relative to hTFAA in an assay of Factor X activation catalyzed by the complex of FVIIa with membrane TF. In addition, these affinity improved hTFAA variants are more potent anticoagulants and have increased antithrombotic activity relative to hTFAA in an ex vivo flow chamber model of thrombosis. The potency trend parallels increases in FVIIa-binding affinity; however, the absolute fold increases in potencies are greater than increases in binding affinity, consistent with kinetic studies of the FVIIa-binding event. Through incorporation of 10 amino acid substitutions (hTFAA-3) we have increased the antithrombotic activity of hTFAA by 20-fold.

The high incidence of complex diseases has become a worldwide threat to human health. Multiple targets and pathways are perturbed during the pathological process of complex diseases. Systematic investigation of complex relationship between drugs and diseases is necessary for new association discovery and drug repurposing. For this purpose, three causal networks were constructed herein for cardiovascular diseases, diabetes mellitus, and neoplasms, respectively. A causal inference-probabilistic matrix factorization (CI-PMF) approach was proposed to predict and classify drug-disease associations, and further used for drug-repositioning predictions. First, multilevel systematic relations between drugs and diseases were integrated from heterogeneous databases to construct causal networks connecting drug-target-pathway-gene-disease. Then, the association scores between drugs and diseases were assessed by evaluating a drug's effects on multiple targets and pathways. Furthermore, PMF models were learned based on known interactions, and associations were then classified into three types by trained models. Finally, therapeutic associations were predicted based upon the ranking of association scores and predicted association types. In terms of drug-disease association prediction, modified causal inference included in CI-PMF outperformed existing causal inference with a higher AUC (area under receiver operating characteristic curve) score and greater precision. Moreover, CI-PMF performed better than single modified causal inference in predicting therapeutic drug-disease associations. In the top 30% of predicted associations, 58.6% (136/232), 50.8% (31/61), and 39.8% (140/352) hit known therapeutic associations, while precisions obtained by the latter were only 10.2% (231/2264), 8.8% (36/411), and 9.7% (189/1948). Clinical verifications were further conducted for the top 100 newly predicted therapeutic associations. As a result, 21, 12, and 32 associations have been studied and many treatment effects of drugs on diseases were investigated for cardiovascular diseases, diabetes mellitus, and neoplasms, respectively. Related chains in causal networks were extracted for these 65 clinical-verified associations, and we further illustrated the therapeutic role of etodolac in breast cancer by inferred chains. Overall, CI-PMF is a useful approach for associating drugs with complex diseases and provides potential values for drug repositioning.

A disease molecular signature is a set of biomolecular features that are prognostic of clinical phenotypes and indicative of underlying pathology. It is of great importance to develop computational approaches for finding more relevant molecular signatures. Based upon the hypothesis that various components in a molecular signature are more likely to share similar patterns, we introduced a novel three step network based approach (TSNBA) to identify the molecular signature and key pathological regulators. Protein-protein interaction (PPI) network and ranking algorithm were integrated in the first step to find pathology related proteins with high accuracy. It was followed by the second step to further screen with co-expression patterns for better pathology enrichment. Context likelihood of relatedness (CLR) algorithm was used in the third step to infer gene regulatory networks and identify key transcription regulators. We applied this approach to study IL-1 (interleukin-1) and TNF-alpha (tumor necrosis factor-alpha) stimulated inflammation. TSNBA identified inflammatory signature with high accuracy and outperformed 5 competing methods namely fold change, degree, interconnectivity, neighborhood score and network propagation based approaches. The best molecular signature, with 80% (40/50) confirmed inflammatory genes, was used to predict inflammation related genes. As a result, 8 out of 10 predicted inflammation genes that were not included in the benchmark Entrez Gene database were validated by literature evidence. Furthermore, 23 of the 32 predicted inflammation regulators were validated by literature evidence. The rest 9 were also validated with TF (transcription factor) binding site analysis. In conclusion, we developed an efficient strategy for disease molecular signature finding and key pathological regulator identification.

Antibody-dependent cell-mediated cytotoxicity (ADCC) is an important mechanism of action implicated in the clinical efficacy of several therapeutic antibodies. In vitro ADCC assays employing effector cells capable of inducing lysis of target cells bound by antibodies are routinely performed to support the research and development of therapeutic antibodies. ADCC assays are commonly performed using peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells or engineered cell lines as effector cells. In this study we evaluated the impact of different effector cell types including primary PBMCs, primary NK cells, engineered NK cell lines, and an engineered reporter cell line, on the in vitro ADCC activity of two glycoforms of a humanized IgG1 antibody. The results of this study show the differential effects on both the efficacy and potency of the antibodies by different effector cells and the finding that both the allotype and the expression level of CD16a affect the potency of effector cells in ADCC assays. Our results also show that engineered NK or reporter cell lines provide reduced variability compared to primary effector cells for in vitro ADCC assays.

Serum response factor (SRF), a member of the MCM1, agamous, deficiens, SRF (MADS) family of transcriptional activators, has been implicated in the transcriptional control of a number of cardiac muscle genes, including cardiac alpha-actin, skeletal alpha-actin, alpha-myosin heavy chain (alpha-MHC), and beta-MHC. To better understand the in vivo role of SRF in regulating genes responsible for maintenance of cardiac function, we sought to test the hypothesis that increased cardiac-specific SRF expression might be associated with altered cardiac morphology and function. We generated transgenic mice with cardiac-specific overexpression of the human SRF gene. The transgenic mice developed cardiomyopathy and exhibited increased heart weight-to-body weight ratio, increased heart weight, and four-chamber dilation. Histological examination revealed cardiomyocyte hypertrophy, collagen deposition, and interstitial fibrosis. SRF overexpression altered the expression of SRF-regulated genes and resulted in cardiac muscle dysfunction. Our results demonstrate that sustained overexpression of SRF, in the absence of other stimuli, is sufficient to induce cardiac change and suggest that SRF is likely to be one of the downstream effectors of the signaling pathways involved in mediating cardiac hypertrophy.

During cyclic deformation, the early stages of dislocation pattern formation and evolution of a single-slip-oriented copper single crystal were simulated by using the method of discrete dislocation dynamics and observed by using the electron channeling contrast (ECC) technique in a scanning electron microscopy (SEM). Computer simulation results agree rather well with the experimental observations. Hence, some new mechanisms were proposed. Such as in the early stages of cyclic deformation, the formation and evolution of dislocation veins could originate from the matrix walls which are composed by the same sign of dislocations, and a dipole matrix wall could be formed by combining two matrix walls with the opposite signs of dislocations. (C) 2001 Elsevier Science B.V. All rights reserved.

It is well established that cardiac remodeling plays a pivotal role in the development of heart failure, a leading cause of death worldwide. Meanwhile, sympathetic hyperactivity is an important factor in inducing cardiac remodeling. Therefore, an in-depth understanding of beta-adrenoceptor signaling pathways would help to find better ways to reverse the adverse remodeling. Here, we reviewed five pathways, namely mitogen-activated protein kinase signaling, Gs-AC-cAMP signaling, Ca(2+)-calcineurin-NFAT/CaMKII-HDACs signaling, PI3K signaling and beta-3 adrenergic signaling, in cardiac remodeling. Furthermore, we constructed a cardiac-remodeling-specific regulatory network including miRNA, transcription factors and target genes within the five pathways. Both experimental and clinical studies have documented beneficial effects of beta blockers in cardiac remodeling; nevertheless, different blockers show different extent of therapeutic effect. Exploration of the underlying mechanisms could help developing more effective drugs. Current evidence of treatment effect of beta blockers in remodeling was also reviewed based upon information from experimental data and clinical trials. We further discussed the mechanism of how beta blockers work and why some beta blockers are more potent than others in treating cardiac remodeling within the framework of cardiac remodeling network.

The effect of a caspase-3 inhibitor on N -methyl- N -nitrosourea (MNU)-induced retinal degeneration was investigated. Sixty mg kg(-1)MNU was given intraperitoneally to 50 day old female Sprague-Dawley rats, and 4000 ng Ac-DEVD-CHO, a caspase-3 inhibitor, was injected intravitreally twice at 0 and 10 hr after MNU. In both peripheral and central retina, an apoptotic index of the photoreceptor cells 24 hr after MNU treatment was calculated by TUNEL labeling, and retinal damage 7 days after MNU treatment was evaluated from retinal thickness and a retinal damage ratio (length of damaged retina : whole retinal length). In MNU-treated rats, the TUNEL index 24 hr post-MNU was 79.5% in the peripheral and 83.7% in the central retina, while the Ac-DEVD-CHO injection significantly reduced it to 59.7 and 71.8%, respectively. Total retinal thickness 7 days after MNU was 38 microm in the peripheral and 75 microm in the central retina. Ac-DEVD-CHO injection increased these values to 72 and 77 microm, respectively. The retinal damage ratio 7 days after MNU was 98.5%. Ac-DEVD-CHO injection significantly reduced this value to 54.4%. The use of a caspase-3 inhibitor was effective in the suppression of MNU-induced retinal apoptosis and may be a therapeutic intervention in human retinitis pigmentosa.

Cataract was induced by a single intraperitoneal injection of 100 mg/kg N-methyl- N-nitrosourea (MNU) to 0-, 5-, 10-, 15-, or 20-day-old male and female Sprague-Dawley rats. In day 0, 5, 10, and 15 MNU-treated rats, mature cataracts were constantly seen 7, 14, 14, and 30 days after dosing, respectively. In the day 20 MNU-treated rats, only subcapsular cataract was seen 30 days after dosing. Therefore, the rats exposed to MNU at an earlier age caused cataract more rapidly and severely. In the day 0 MNU-treated rats, 7-methyldeoxyguanosine DNA adduct was detected in the lens epithelial nuclei 12 hours after MNU dosing, followed by apoptosis, which was confirmed by morphology, by TUNEL signals, and by DNA ladder and peaked 3 days after MNU dosing. In the apoptosis cascade, upregulation of Bax, downregulation of Bcl-2, and increased CPP32 protease (caspase-3) activity were seen 12 hours after MNU dosing. Therefore, the pathogenesis of MNU-induced cataract was associated with DNA adduct formation in the lens epithelial cell nuclei leading to apoptosis by upregulation of Bax protein, downmodulation of Bcl-2 protein, and activation of caspase-3.

Alopecia was induced in male and female neonatal C57BL mice by a single intraperitoneal injection of 60 mg/kg N-methyl-N-nitrosourea (MNU). MNU administration was most effective in the 8-day-old mice and less effective in the 5-day-old mice (at active and early anagen stages of the first hair cycle, respectively). No alopecia was seen in the day 14 MNU-treated animals (at telogen stage of the first hair cycle). MNU effectively induced hair follicular cell apoptosis at the anagen stage by up-regulation of Bax protein without down-modulation of Bcl-2 protein. In day 8 MNU-treated mice, the immunosuppressive agent 0.01% tacrolimus hydrate (FK506), when topically applied for 5 days from 1 day after MNU treatment (before the occurrence of alopecia), decreased the severity of alopecia. However, it did not stimulate hair growth when applied for 5 days from 20 days of age (after occurrence of alopecia).

6 páginas, 2 figuras., [Background]: Remodeling of the extracellular matrix plays an important role during the pathogenesis of atherosclerosis and restenosis. The matrix glycoprotein thrombospondin-1 (TSP1) inhibits endothelial cell proliferation and migration in vitro. In contrast, TSP1 facilitates the growth and migration of cultured vascular smooth muscle cells. Accordingly, we investigated the hypothesis that administration of anti-TSP1 antibody could facilitate reendothelialization and inhibit neointimal thickening in balloon-injured rat carotid artery. [Methods and Results]: Sprague-Dawley rats were subjected to left common carotid artery denudation, after which arteries were treated with C6.7 anti-TSP1 or control antibody. Evans blue dye staining 2 weeks after injury disclosed significantly increased reendothelialization in arteries treated with C6.7 antibody compared with the control group, and this effect was associated with increased number of proliferating cell nuclear antigen–positive endothelial cells. In contrast, treatment with C6.7 antibody decreased the number of proliferating cell nuclear antigen–positive vascular smooth muscle cells in the injured arterial wall. Neointimal thickening was correspondingly attenuated to a statistically significant degree in arteries receiving C6.7 antibody versus the control group at both the 2-week and 4-week time points. [Conclusions]: Intra-arterial delivery of antibody against TSP1 facilitated reendothelialization and reduced neointimal lesion formation after balloon denudation., This study was supported by National Institutes of Health grants HL-57519 and AG-15227 (V.A.); HL-40518, HL-57516, and HL-53354 (J.M.I.); and CA-65872 (W.A.F.).

Little information is available on the stability of three-center hydrogen bonds relative to two-center hydrogen bonds. In an XHX three-center hydrogen bond, two donors interact with one acceptor; here, we compare an HXH interaction with the component two-center hydrogen bonds (H−X and X−H) in a small depsipeptide, 1. The results show that there is negative cooperativity between the two components of the HXH interaction.

Rotavirus Group A G9, China, Of 322 stool specimens collected from children with diarrhea from October 2005 through September 2006 in Wuhan, China, group A rotavirus was identified in 101 (31.4%). The most prevalent group A rotavirus genotype was G3P[8] (62.6%), followed by G1P[8](17.6%), G1+G3P[8](8.8%), G3P[4](6.6%), G1P[4](2.2%), and G9P[8](2.2%). The G9 strains were first detected in Wuhan.

The morphologic response of the rat retina at different ages to the various doses of the alkylating agent N-methyl-N-nitrosourea (MNU) was examined. MNU at the dose of 35, 50, 75, 90, and/or 105 mg/kg was injected intraperitoneally (ip) to male and female Sprague-Dawley and Lewis rats at 0, 21, 50, and 150 days of age, and the retinas were examined 7 or 21 days after the treatment. In both strains of rats, regardless of gender, MNU evoked age-specific and dose-dependent retinal lesions. When ≥ 50 mg/kg MNU were administered at day 0, retinal dysplasia characterized by rosette formation was found in the outer neuroblastic/nuclear layer above the continuous lining single layer of pigment epithelial cells. The severity of the lesion was not dose-related. When > 90 mg/kg, > 50 mg/kg and ≥ 35 mg/kg MNU were administered at day 21, 50, and 150, respectively, retinal degeneration characterized by selective photoreceptor cell loss due to apoptosis with disorganized pigment epithelial cells was found in all rats. Thus, the sensitivity of photoreceptor cells in the development of retinal degeneration induced by MNU increased in parallel with aging.

Coronary heart disease (CHD), the leading cause of global morbidity and mortality in adults, has been reported to be associated with hundreds of genes. A comprehensive understanding of the CHD-related genes and their corresponding interactions is essential to advance the translational research on CHD. Accordingly, we construct this knowledgebase, CHD@ZJU, which records CHD-related information (genes, pathways, drugs and references) collected from different resources and through text-mining method followed by manual confirmation. In current release, CHD@ZJU contains 660 CHD-related genes, 45 common pathways and 1405 drugs accompanied with >8000 supporting references. Almost half of the genes collected in CHD@ZJU were novel to other publicly available CHD databases. Additionally, CHD@ZJU incorporated the protein–protein interactions to investigate the cross-talk within the pathways from a multi-layer network view. These functions offered by CHD@ZJU would allow researchers to dissect the molecular mechanism of CHD in a systematic manner and therefore facilitate the research on CHD-related multi-target therapeutic discovery. Database URL: http://tcm.zju.edu.cn/chd/

Pharmacokinetic (PK) and immunohistochemistry (IHC) assays are essential to the evaluation of the safety and efficacy of therapeutic monoclonal antibodies (mAb) during drug development. These methods require reagents with a high degree of specificity because low concentrations of therapeutic antibody need to be detected in samples containing high concentrations of endogenous human immunoglobulins. Current assay reagent generation practices are labor-intensive and time-consuming. Moreover, these practices are molecule-specific and so only support one assay for one program at a time. Here, we describe a strategy to generate a unique assay reagent, 10C4, that preferentially recognizes a panel of recombinant human mAbs over endogenous human immunoglobulins. This “panel-specific” feature enables the reagent to be used in PK and IHC assays for multiple structurally-related therapeutic mAbs. Characterization revealed that the 10C4 epitope is conformational, extensive and mainly composed of non-CDR residues. Most key contact residues were conserved among structurally-related therapeutic mAbs, but the combination of these residues exists at low prevalence in endogenous human immunoglobulins. Interestingly, an indirect contact residue on the heavy chain of the therapeutic appears to play a critical role in determining whether or not it can bind to 10C4, but has no affect on target binding. This may allow us to improve the binding of therapeutic mAbs to 10C4 for assay development in the future. Here, for the first time, we present a strategy to develop a panel-specific reagent that can expedite the development of multiple clinical assays for structurally-related therapeutic mAbs.

Chinese medicine has evolved from thousands of years of empirical applications and experiences of combating diseases. It has become widely recognized that the Chinese medicine acts through complex mechanisms featured as multicompound, multitarget and multipathway. However, there is still a lack of systematic experimental studies to elucidate the mechanisms of Chinese medicine. In this study, the differentially expressed genes (DEGs) were identified from myocardial infarction rat model treated with Xuesaitong Injection (XST), a Chinese medicine consisting of the total saponins fromPanax notoginseng(Burk.) F. H. Chen (Chinese Sanqi). A network-based approach was developed to combine DEGs related to cardiovascular diseases (CVD) with lines of evidence from the literature mining to investigate the mechanism of action (MOA) of XST on antimyocardial infarction. A compound-target-pathway network of XST was constructed by connecting compounds to DEGs validated with literature lines of evidence and the pathways that are functionally enriched. Seventy potential targets of XST were identified in this study, of which 32 were experimentally validated either by ourin vitroassays or by CVD-related literatures. This study provided for the first time a network view on the complex MOA of antimyocardial infarction through multiple targets and pathways.

Continuous improvement in bioanalytical method development is desired in order to ensure the quality of the data and to better support pharmacokinetic (PK) and safety studies of biotherapeutics. One area that has been getting increasing attention recently is in the assessment of “free” and “total” analyte and the impact of the assay format on those assessments. To compliment these considerations, the authors provide a critical review of available literature and prospectively explore methods to mitigate the potential impact of anti-drug antibody on PK assay measurement. This challenge is of particular interest and importance since biotherapeutic drugs often elicit an immune response, and thus may have a direct impact on quantification of the drug for its PK and safety evaluations.

Type 2 diabetes mellitus (T2D), affecting >90% of the diabetic patients, is one of the major threats to human health. A comprehensive understanding of the mechanisms of T2D at molecular level is essential to facilitate the related translational research. Here, we introduce a comprehensive and up-to-date knowledgebase for T2D, i.e. T2D@ZJU. T2D@ZJU contains three levels of heterogeneous connections associated with T2D, which is retrieved from pathway databases, protein–protein interaction databases and literature, respectively. In current release, T2D@ZJU contains 1078 T2D related entities such as proteins, protein complexes, drugs and others together with their corresponding relationships, which include 3069 manually curated connections, 14 893 protein–protein interactions and 26 716 relationships identified by text-mining technology. Moreover, T2D@ZJU provides a user-friendly web interface for users to browse and search data. A Cytoscape Web-based interactive network browser is available to visualize the corresponding network relationships between T2D-related entities. The functionality of T2D@ZJU is shown by means of several case studies. Database URL: http://tcm.zju.edu.cn/t2d

Threshold selection is a critical step in using binary change detection methods. The threshold determines the accuracy of change detection results but is highly subjective and scene-dependent, depending on the familiarity with the study area and the analyst’s skill. Nearest neighbor classification is a non-parametric classifier, which was applied to remove the threshold. In order to find the most suitable feature to detect construction and farmland changes, a variety of single and multiple variables were explored. They were regional similarity (RSIM), brightness difference images (BDIs), multi-band difference images (MDIs), multi-band ratio difference images (MRDIs), a combination of RSIM and BDIs (RSIMBD), a combination of RSIM and a optimum band difference and a optimum band ratio difference (RSIMDR), MDIs and MRDIs multiple variable groups. All were tested for two study sites of the bi-temporal SPOT 5 imagery, the results indicated that RSIM, RSIMDR, RSIMBD were significantly better than other single and multiple variables.

Cell kinetics (cell proliferation and cell death) and the expression of progesterone receptor (PgR) during growth and regression of pregnancy-dependent mammary tumors (PDMT) of female GRS/A mice were investigated on the histologic level. Cell proliferation was determined using monoclonal anti-proliferating cell nuclear antigen (PCNA) antibody (PC10) and cell death was detected using the TUNEL method. PgR expression was determined using monoclonal anti-PgR antibody (10A9). In growing PDMT (type P tumor) obtained during days 16-20 of pregnancy, numerous PCNA labeling was observed in both the epithelial and mesenchymal cells, whereas PgR was found only in epithelial cells and no TUNEL signal was detected. In regressing PDMT obtained during days 0-5 of lactation, the level of PCNA labeling was low and the PgR-positive cells were preferentially labeled by the TUNEL staining, which led to microcyst formation (cystic degeneration). Pale cell carcinoma was shown to be pregnancy-dependent, since the tumor cells were universally PgR-positive, and TUNEL-positive signal with low PCNA-labeling was detected after the delivery.

TonB is known to be a bacterial periplasmic protein that transduces proton from the inner membrane to the outer membrane receptor in complex with the ExbB and ExbD proteins. Actinobacillus pleuropneumoniae TonB2 protein is the second TonB protein that is important for iron acquisition and virulence. The TonB2 protein was verified to be immunogenic and could afford partial protection for animals from lethal infection. In the present study, the recombinant TonB2 (rTonB2) was overexpressed in Escherichia coli BL21(DE3) and purified. The rTonB2 was then used as antigen to immunize BALB/c mice for the production of monoclonal antibodies (MAb). Four clones of TonB2-specific MAb secretion hybridomas--2F2, 2G8, 3D2, and 6F10--were selected. The MAbs 2F2, 3D2, and 6F10 were classified as IgG1 isotype and 2G8 was of IgG2a isotype. Western blot and ELISA results indicated that MAbs had specific binding activity to rTonB2. The MAbs generated here will be used for further functional analyses of the TonB2 protein.

The G10P[15] rotavirus CC0812-1 isolated from a diarrheal woman in Wuhan, China, in 2008 is phylogenetically close to the Lanzhou lamb rotavirus (LLR) of a monovalent human rotavirus vaccine produced by the Lanzhou Institute of Biological Products, China, and rotavirus Lamb-NT. This rotavirus can be used as the backbone of the attenuated rotavirus reassortant as a rotavirus vaccine candidate. In this study, rotavirus CC0812-1 was purified from the culture supernatant of CC0812-1-infected MA104 cells and used as antigen to immunize BALB/c mice. Four hybridoma clones were developed secreting antibodies that reacted with CC0812-1, designated as 1B1, 1B8, 1F11, and 1G10, respectively. Western blot analysis indicated that the four monoclonal antibodies (MAbs) were all specific for VP4 of rotavirus CC0812-1. Isotyping revealed that MAbs 1B1, 1B8, and 1G10 belonged to the IgM class, while MAb 1F11 belonged to the IgG1 subclass. A neutralization test demonstrated that the four MAbs all had the capacity to neutralize rotavirus CC0812-1. The neutralizing titers of the BALB/c mice ascites were 1:2048, 1:1024, 1:512, and 1:512 for MAbs 1B1, 1B8, 1F11, and 1G10, respectively.

Infrared heating was recently used to develop a more efficient roasting technology than traditional hot air roasting. Therefore, in this study, we evaluated the shelf-life of almonds roasted with three different approaches, namely infrared (IR), sequential infrared and hot air (SIRHA) and regular hot air (HA). Nine medium roasted almond samples produced by the aforementioned heating methods were processed at three different temperatures (130, 140 and 150 C), packed in paper bags and then stored at 37 C for three, six or eight months. Shelf-life of the roasted almonds was determined by measuring the changes in colour, peroxide value, moisture content, water activity, volatile components and sensory quality. No significant difference was observed in moisture content and water activity among the almond samples processed with different roasting methods and stored under the same conditions. GC/MS analysis showed that aldehydes, alcohols, and pyrazines were the main volatile components of almonds. Aliphatic alde- hydes such as hexanal, (E)-2-octenal, and nonanal were produced as off-odours during storage. Although the overall quality of roasted almonds produced with SIRHA and HA heating was similar during the first three months of storage, their peroxide value and concentration of aliphatic aldehydes differed signifi- cantly for different roasting methods and increased significantly in all roasted samples during storage. We postulate that hexanal and nonanal might be better indicators of the shelf life of roasted almonds than the current standard, peroxide value.

DNA ladder fragments, regarded as a biochemical hallmark of apoptosis, have been separated quickly and successfully by capillary electrophoresis. Inter-nucleosomal DNA fragmentations induced by xylitol selenite were determined for the first time, while hydroxypropylmethylcellulose (HPMC) was served as the sieving matrix in dynamic sieving capillary electrophoresis. The calibration curve (r(2) = 0.991) was established and multiples of two different nucleosomes (140 and 180 bp) were formed in the presence of xylitol selenite. Selenium compounds inhibited carcinogenesis in animal models, SMMC-7221 cells and several other cells by increasing apoptosis. The described method was useful in elucidating the anticancer activities of xylitol selenite and other selenium compounds, which was more effective to detect small fragments than slab gel electrophoresis.

The relation between quantified emphysema and measured lung content of coal and silica was investigated in the lungs of 264 deceased underground coalworkers who had been exposed to mixed coal and silica dust. Lung specimens obtained at postmortem and inflated and fixed under standard conditions were used to quantify the extent of emphysema and then to measure the amount of coal and silica present in the lungs at the time of death. These data were combined with clinical and other pathological information from the subjects. Multiple regression analysis showed that the extent of emphysema (E score) had a strong positive quantitative relation with coal content of the lungs (p < 0.0003), age (p < 0.0001), and smoking (p < 0.0001). There was a significant negative interaction of uncertain biological importance between coal content of the lungs and smoking (p < 0.004; E score = -1.79 + 0.62 coal + 0.06 age + 0.21 smoking -0.17 coal x smoking; adjusted R2 = 0.25). In lifelong non-smokers emphysema was particularly strongly related to coal content and age (coal: p < 0.001; age: p < 0.002; E score = -1.56 + 0.78 coal + 0.06 age; adjusted R2 = 0.66). The relation was basically unchanged by adding a lung silica content term. Emphysema score was highly negatively correlated with forced expiratory volume in one second (FEV1; % predicted, obtained within five years of death) (r = -0.44, p < 0.0001). Degree of lung fibrosis was highly positively associated with lung silica content (chi 2(1) = 12.9, p < 0.0003). These results provide strong evidence that emphysema in coalworkers is actually related to lung coal content. The role silica in development of emphysema, however remains unclear.

In this paper, a method of estimating the parameters of the model of the clutch driven disc detection system is proposed. After analyzing the parts of the clutch driven disc detection system, the simplified mathematical model is built. The model parameters were estimated by the Least Squares method. Based on the virtual instrument technology of NI company, the interface and data acquisition programs are developed. The simulation results and the experiments show that the characteristics of the identified model are similar to the characteristics of the open loop control system and the calculating speed is improved by using the complex method.

Numerous observations implicate interferon-α (IFNα) in the pathophysiology of systemic lupus erythematosus (SLE); however, the potential impact of endogenous anti-IFNα autoantibodies (AIAAs) on IFN-pathway and disease activity is unclear. The aim of this study was to characterize IFN-pathway activity and the serologic and clinical profiles of AIAA-positive patients with SLE.Sera obtained from patients with SLE (n = 49), patients with rheumatoid arthritis (n = 25), and healthy control subjects (n = 25) were examined for the presence of AIAAs, using a biosensor immunoassay. Serum type I IFN bioactivity and the ability of AIAA-positive sera to neutralize IFNα activity were determined using U937 cells. Levels of IFN-regulated gene expression in peripheral blood were determined by microarray, and serum levels of BAFF, IFN-inducible chemokines, and other autoantibodies were measured using immunoassays.AIAAs were detected in 27% of the serum samples from patients with SLE, using a biosensor immunoassay. Unsupervised hierarchical clustering analysis identified 2 subgroups of patients, IFN(low) and IFN(high) , that differed in the levels of serum type I IFN bioactivity, IFN-regulated gene expression, BAFF, anti-ribosomal P, and anti-chromatin autoantibodies, and in AIAA status. The majority of AIAA-positive patients had significantly lower levels of serum type I IFN bioactivity, reduced downstream IFN-pathway activity, and lower disease activity compared with the IFN(high) patients. AIAA-positive sera were able to effectively neutralize type I IFN activity in vitro.Patients with SLE commonly harbor AIAAs. AIAA-positive patients have lower levels of serum type I IFN bioactivity and evidence for reduced downstream IFN-pathway and disease activity. AIAAs may influence the clinical course in SLE by blunting the effects produced by IFNα.

The predominant driver of bioanalysis in supporting drug development is the intended use of the data. Ligand-binding assays (LBA) are widely used for the analysis of protein biotherapeutics and target ligands (L) to support pharmacokinetics/pharmacodynamics (PK/PD) and safety assessments. For monoclonal antibody drugs (mAb), in particular, which non-covalently bind to L, multiple forms of mAb and L can exist in vivo, including free mAb, free L, and mono- and/or bivalent complexes of mAb and L. Given the complexity of the dynamic binding equilibrium occurring in the body after dosing and multiple sources of perturbation of the equilibrium during bioanalysis, it is clear that ex vivo quantification of the forms of interest (free, bound, or total mAb and L) may differ from the actual ones in vivo. LBA reagents and assay formats can be designed in principle to measure the total or free forms of mAb and L. However, confirmation of the forms being measured under the specified conditions can be technically challenging. The assay forms and issues must be clearly communicated and understood appropriately by all stakeholders as the program proceeds through the development process. This paper focuses on monoclonal antibody biotherapeutics and their circulatory L that are either secreted as soluble forms or shed from membrane receptors. It presents an investigation into the theoretical and practical considerations for total/free analyte assessment to increase awareness in the scientific community and offer bioanalytical approaches to provide appropriate PK/PD information required at specific phases of drug development.

Antibody charge variants have gained considerable attention in the biotechnology industry due to their potential influence on stability and biological activity. Subtle differences in the relative proportions of charge variants are often observed during routine biomanufacture or process changes and pose a challenge to demonstrating product comparability. To gain further insights into the impact on biological activity and pharmacokinetics (PK) of monoclonal antibody (mAb) charge heterogeneity, we isolated the major charge forms of a recombinant humanized IgG1 and compared their in vitro properties and in vivo PK. The mAb starting material had a pI range of 8.7-9.1 and was composed of about 20% acidic variants, 12% basic variants, and 68% main peak. Cation exchange displacement chromatography was used to isolate the acidic, basic, and main peak fractions for animal studies. Detailed analyses were performed on the isolated fractions to identify specific chemical modification contributing to the charge differences, and were also characterized for purity and in vitro potency prior to being administered either subcutaneously (SC) or intravenously (IV) in rats. All isolated materials had similar potency and rat FcRn binding relative to the starting material. Following IV or SC administration (10 mg/kg) in rats, no difference in serum PK was observed, indicating that physiochemical modifications and pI differences among charge variants were not sufficient to result in PK changes. Thus, these results provided meaningful information for the comparative evaluation of charge-related heterogeneity of mAbs, and suggested that charge variants of IgGs do not affect the in vitro potency, FcRn binding affinity, or the PK properties in rats.

The immunogenicity of SARS-CoV nucleocapsid DNA vaccine and the immunoregulatory activity of interleukin-2 (IL-2) were investigated. DNA vaccine plasmids, pcDNA-N and pcDNA-IL2, were constructed and inoculated into BALB/c mice with or without pcDNA-IL2 by intramuscular injection. Cellular and humoral immune responses were assessed by indirect ELISA, lymphocyte proliferation assays, ELISPOT and FACS. The nucleocapsid DNA vaccine had good immunogenicity and can induce specific humoral and cellular immunity in BALB/c mice, while IL-2 plays an immunoadjuvant role and enhances specific immune responses. This study provides a frame of reference for the design of DNA vaccines against SARS-CoV.