Development of a robust and semi-automated two-step antibody purification process.

Advances in antibody discovery technologies, especially with the availability of humanized mice and phage/yeast library approaches, enable the generation of a large diversity of antibodies against nearly any target of interest. As a result, there is an increasing demand for the production of larger numbers of purified antibodies at quantities (10s-100s of milligrams) sufficient for functional screening assays, drug-ability/develop-ability studies and immunogenicity assessments. To accommodate this need, new methods are required that bridge miniature high throughput/plate-based purification and conventional, one at a time, two-step purification at much larger scales. Thus, we developed a semi-automated, mid-scale (i.e., 1-75 mg) purification process that uses a combination of parallel affinity capture and automated sequential polishing to provide substantially improved throughput while delivering high purity. We optimized the affinity capture step to perform 24 monoclonal antibody purifications in parallel using a Protein Maker for 20-200 mL culture media. The eluant is transferred directly to an AKTA pure system equipped with an autosampler for sequential preparative size exclusion chromatography to remove aggregates and undesirable impurities, as well as exchange the antibody into a buffer suitable for most uses, including cell-based assays. This two-step purification procedure, together with plate-based protein analytical methods, can purify 24-48 monoclonal antibodies in <20 hours and generate up to 80 mg per sample. A stringent clean-in-place protocol for both systems and column maintenance was designed and established to minimize endotoxin contamination. This process has proven to be very reliable and robust, enabling the production of thousands of antibodies of sufficient quality and quantity that are suitable for cell-based assays, biochemical/biophysical characterization, and in vivo animal models.

The Gut Microbiome Modulates the Changes in Liver Metabolism and in Inflammatory Processes in the Brain of Chronic Unpredictable Mild Stress Rats.

Generally, depression is the result of complex gene-environment interactions. Recent studies have showed that the gut microbiota can affect brain function through the microbiota-gut-brain axis. However, the underlying mechanism of the microbiota and potential influence of depression remain elusive. We aimed to determine how gut microbiome contributes to the process of depression and further influences the host. Chronic unpredictable mild stress (CUMS) is used to establish a depression model. Fecal microbiota transplant (FMT) is applied to illustrate that depression can be transmitted via microbiota, and metabolism of liver analysis is applied to demonstrate further influence to the liver. We also analyzed the astrocyte activation in the brain by immunofluorescence (IF). Here, we show that the structure of the gut microbiome changes markedly after rats undergo CUMS. Notably, we found that the ratio of Lactobacillus to Clostridium can be a vital index for the development of depression. Depression-like behavior can be duplicated through FMT. Moreover, increased zonulin and fatty acid binding protein-2 indicates that gut barrier integrity is broken after FMT. Subsequently, metabolomics shows that liver metabolic disorder occurs and leads to liver coagulative necrosis. In addition, increased inflammatory cytokine expression and higher astrocyte activation indicate an inflammatory process in the brain. These findings suggest that dysbiosis gut microbiome contributes to development of depression and further causes liver metabolic disorders in a way that may be relevant to the Lactobacillus to Clostridium ratio.

Long-term acupuncture treatment has a multi-targeting regulation on multiple brain regions in rats with Alzheimer's disease: a positron emission tomography study.

The acute effect of acupuncture on Alzheimer's disease, i.e., on brain activation during treatment, has been reported. However, the effect of long-term acupuncture on brain activation in Alzheimer's disease is unclear. Therefore, in this study, we performed long-term needling at Zusanli (ST36) or a sham point (1.5 mm lateral to ST36) in a rat Alzheimer's disease model, for 30 minutes, once per day, for 30 days. The rats underwent 18F-fluorodeoxyglucose positron emission tomography scanning. Positron emission tomography images were processed with SPM2. The brain areas activated after needling at ST36 included the left hippocampus, the left orbital cortex, the left infralimbic cortex, the left olfactory cortex, the left cerebellum and the left pons. In the sham-point group, the activated regions were similar to those in the ST36 group. However, the ST36 group showed greater activation in the cerebellum and pons than the sham-point group. These findings suggest that long-term acupuncture treatment has targeted regulatory effects on multiple brain regions in rats with Alzheimer's disease.

A Comparative Evaluation of the Mechanical Properties of Two Calcium Phosphate/Collagen Composite Materials and Their Osteogenic Effects on Adipose-Derived Stem Cells.

Adipose-derived stem cells (ADSCs) are ideal seed cells for use in bone tissue engineering and they have many advantages over other stem cells. In this study, two kinds of calcium phosphate/collagen composite scaffolds were prepared and their effects on the proliferation and osteogenic differentiation of ADSCs were investigated. The hydroxyapatite/ß-tricalcium phosphate (HA/ß-TCP) composite scaffolds (HTPSs), which have an additional ß-tricalcium phosphate, resulted in better proliferation of ADSCs and showed osteogenesis-promoting effects. Therefore, such composite scaffolds, in combination with ADSCs or on their own, would be promising for use in bone regeneration and potential clinical therapy for bone defects.

[Study of Spectral Property and Measurement of Linear Variable Filters].

Linear variable filters (LVF) are widely used in a variety of small rapid spectrometric detecting devices. In the present paper, the splitting property of LVF was studied, and the spectral-property expression in Gaussian form was given, and the relationship between indexes of expression and LVF center transmission, width of spectrum, and linear dispersion was specified. Measurement based on monochromator for imaging spectrometers calibration was introduced, the sensitivity for detection system was discussed, and the tolerance was analyzed. It was shown that the slit displacement of monochromator relative to the optical axle and the slope of LVF affect the detecting accuracy most, and the tolerance can be reduced by adjustment of optical path and structure to reach requirement. System was built and the detection for spectral property of sample LVF was made. The result of data processing indicates that MSE for center transmission detection was lower than 0.05%, and the accuracy of this method was proved. Reference-parameters of relevant system designing and calibrating based on the LVF can be provided by this method.

Acupuncture for ischemic stroke: cerebellar activation may be a central mechanism following Deqi.

The needling sensation of Deqi during acupuncture is a key factor of influencing acupuncture outcome. Recent studies have mainly focused on the brain function effects of Deqi in a physiological state. Functional magnetic resonance imaging (fMRI) on the effects of acupuncture at Waiguan (SJ5) in pathological and physiological states is controversial. In this study, 12 patients with ischemic stroke received acupuncture at Waiguan (SJ5) and simultaneously underwent fMRI scanning of the brain, with imaging data of the activated areas obtained. Based on the patient's sensation, imaging data were allocated to either the Deqi group or non-Deqi group. In the Deqi group, the activated/deactivated areas were the left superior temporal gyrus (BA39)/right anterior lobe of the cerebellum and left thalamus. In the non-Deqi group, the activated areas included the medial frontal gyrus of the right frontal lobe (BA11), right limbic lobe (BA30, 35), and left frontal lobe (BA47), while the only deactivated area was the right parietal lobe (BA40). Compared with the non-Deqi group, the Deqi group exhibited marked activation of the right anterior lobe of the cerebellum and right limbic lobe (BA30). These findings confirm that the clinical effect of Deqi during acupuncture is based on brain functional changes. Cerebellar activation may be one of the central mechanisms of acupuncture in the treatment of ischemic stroke.

Self-assembled targeted folate-conjugated eight-arm-polyethylene glycol-betulinic acid nanoparticles for co-delivery of anticancer drugs.

In this study, a targeted nanoparticle platform for co-delivery of anticancer drugs based on folate-conjugated eight-arm-polyethylene glycol-betulinic acid (F-8arm-PEG-BA) was first presented. F-8arm-PEG-BA was synthesized by introducing target molecules (folate) and drug molecules (betulinic acid, BA) to hydrophilic molecules (8arm-PEG). Then another anticancer drug, hydroxycamptothecin (HCPT), was encapsulated into the self-assembled nanoparticles from the conjugate by a simple nanoprecipitation method. These F-8arm-PEG-BA/HCPT nanoparticles (NPs) had a small size (∼120 nm), acceptable critical aggregation concentration (∼64.8 µg mL-1), and high drug loading (∼18 wt% BA and ∼16 wt% HCPT). Compared to the free drugs, the nanoparticles significantly improved the cellular cytotoxicity and exhibited an obvious synergistic effect by the co-delivery of two different anticancer drugs, BA and HCPT. Pharmacokinetics study revealed the nanoparticles could prolong the circulation of BA and HCPT in the blood. In vivo studies indicated that the nanoparticles enhanced tumor targeting and antitumor activity with lower systemic toxicity. In conclusion, F-8arm-PEG-BA/HCPT NPs have great potential for targeted chemotherapy for cancer.

[Multi-DSP parallel processing technique of hyperspectral RX anomaly detection].

To satisfy the requirement of high speed, real-time and mass data storage etc. for RX anomaly detection of hyperspectral image data, the present paper proposes a solution of multi-DSP parallel processing system for hyperspectral image based on CPCI Express standard bus architecture. Hardware topological architecture of the system combines the tight coupling of four DSPs sharing data bus and memory unit with the interconnection of Link ports. On this hardware platform, by assigning parallel processing task for each DSP in consideration of the spectrum RX anomaly detection algorithm and the feature of 3D data in the spectral image, a 4DSP parallel processing technique which computes and solves the mean matrix and covariance matrix of the whole image by spatially partitioning the image is proposed. The experiment result shows that, in the case of equivalent detective effect, it can reach the time efficiency 4 times higher than single DSP process with the 4-DSP parallel processing technique of RX anomaly detection algorithm proposed by this paper, which makes a breakthrough in the constraints to the huge data image processing of DSP's internal storage capacity, meanwhile well meeting the demands of the spectral data in real-time processing.

[Impact and improvement of the mechanical shutter on large-array filter-type multispectral imaging system].

The present paper analyzes the impact of mechanical shutter on the spectral image acquisition and processing of large-array filter-type multispectral imaging system. The final image quality relies highly on the mechanical shutter due to the fluctuation at exposure time. The conventional mechanical shutter's structure and driving method was analyzed to find out the key fact for its poor stability. An improved method of mechanical transmission and circuit driving was proposed. Laboratory experiments showed that with the improved design strategy, the maximum rate of change between adjacent exposures was reduced from 15.05% to 0.96%, which is a great improvement of the exposure time stability. Field test was also carried out and the results show that the combined color images are closer to the realistic targets and no abrupt color change iwas found. It's of great significance for practical application in multispectral image process, interpretation and target identification.

[Isolation and mass spectrometric analysis of antioxidant peptides from enzymatic hydrolysates of roasted pills of Asini Corii Colla].

A fraction named GFC-1 with high antioxidant activities in vitro was isolated from the enzymatic hydrolysates of roasted pills of Asini Corii Colla, and the peptides in this fraction were identified. The enzymatic hydrolysates were isolated and purified with anion exchange chromatography and Sephadex G-25 filtration chromatography successively. GFC-1, a fraction isolated from the hydrolysates, exhibited the highest DPPH and ABTS scavenging capacity (DPPH 47. 95% at 2.0 g x L(-1) and ABTS 97.20% at 0.40 g x L(-1). Nine peptides from GFC-1 were identified by LC-ESI-MS/MS coupled with TurboSEQUEST search software and Swiss-Prot data base, and a high repetition core sequence GPAGPP*GPP* was also found.

[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.

Brain areas involved in acupuncture needling sensation of de qi: a single-photon emission computed tomography (SPECT) study.

BACKGROUND: De qi is a sensory response elicited by acupuncture stimulation. According to traditional Chinese medicine (TCM), de qi is essential for clinical efficacy. However, the understanding of the neurobiological basis of de qi is still limited. OBJECTIVE: To investigate the relationship between brain activation and de qi by taking a single-photon emission computed tomography (SPECT) scan while applying acupuncture at TE5. METHODS: A total of 24 volunteers were randomly divided into 4 groups, and received verum or sham acupuncture at true acupuncture point TE5 or a nearby sham point according to grouping. All subjects then received a (99m)Tc-ethylcysteinate dimer (ECD) SPECT scan. RESULTS: All six subjects in the verum acupuncture at true acupuncture point group experienced de qi sensation; in contrast, all six subjects in the sham acupuncture at the sham point group responded with nothing other than non-sensation. Compared to the scan results from subjects who experienced non-sensation, SPECT scans from subjects with de qi sensation demonstrated significant activated points mainly located in brodmann areas 6, 8, 19, 21, 28, 33, 35, 37, 47, the parahippocampal gyrus, lentiform nucleus, claustrum and red nucleus; deactivated points were seen in brodmann areas 9 and 25. CONCLUSIONS: Verum acupuncture at true acupuncture points is more likely to elicit de qi sensation. De qi sensations mainly resulted in brain area activations, but not deactivations. These brain areas are related to the curative effect of Te5. The acupuncture needle sensations of de qi and sharp pain are associated with different patterns of activations and deactivations in the brain.

[Modeling and correction of the effect of detector lateral fringe error in the large aperture static imaging spectrometer].

When the large aperture static imaging spectrometry (LASIS) was used in spaceborne remote sensing, the spectrum could not be well recovered from the interferogram with detector lateral fringe error. In order to obtain the more accurate spectrum, the error must be corrected. According to the imaging principle analysis of LASIS, the imaging model with lateral fringe error was given, and the error correction method was presented. Finally, the error correction method was validated with the data acquired by LASIS system. The results indicated that the error was commendably corrected by the method mentioned above, and the accuracy of recovered spectrum was evidently improved.

[Characteristics of PET cerebral functional imaging during "Deqi" of acupuncture in healthy volunteers].

OBJECTIVE: To observe the characteristics of needling sensation of "Deqi" (feelings of soreness, numbness, distending and heaviness, SNDH) by using positron emission tomography (PET) based on changes of glucose metabolism in different functional brain areas. METHODS: Eighteen normal volunteers [9 men and 9 women, mean age (23.23-1- 3. 32) years] were randomly divided into control, Waiguan (SJ 5) and non-acupoint groups (n=6 in each group). SJ 5 and non-acupoint (the midpoint between SJ 5 and the running course of the Small Intestine Meridian on the right forearm) were punctured by using a sterilized filiform needle. PET scan of the brain began 40 min after intravenous 18 F-fluorodeoxyglucose (FDG) injection (0. 11 mCi/kg body weight, left opisthenar vein). Needling sensations including "Deqi"(n= 5), tingling (n 5) and no-apparently-specific-feeling (NASF) were acquired by acupuncture stimulation and grouped. The needling sensations were evaluated by using Visual Analog Scale(VAS). The acquired image data of different needling-sensation groups were analyzed using SPM 2. 0 software in the Matlab Platform. RESULTS: After receiving acupuncture stimulation of SJ 5, 5 volunteers in the Waiguan (SJ 5) group experienced fee- lings of SNDH, with the mean VAS score being 4.23 +/- 1. 50, and 5 volunteers of the non-acupoint group had a tingling feeling, with the mean VAS score being 5.73 2.40. The VAS score of the tingling group was significantly higher than that of SNDH group (P<0. 05). Compared with the NASF control group, the activated cerebral areas were Brodmann area (BA) 7, 13, 20, 22, 39, 42 and BA 45 in the SNDH group, mainly involving the left temporal lobe, superior temporal gyrus, etc. and being obviously different to those of the control group (P<0. 001,k>10 voxels). The activated cerebral areas in the tingling group were BA 18, 19, 22, 24, 25, 32, 36, 40 and BA 45, predominantly involving the left limbic lobe, hippocampal gyrus, etc. and being apparently different to those of the control group (P<0. 001,k> 10 voxels). CONCLUSION: Acupuncture of Waiguan (SJ 5) mainly produces feelings of soreness, numbness, distending and heaviness, and the activated cerebral areas mainly involve the left temporal lobe, superior temporal gyrus, etc. ; while acupuncture of its neighboring non-acupoint chiefly induces a feeling of tingling in the healthy subjects, and the activated regions predominately involve the left limbic lobe, hippocampal gyrus, etc.

PEGylation markedly enhances the in vivo potency of recombinant human non-glycosylated erythropoietin: a comparison with glycosylated erythropoietin.

Recombinant human non-glycosylated erythropoietin (rh-ngEpo) expressed in E. coli was attached to polyethylene glycol (PEG) chains with different sizes and structures. The pharmacokinetic properties and in vivo potency of the PEGylated protein were investigated and comparisons were drawn between the conjugates and glycosylated recombinant Epo (rhEpo). The rh-ngEpo was modified with linear PEG-aldehyde (PEG-ALD, 20 kDa, 30 kDa, and 40 kDa) and a branched N-hydroxysuccinimide activated PEG (PEG(2)-NHS, 40 kDa). The monoPEGylated proteins were isolated by ion-exchange chromatography. The purified monoPEGylated conjugates suffered 6.5-86.1% loss of in vitro bioactivity compared to the unmodified rh-ngEpo. In addition, PEGylation remarkably increased the resistance of rh-ngEpo against plasma degradation. Pharmacokinetic studies showed that the plasma half-life of rh-ngEpo was increased 9.7-17.4 times by PEGylation, with the two 40k-PEG-rh-ngEpos-treated groups exhibiting better pharmacokinetic performances than rhEpo. Moreover, all the conjugates resulted in markedly enhanced Ret% (the percentage of reticulocyte count in red blood cells) compared with rh-ngEpo after subcutaneous injection. The two 40k-PEG conjugates demonstrated comparable in vivo efficacies compared with rhEpo. Overall, this research provides opportunities for the development of more cost-effective erythropoiesis-stimulating protein drugs.

[Primary structure determination of hirudin and reteplase fusion protein by LC/ESI-MS/MS spectrometry].

The aim is to determine the primary structure of a new hirudin and reteplase fusion protein (HV12p-rPA) by LC-ESI-MS/MS spectrometry. The molecular weight of the hirudin and reteplase fusion protein (HV12p-rPA) was measured by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The HV12p-rPA was digested with trypsin and chymotrypsin separately and the peptides in the digest mixtures were identified by LC-ESI-MS/MS. The molecular weight of HV12p-rPA was 41,472 Da, which was in accordance with the theoretical value. The peptide fragments of HV12p-rPA digested with trypsin were identified by LC-ESI-MS/MS spectrometry and the results indicated that the fusion protein contained r-PA. Then, the peptides of HV12p-rPA digested with chymotrypsin were identified by the same method. The results indicated that the fusion protein contained HV12p and the linker-containing peptide, DEGGGSY. MASDF and LDWIRDNMRP were identified as the N-terminal and C-terminal containing peptides in the chymotryptic digest mixture of the fusion protein. All of the Xcorr values exceeded 1.5, some of which were above 3.0, showing that the results were correct and credible and a sequence coverage of 85% was achieved. HPLC/MS analysis coupled with uncompleted digestion indicated that all these peptides were arranged with the correct order as expected. Thus, sequence of the fusion protein was confirmed and it was consistent with our design in upstream construction.

Identification of an oxidative refolding intermediate of recombinant consensus interferon from inclusion bodies and design of a two-stage strategy to promote correct disulfide-bond formation.

Dilution refolding of recombinant consensus IFN (interferon) from inclusion bodies suffers from low yield. A stable intermediate was found to mix with the correct product and to have an antiviral activity of less than 10% of the latter. This intermediate would form precipitates upon removal of the precipitation inhibitor arginine. Compared with the native protein, the intermediate moved more slowly on non-reducing SDS/PAGE. The CD and fluorescence spectra indicated that it had formed a native-like structure, but had only one disulfide bond: Cys(29)-Cys(139). Further evidence showed that the formation of Cys(29)-Cys(139) is specific and very likely to happen, even in the presence of a high concentration of reducing agent, whereas pairing of the other disulfide (Cys(1)-Cys(99)) needed a stronger oxidative condition. It competed with intermolecular disulfide bonding to form covalent oligomers. On the basis of this discovery, a two-stage refolding step strategy was designed that employed a modified dilution refolding step followed by a dialysis refolding step. The first stage used a high concentration of reducing agent together with the precipitation inhibitor arginine. The purpose was to hinder any reaction through Cys(1) or Cys(99) but allow the intramolecular disulfide bonding of Cys(29)-Cys(139). The second stage was a dialysis step that gradually increased the oxidative agent concentration and simultaneously decreased the arginine concentration. The refolding yield was increased from 35 to 82%, while the mass recovery was increased from 60 to 96%. Moreover, this strategy could suppress precipitation even after arginine was completely removed.

A cyanopropyl reversed phase chromatography enables resolution of huperzine A and B on analytical and preparative scales.

Huperzine A (HupA) and huperzine B (HupB) are two medically important components of Huperzia serrata. It is difficult to obtain high yields of the separation from the plant using conventional liquid extraction and chromatography. However, this study has found that RP chromatography with cyanopropyl (CN) medium was able to separate these two analogs simultaneously from the plant extract with higher resolution. Comparison between a CN medium and a popular C18 medium demonstrated the superiority of the CN over the C18 in resolution for both analytical and preparative separation of HupA and HupB. A preparative process was developed for simultaneous purification of HupA and HupB from H. serrata. The yields on the basis of the mass of the herbal powder for HupA and HupB were 0.019 and 0.008% respectively, which were about 1.9 and 10 times of those reported in the literature.