[Peptides analysis in digested edible bird's nest by HPLC-MS].

Edible bird's nest contains lots of glycoproteins. The glycosylation inhomogeneity for glycoprotein often results in wide range of molecular weight and the difficulty for protein separation and charaterization. In this paper, proteins in the edible bird's nest were extracted using multiple extractions, and then digested by PNgase F and trypsin. The digest mixture was separated with HPLC, and peptides were identified based on MS/MS data searching. The results indicated that the extracted proteins were amount to 79.7% of total protein in the edible bird's nest. More than 20 species of peptides in the digested mixture were identified. The sequences of these peptides showed similarity with some proteins from Swiss-prot. The research indicated that deglycosylation, tryptic digestion coupled with HPLC-MS/MS is a proper strategy for characterization of proteins in the edible bird's nest.

Age-related changes in the ratio of Type I/III collagen and fibril diameter in mouse skin.

The content of type I collagen (COL-I) and type III collagen (COL-III) and the ratio between them not only affect the skin elasticity and mechanical strength, but also determine the fibril diameter. In this research, we investigated the age-related changes in COL-I/COL-III ratio with their formed fibril diameter. The experimental result was obtained from high performance liquid chromatography-mass spectrometer, hydroxyproline determination, picrosirius red staining and transmission electron microscopes (TEM), respectively. The result indicated that the COL-I/COL-III ratio in mouse skin increased with aging. From the 0th to 9th week, the COL-I/COLIII ratio increased from 1.3:1 to 4.5:1. From the 9th to the 18th week, it remained between 4.5:1 and 4.9:1. The total content of COL-I and COL-III firstly increased and then decreased with aging. The TEM result showed that the fibril diameter increased with aging. From the 0th to 9th week, the average fibril diameter increased from 40 to 112 nm; From the 9th to 18th weeks, it increased from 112 to 140 nm. After the 9th week. The fibril diameter showed obvious uneven distribution. Thus, the COL-I/COLIII ratio was proportional to the fibril diameter, but inversely proportional to the uniformity of fibril diameter.

Identification and Characterization of Fibronectin-Binding Peptides in Gelatin.

Collagen and fibronectin (FN) are important components in the extracellular matrix (ECM). Collagen-FN binding belongs to protein-protein interaction and plays a key role in regulating cell behaviors. In this study, FN-binding peptides were isolated from gelatin (degraded collagen) using affinity chromatography, and the amino acid sequences were determined using HPLC-MS. The results indicated that all FN-binding peptides contained GPAG or GPPG. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and dual-polarization interferometry (DPI) were used to analyze the effects of hydroxylation polypeptide on FN binding activity. DPI analysis indicated that peptides with molecular weight (MW) between 2 kDa and 30 kDa showed higher FN-binding activity, indicating MW range played an important role in the interaction between FN and peptides. Finally, two peptides with similar sequences except for hydroxylation of prolines were synthesized. The FN-binding properties of the synthesized peptides were determined by MALDI-TOF MS. For peptide, GAPGADGP*AGAPGTP*GPQGIAGQR, hydroxylation of P8 and P15 is necessary for FN-binding. For peptide, GPPGPMGPPGLAGPPGESGR, the FN-binding process is independent of proline hydroxylation. Thus, FN-binding properties are proline-hydroxylation dependent.

Bacteriophage therapy for empyema caused by carbapenem-resistant Pseudomonas aeruginosa.

Pseudomonas aeruginosa is a frequent causative agent of post-pneumonectomy empyema-associated broncho-pleural fistula (BPF) and it has a high mortality rate. In recent years, the therapeutic potential of bacteriophage therapy has recognized anew as antimicrobial resistance increases globally. Studies are increasingly reporting the efficacy and safety of bacteriophage therapy for the treatment of multidrug-resistant bacterial infections. However, the clinical efficacy of bacteriophage therapy in empyema has seldom been studied. The current study reports the authors' experience with bacteriophage therapy for a 68-year-old Chinese man who suffered BPF-associated empyema and pneumonia caused by carbapenem-resistant P. aeruginosa. A personalized lytic pathogen-specific two-phage preparation was administered to the patient continuously for 24 days in combination with conventional antibiotics. The treatment was well-tolerated, resulting in clearance of the pathogen and improvement of the clinical outcome. This experience shows that a combined conventional antibiotic treatment with bacteriophage therapy may be effective at alleviating a multidrug-resistant bacterial infection in BPF-associated empyema.

Clinical status of patients 1 year after hospital discharge following recovery from COVID-19: a prospective cohort study.

BACKGROUND: The long-term clinical status of coronavirus disease 2019 (COVID-19) in recovered patients remains largely unknown. This prospective cohort study evaluated clinical status of COVID-19 and explored the associated risk factors. METHODS: At the outpatient visit, patients underwent routine blood tests, physical examinations, pulmonary function tests, 6-min walk test, high-resolution computed tomography (CT) of the chest, and extrapulmonary organ function tests. RESULTS: 230 patients were analyzed. Half (52.7%) reported at least one symptom, most commonly fatigue (20.3%) and sleep difficulties (15.8%). Anxiety (8.2%), depression (11.3%), post-traumatic symptoms (10.3%), and sleep disorders (26.3%) were also reported. Diffusion impairments were found in 35.4% of the patients. Abnormal chest CT scans were present in 63.5% of the patients, mainly reticulation and ground-glass opacities. Further, a persistent decline in kidney function was observed after discharge. SARS-CoV-2-specific antibodies of IgA, IgG, and IgM were positive in 56.4%, 96.3%, and 15.2% of patients, respectively. Multivariable logistic regression showed that disease severity, age, and sex were closely related to patient recovery. CONCLUSIONS: One year after hospital discharge, patients recovered from COVID-19 continued to experience both pulmonary and extrapulmonary dysfunction. While paying attention to pulmonary manifestations of COVID-19, follow-up studies on extrapulmonary manifestations should be strengthened.

[Changes of collagen and MMP-1 in liver, lung and kidney during growth of mice].

The high performance liquid chromatography (HPLC) and enzyme-linked immunoassay (ELISA) were used to investigate the changes of collagen and matrix metalloproteinase-1 (MMP-1) in liver, lung and kidney during growth process of mice. The mice from 0 to 18 weeks were used as the research objects. The contents and proportions of hydroxyproline (Hyp), which were used to calculate the collagen contents, in liver, lung and kidney of different weeks were analyzed with HPLC. The contents and activity of MMP-1 in liver, lung and kidney of different weeks were analyzed with ELISA. The results showed that the collagen contents in liver, lung, and kidney were different (Lung(COL)>Kidney(COL)>Liver(COL)), and they all increased first and then decreased with weeks. The collagen contents in liver, lung, and kidney reached the highest level in the ninth (5.52 ng/mg), sixth (54.10 ng/mg) and ninth (19.20 ng/mg) week, respectively. Then it declined slowly from 9 to 18 weeks. The result of ELISA showed that the MMP-1 contents in liver, lung and kidney decreased first and then increased with weeks, and the trend of MMP-1 activity was opposite. It indicated that the increase of collagen contents in the tissues will inhibit the secretion of MMP-1.

Three dimensional polyvinyl alcohol scaffolds modified with collagen for HepG2 cell culture.

The creation of in vitro functional hepatic tissue simulating micro environmental niche of the native liver is a keen area of research due to its demand in bioartificial liver. However, it is still unclear how to maintain benign cell function while achieving the sufficient cell quantity. In this work, we aim to prepare a novel scaffold for the culture of HepG2 cells, a liver cell line, by modifying polyvinyl alcohol (PVA) scaffold with collagen (COL). PVA is a kind of synthetic biostable polymer with high hydrophilicity in the human body, has been widely used in the biomedical field. However, the use of PVA is limited in cell cultures due to lack of biologically active functional groups. In this study, amino silane (KH-550), glutaraldehyde and native type I collagen were used to modify three-dimensional PVA scaffold to establish a suitable composite scaffold for hepatocyte culture. Three types of composite scaffolds were prepared for different collagen content, named as PVA/COL (0.2%), PVA/COL (0.5%) and PVA/COL (0.8%), respectively. The composite scaffolds were characterized by SEM, XPS, FTIR, MS, porosity estimation and water contact angle measurement. The PVA/COL (0.8%) scaffolds had the highest collagen content of 12.13%. The composite scaffold showed high porosity with interconnected pores. Furthermore, the biocompatibility between HepG2 cells and scaffolds was evaluated by the ability of cell proliferation, albumin secretion, as well as urea synthesis. The coating of collagen on PVA scaffolds promoted hydrophilicity and HepG2 cell adhesion. Additionally, enhanced cell proliferation, increased albumin secretion and urea synthesis were observed in HepG2 cells growing on collagen-coated three-dimensional PVA scaffolds.

Detection of Type I and III collagen in porcine acellular matrix using HPLC-MS.

Acellular matrix (ACM) has been widely used as a biomaterial. As the main component of ACM, collagen type and content show influence on the material properties. In this research, the collagen in ACM from different tissues of pig were determined by detection of marker peptides. The marker peptides of Type I and III collagen were identified from the digested collagen standards using ions trap mass spectrometry (LCQ). The relationship between the abundance of marker peptide and collagen concentration was established using triple quadrupole mass spectrometer (TSQ). The contents of Type I and III collagen in ACM from different tissues were determined. The method was further verified by hydroxyproline determination. The results showed that, the sum of Type I and III collagen contents in the ACM from small intestinal submucosa, dermis and Achilles tendon of pig were about 87.59, 81.41 and 61.13%, respectively, which were close to the total collagen contents in these tissues. The results proved that this method could quantitatively detect the collagen with different types in the ACM of various tissues.

A novel thrombin inhibitory peptide discovered from leech using affinity chromatography combined with ultra-high performance liquid chromatography-high resolution mass spectroscopy.

Thrombin (THR) inhibitors play an important role in the treatment of thrombotic diseases. This study established a THR-based bio-specific extraction coupled with affinity chromatography and ultra-high performance liquid chromatography-high resolution mass spectroscopy (UPLC-HR-MS) analysis method to screen and identify THR ligands in Leech. After evaluating the reliability of the screening method using positive control drug (hirudin), it was successfully used to screen the potential active constituents in leech. And a comprehensive analysis of the peptides in leech elution was performed by UPLC-HR-MS, a total of 34 peptides were identified. At the same time, anti-THR activity was explored and inferred by searching databases and published literature. As a result, six peptides were discovered to be potential active compounds in leech. Further, the six peptides were synthesized and in vitro enzymatic activity assay was performed. Finally, SYELPDGQVITIGNER was screened as an anti-THR peptide with an IC50 value of 255.75 µM and it was discovered for the first time from Whitmania pigra Whitman and Hirudo nipponica Whitman. The molecular docking study showed that THR inhibitory activity of the polypeptide was mainly attributed to the hydrogen bond interactions, van der Waals forces and electrostatic interactions interaction between polypeptide and THR. These results suggest that the polypeptide is a potential natural THR inhibitor that can be used as anticoagulant.

Polymeric Micelles Encapsulating a Small Molecule SO2 Fluorescent Probe Exhibiting Novel Analytical Performance and Enhanced Cellular Imaging Ability.

Because of the limited knowledge on the relationship between molecular structure and analytical performance, developing a small molecule fluorescent probe with desirable response properties is usually a laborious work. On the other hand, the application of small molecule fluorescent probe in biological samples is always limited due to the unwanted interaction between dyes and biomacromolecules. Polymer micelles, thanks to its unique core-shell structure, may have the potential to improve these situations. However, utilization of polymer micelles to improve these situations is rarely explored. Herein, we engineered the first micellar SO2 nanoprobe Nano-Cz by self-assembly of a carbazole-based SO2 small molecule probe and an amphiphilic copolymer (DSPE-mPEG2000). The optical and cell imaging experiments revealed that Nano-Cz can work in 100% aqueous environment and act as an effective mitochondrial-targeting ratio SO2 nanoprobe. Compared with the single small molecule probe, Nano-Cz showed extraordinary large dynamic response range (0-0.7 mM vs 0-50 µM), eliminated signal interference from DNA and superior cellular imaging performance. These results clearly show the ability of polymer micelles in modulating sensors' analytical performance and reducing the signal interference from the unwanted interaction between small molecule probe and biomacromolecule, indicating that polymer micelles encapsulating single small molecule probe can provide us an alternative strategy to explore sensors with various performance and promote the biological application of fluorescent sensors. In addition, we hope that more and more polymer micelles would be used to construct biosensors in the future.

Site-specific immobilization of lysozyme upon affinity chromatography resin by forecasting lysine activity and controlling pH and epoxy group density.

Here, a site-specific immobilization strategy for lysozyme was discussed. First, we calculated the surface micro-environment of lysozyme as a model protein and forecasted the nucleophilic attack activity order of six lysine residues within lysozyme, namely, K96> K97 and K33>K1, K13, and K116. Second, lysozyme was immobilized on agarose resin (epoxy group density 22.34 µmol/g) and incubated at pH 9.5 for 12 h. We then compared the peptide maps of free and immobilized lysozyme and established a quantitative detection method for immobilization sites. Third, we studied the effect of immobilization conditions such as epoxy group density and pH upon immobilization sites. When lysozyme was immobilized upon resin with a high epoxy group density (22.34 µmol/g) at pH 9.5 for 12 h, multiple lysozyme immobilization sites were found, including K33, K96 and K97; when lysozyme was immobilized upon resin with a low epoxy group density (11.36 µmol/g) at pH 9.5 for 12 h, only one lysozyme immobilization site was found, K96. The pH also had an effect upon immobilization sites. When immobilization was performed at pH≥10.5 with low epoxy group density resin (11.36 µmol/g) for 12 h, the immobilization sites included at least K33, K96 and K97; when immobilization was performed at pH 9.5 with other conditions remaining unchanged, the only lysozyme immobilization site found was at K96. These experimental results were highly consistent with the forecasted results, revealing some regularities of immobilization site control for lysozyme upon affinity chromatography resin.

[Relationships between the expressions of intercellular adhesion molecule-1 and tissue inhibitor of metalloproteinase-1 and matrix metalloproteinase-9 in lung tissues of patients with chronic obstructive pulmonary disease].

OBJECTIVE: To evaluate the correlation between the expressions of intercellular adhesion molecule-1 (ICAM-1) and tissue inhibitor of metalloproteinase-1 (TIMP-1) and matrix metalloproteinase-9 (MMP-9) in lung tissues of patients with COPD. METHODS: Lung tissues from patients with COPD (COPD group, n = 19) and those without COPD (smokers and nonsmokers with normal lung function, n = 11 and 9, respectively) were obtained from surgical excisions of lung cancer patients. The mRNA expression of ICAM-1, TIMP-1 and MMP-9 was detected using semi-quantitative RT-PCR. The protein expression of ICAM-1, TIMP-1 and MMP-9 was detected by using immunohistochemistry method. RESULTS: There were significant differences in FEV1% and FEV1/FVC% among smokers without COPD, nonsmokers without COPD and COPD patients. MMP-9 was highly expressed in alveolar epithelial cells, bronchial epithelial cells, vascular smooth muscle cells, alveolar macrophages, and interstitial cells in the COPD group, compared with smokers without COPD group and nonsmokers without COPD group (54.0 +/- 15.0), (1.2 +/- 0.7) and (1.4 +/- 0.8). Low level expression of TIMP-1 was detected in alveolar macrophages, alveolar epithelial cells and vascular smooth muscle cells in the COPD group, but no expression in smokers and nonsmokers without COPD. High level expression of ICAM-1 was detected in alveolar epithelial cells, and the expression was higher in the COPD group (52.1 +/- 13.4), (2.1 +/- 1.1) and (4.5 +/- 2.4). The mRNA level of MMP-9 showed significant difference among patients with COPD, smokers without COPD and nonsmokers without COPD (0.71 +/- 0.16), (0.20 +/- 0.08) and (0.17 +/- 0.05). The mRNA level of TIMP-1 was also significantly different among patients with COPD, smokers without COPD and nonsmokers without COPD (0.47 +/- 0.10), (0.26 +/- 0.08) and (0.20 +/- 0.06). ICAM expression was also significantly higher in patients with COPD as compared with smokers without COPD and nonsmokers without COPD (0.62 +/- 0.15), (0.44 +/- 0.12) and (0.37 +/- 0.11). Both the mRNA and the protein levels of MMP-9 were inversely correlated with FEV1 % and FEV1/FVC% (r= -0.759, -0.756, -0.772, -0.725, respectively, P <0.01). TIMP-1 mRNA level was inversely correlated with FEV1% and FEV1/FVC% (r = -0.675, -0.623, respectively P <0.01). Negative correlations were also noted between ICAM-1 expressions (both mRNA and protein) and FEV1% or FEV1/FVC% (r = -0.580, -0.531, -0.739, -0.756, respectively P <0.01). Interestingly, the mRNA expression of TIMP-1, MMP-9 and ICAM-1 was positively correlated (r = 0.576, 0.524, P < 0.01), while the protein levels of MMP-9 and ICAM-1 were positively correlated (r = 0.964, P <0.01). CONCLUSION: There was a significant correlation between over-expression of ICAM-1 and TIMP-land MMP-9 in lung tissues from COPD patients. Over-expressions of ICAM-1 in the lung may result in accumulation of inflammatory cells releasing certain inflammatory factors that could destroy the normal lung structure. In addition, highly expressed TIMP-1 and MMP-9 in lung tissues may also contribute to the destruction and reconstitution of the bronchial or/and alveolar wall, which is likely to play a major role in airway obstruction.

CD70, a novel target of CAR T-cell therapy for gliomas.

Background: Cancer immunotherapy represents a promising treatment approach for malignant gliomas but is hampered by the limited number of ubiquitously expressed tumor antigens and the profoundly immunosuppressive tumor microenvironment. We identified cluster of differentiation (CD)70 as a novel immunosuppressive ligand and glioma target. Methods: Normal tissues derived from 52 different organs and primary and recurrent low-grade gliomas (LGGs) and glioblastomas (GBMs) were thoroughly evaluated for CD70 gene and protein expression. The association between CD70 and patients' overall survival and its impact on T-cell death was also evaluated. Human and mouse CD70-specific chimeric antigen receptors (CARs) were tested respectively against human primary GBMs and murine glioma lines. The antitumor efficacies of these CARs were also examined in orthotopic xenograft and syngeneic models. Results: CD70 was not detected in peripheral and brain normal tissues but was constitutively overexpressed by isocitrate dehydrogenase (IDH) wild-type primary LGGs and GBMs in the mesenchymal subgroup and recurrent tumors. CD70 was also associated with poor survival in these subgroups, which may link to its direct involvement in glioma chemokine productions and selective induction of CD8+ T-cell death. To explore the potential for therapeutic targeting of this newly identified immunosuppressive axis in GBM tumors, we demonstrate that both human and mouse CD70-specific CAR T cells recognize primary CD70+ GBM tumors in vitro and mediate the regression of established GBM in xenograft and syngeneic models without illicit effect. Conclusion: These studies identify a previously uncharacterized and ubiquitously expressed immunosuppressive ligand CD70 in GBMs that also holds potential for serving as a novel CAR target for cancer immunotherapy in gliomas.

[Expressions of and the effect of smoking on vascular endothelial growth factor and induced nitric oxide synthase in lung tissues of chronic obstructive pulmonary disease patients].

OBJECTIVE: To investigate the expressions of and the effect of smoking on vascular endothelial growth factor (VEGF) and induced nitric oxide synthase (iNOS) in lung tissues of chronic obstructive pulmonary disease (COPD) patients. METHODS: The peripheral lung tissues were obtained from 46 patients with lung carcinoma. They were divided into three groups according to their habit of smoking and lung function, 19 smokers with moderate COPD, 12 smokers and 15 nonsmokers with normal lung function. The expression of VEGF and iNOS was detected by immunohistochemistry. RESULTS: Expressions of VEGF and iNOS were increased in lung tissues of smokers without COPD (1.50 +/- 0.39, 1.45 +/- 0.41) compared with nonsmokers without COPD (1.18 +/- 0.33, 1.09 +/- 0.41) (each P < 0.05), and were significantly increased in lung tissues of smokers with moderate COPD (2.19 +/- 0.51, 2.39 +/- 0.45) compared with nonsmokers without COPD (each P < 0.01). The expression of VEGF in lung tissues was significantly correlated with the expression of iNOS (r = 0.78, P < 0.01), but was inversely correlated with FEV(1) (r = -0.67, P < 0.01). CONCLUSIONS: Expressions of VEGF and iNOS were upregulated in lung tissues of smokers and patients with moderate COPD. Overexpression of iNOS and VEGF may participate in the mechanism of airway and vascular remodeling, and airflow limitation in COPD.

[Collagen quantitation by detection of marker peptides with HPLC-MS].

A method for quantitation of collagen was established by detecting marker peptide with high performance liquid chromatography-mass spectrometry (HPLC-MS). Theoretical marker peptides were selected by sequence comparison. Bovine collagen type I was digested with trypsin. Marker peptides typical for collagen type I were identified with HPLC-MS. The relationship between the abundance of marker peptides and collagen concentration was established. The results show that GEAGPSGPAGPTGAR and the other 5 peptides showed high resolution during chromatographic separation and high signal intensity during MS analysis. Peptide signal intensity and collagen concentration showed a good linear relationship in the range from 0.1 to 3 mg/mL. Bovine tendon and collagen sponge were used as actual samples and collagen contents were determined as 90.2% and 93.4% respectively. Quantitation of marker peptides of collagen was a feasible method to identify and quantify collagens in medical device research and development.

Aggregation and antigenicity of virus like particle in salt solution--A case study with hepatitis B surface antigen.

The phenomenon of aggregation of virus-like particles (VLPs) in salt solution and the corresponding effect upon antigenicity was reported. Asymmetrical flow field-flow fractionation (AF4) combined with multi-angle laser light scattering (MALLS) was used to characterize the size and the aggregation behavior of hepatitis B surface antigen (HBsAg). The average diameter of HBsAg VLP was 22.8±0.4 nm and it tended to aggregate in salt solution to form large particles and the antigenicity changed accordingly. In 0-4 M NaCl solution, part of HBsAg molecules aggregated rapidly into oligomeric particles (OP), whose diameter distributed from 25 to 40 nm, and the antigenicity slightly decreased about 10%. The aggregation reaction is reversible. After removing NaCl, both size and antigenicity could recover to normal level (92-96%). By contrast, the aggregation process is more complicated in (NH4)2SO4 solution. Most of HBsAg particles aggregated into OP and further aggregated into polymeric particles (PP). The diameter of the PP could reach 40 to 140 nm. The concentration of (NH4)2SO4 had remarkable influence upon the rate of aggregation. When concentration of (NH4)2SO4 was below 1 M, most of HBsAg aggregated only into OP in 1 h. While with concentration of (NH4)2SO4 above 1 M, most of particles formed PP within 1 h. The aggregation process to PP was irreversible. After removing (NH4)2SO4, the large aggregates could not recover to normal particles and the remaining antigenicity was below 30%.

[The breeding of high glutathione-producing strain and optimization of culture condition].

High glutathione-producing strain ZJF-71 was constructed by primary screening,isolation of haploid, mutagenesis and protoplasts fusion. The yield of glutathione of the fusant ZJF-71 is 1.59 and 1.42 times that of the parental strains Y64 and Y247, respectively. The factors that affected the biomass and glutathione content of the fusant ZJF-71 were also tested. The highest level of glutathione was obtained in 32 h at 30 degrees C and 200 r/min, when 30 mL of culture in 250- mL shake flasks was incubated in fermentation medium which contained (w/v): 6% cane sugar, 1% peptone, 1% yeast extract, and 2mmol/L cysteine. Glutathione yield under the optimal fermentation condition showed a 2.8-fold improvement over that of the initial condition. The fusant ZJF-71 is stable in genetic by analysis of genetic stability.

[Purification of recombinant human antithrombin III expressed in a goat mammary bioreactor].

Antithrombin III (AT III) is the most important anti-clotting substance. Recombinant human antithrombin III (rhAT III) expressed in transgenic goat milk attracts more and more attention. Develop an effective purification route for rhAT III is vital to its industrial production. An efficient purification method was developed for the rapid purification of rhAT III by isoelectric precipitation and heparin affinity chromatography. First, casein was effectively removed by isoelectric precipitation. rhAT III was further purified by heparin affinity chromatography. In the process of heparin affinity chromatography, the effects of pH and temperature on the stability of rhAT III were studied, and the effects of operating conditions, elution gradient, flow rate and sample loaded, on the purification efficiency were also studied. Under the optimized conditions, the protein recovery of rhAT III was about 90% with purity over 99%, while its activity recovery was about 50%. Such a purification process is very simple and effective, and it would provide a valuable reference for the further scaling-up of industrial production.

An automatic system for multidimensional integrated protein chromatography.

An automatic system for multidimensional integrated protein chromatography was designed for simultaneous separation of multiple proteins from complex mixtures, such as human plasma and tissue lysates. This computer-controlled system integrates several chromatographic columns that work independently or cooperatively with one another to achieve efficient high throughputs. The pipelines can be automatically switched either to another column or to a collection container for each UV-detected elution fraction. Environmental contamination is avoided due to the closed fluid paths and elimination of manual column change. This novel system was successfully used for simultaneous preparation of five proteins from the precipitate of human plasma fraction IV (fraction IV). The system involved gel filtration, ion exchange, hydrophobic interaction, and heparin affinity chromatography. Human serum albumin (HSA), transferrin (Tf), antithrombin-III (AT-III), alpha 1-antitrypsin (α1-AT), and haptoglobin (Hp) were purified within 3 h. The following recovery and purity were achieved: 95% (RSD, 2.8%) and 95% for HSA, 80% (RSD, 2.0%) and 99% for Tf, 70% (RSD, 2.1%) and 99% for AT-III, 65% (RSD, 2.0%) and 94% for α1-AT, and 50% (RSD, 1.0%) and 90% for Hp. The results demonstrate that this novel multidimensional integrated chromatography system is capable of simultaneously separating multiple protein products from the same raw material with high yield and purity and it has the potential for a wide range of multi-step chromatography separation processes.

Greatest International ANtiinfective Trial (GIANT) with moxifloxacin in the treatment of acute exacerbation of chronic bronchitis: subanalysis of Chinese data of a global, multicenter, noninterventional study.

BACKGROUND AND OBJECTIVE: A single infective acute exacerbation of chronic bronchitis (AECB) has a sustained effect on health status. Although a number of clinical investigations have demonstrated the efficacy of antibiotics in AECB, increased bacterial resistance has caused concern about the efficacy of currently available antibiotic therapies. This subanalysis of a global noninterventional study aimed to evaluate the impact of AECB on the patient and the community and the effectiveness and safety of a treatment with moxifloxacin (MXF) tablets in daily life clinical practice in China. METHODS: This prospective, noninterventional, noncontrolled, multicenter observational study, which started in China in April 2004 and ended in February 2007, was part of the global GIANT study. Patients with a diagnosis of mild to severe AECB were treated with MXF tablets 400 mg for a period at the physician's discretion. The observation period for each patient covered a complete treatment period with MXF. For each patient, the physician documented data at an initial visit (baseline) and at least one follow-up visit. Data were collected on demography, diagnosis of infection, pretreatment, concomitant diseases and medications, MXF therapy, course of symptoms during investigations, and final assessment of therapy with respect to MXF. RESULTS: In the Chinese subset of the GIANT study, a total of 11,377 patients were included in the intention-to-treat/safety population. At the end of the initial treatment period, improvement and recovery from infection was observed for 98.6% (n = 11,217/11,377) and 92.6% (n = 10,540/11,377) of all patients. After 1 week of treatment, 76.3% (n = 8681/11,377) of patients had recovered. Median time until improvement and recovery was 3.0 and 6.0 days, respectively. Correspondingly, in 95.8% (n = 10,903/11,377) of all patients, overall effectiveness during the initial treatment period with MXF was assessed as "very good" or "good". Compared with the last AECB, the number of days with impact on daily-life activities and the number of nights with sleep disturbances decreased from 3.0 to 2.0 (median) and from 2.0 to 1.0 (median), respectively. In general, MXF treatment was very well tolerated, with physician's overall assessment of tolerability as "good" or "very good" in 95.2% (n = 10,834/11,377) of patients. The incidence rate of adverse events and adverse drug reactions was 0.82% (n = 93) and 0.67% (n = 76), respectively. The most frequent adverse events were gastrointestinal disorders such as nausea (0.31%, n = 35) and vomiting (0.19%, n = 22), which were mostly drug-related. One individual serious adverse event (dyspnea) occurred during the observation period, which was assessed as drug-related. CONCLUSION: MXF was effective and well tolerated in patients suffering from AECB. The fast speed of the drug's onset of action was associated with rapid improvement of clinical parameters.