Optimization of fermentation medium for xylanase-producing strain Xw2

To improve the fermentation yield of xylanase by optimizing the fermentation conditions for strain Xw2, a Plackett-Burman design was used to evaluate the effects of eight variables on xylanase production by strain Xw2. The steepest ascent （descent） method was used to approach the optimal response surface experimental area. The optimal fermentation conditions were obtained by central composite design and response surface analysis. The results showed that the composition of the optimal fermentation medium was corn cob ＋ 1.5% wheat bran （1：1）, 0.04% MnSO4, 0.04% K2HPO4. 3H2O, and an inoculum size of 6% in 50 mL liquid volume （pH = 6.0）. The optimal culture conditions were 28oc at 150 r/min for 54.23 h. The results of this study can serve as the basis for the industrial production and application of xylanase.     

Molecular Modeling-Based Inclusion Mechanism and Stability Studies of Doxycycline and Hydroxypropyl-β-Cyclodextrin Complex for Ophthalmic Delivery

The aim of the present study was to prepare a stable complex of doxycycline (Doxy) and hydroxypropyl-β-cyclodextrin (HPβCD) for ophthalmic delivery and investigate the inclusion mechanism and the inclusion effects on the stability of Doxy. The Doxy/HPβCD complex was prepared by solution stirring and then characterized by scanning electron microscopy and ultraviolet spectroscopy. Based on results of nuclear magnetic resonance, molecular model of Doxy/HPβCD complex was established using computational simulation of PM3 method implemented in Gaussian 03. Stabilities of Doxy/HPβCD complex in both aqueous solution and solid state at 25°C were evaluated by HPLC. Finally, in vitro antibacterial activity of the Doxy/HPβCD complex was evaluated by disk diffusion test. It was found that the stabilities of Doxy/HPβCD complex in both aqueous solution and solid state were improved obviously as compared with Doxy alone. This stability enhancement is consistent with the inclusion mechanism between HPβCD and Doxy, which showed that the unstable site of Doxy molecule at 6-CH₃ was protected in the hydrophobic cavity of HPβCD, additionally, the chelation of Mg²⁺ provided a synergetic protection of the other unstable site of Doxy at 4-N(CH₃)₂. The antibacterial activity results indicated that Doxy/HPβCD complex might have potential for clinical applications.     

A Fast Workflow for Identification and Quantification of Proteomes

The current in-depth proteomics makes use of long chromatography gradient to get access to more peptides for protein identification, resulting in covering of as many as 8000 mammalian gene products in 3 days of mass spectrometer running time. Here we report a fast sequencing (Fast-seq) workflow of the use of dual reverse phase high performance liquid chromatography - mass spectrometry (HPLC-MS) with a short gradient to achieve the same proteome coverage in 0.5 day. We adapted this workflow to a quantitative version (Fast quantification, Fast-quan) that was compatible to large-scale protein quantification. We subjected two identical samples to the Fast-quan workflow, which allowed us to systematically evaluate different parameters that impact the sensitivity and accuracy of the workflow. Using the statistics of significant test, we unraveled the existence of substantial falsely quantified differential proteins and estimated correlation of false quantification rate and parameters that are applied in label-free quantification. We optimized the setting of parameters that may substantially minimize the rate of falsely quantified differential proteins, and further applied them on a real biological process. With improved efficiency and throughput, we expect that the Fast-seq/Fast-quan workflow, allowing pair wise comparison of two proteomes in 1 day may make MS available to the masses and impact biomedical research in a positive way.The performance of mass spectrometry has been improved tremendously over the last few years (1–3), making mass spectrometry-based proteomics a viable approach for large-scale protein analysis in biological research. Scientists around the world are striving to fulfill the promise of identifying and quantifying almost all gene products expressed in a cell line or tissue. This would make mass spectrometry-based protein analysis an approach that is compatible to the second-generation mRNA deep-seq technique (4, 5).Two liquid chromatography (LC)-MS strategies have been employed to achieve deep proteome coverage. One is a single run with a long chromatography column and gradient to take advantage of the resolving power of HPLC to reduce the complexity of peptide mixtures; the other is a sequential run with two-dimensional separation (typically ion-exchange and reverse phase) to reduce peptide complexity. It was reported by two laboratories that 2761 and 4500 proteins were identified with a 10 h chromatography gradient on a dual pressure linear ion-trap orbitrap mass spectrometer (LTQ Orbitrap Velos)(6–8). Similarly, 3734 proteins were identified using a 8 h gradient on a 2 m long column with a hybrid triple quadrupole - time of flight (Q-TOF, AB sciex 5600 Q-TOF)(9) mass spectrometer. The two-dimensional approach has yielded more identification with longer time. For example, 10,006 proteins (representing over 9000 gene products, GPs)¹ were identified in U2OS cell (10), and 10,255 proteins (representing 9207 GPs) from HeLa cells (11). It took weeks (for example, 2–3 weeks) of machine running time to achieve such proteome coverage, pushing proteome analysis to the level that is comparable to mRNA-seq. With the introduction of faster machines, human proteome coverage now has reached the level of 7000–8500 proteins (representing 7000–8000 GPs) in 3 days (12). Notwithstanding the impressive improvement, the current approach using long column and long gradient suffers from inherent limitations: it takes long machine running time and it is challenging to keep reproducibility among repeated runs. Thus, current throughput and reproducibility have hindered the application of in-depth proteomics to traditional biological researches. A timesaving approach is in urgent need.In this study, we used the first-dimension (1D) short pH 10 RP prefractionation to reduce the complexity of the proteome (13), followed by sequential 30 min second-dimension (2D) short pH 3 reverse phase-(RP)-LC-MS/MS runs for protein identification (14). The results demonstrated that it is possible to identify 8000 gene products from mammalian cells within 12 h of total MS measurement time by applying this dual-short 2D-RPLC-MS/MS strategy (Fast sequencing, Fast-seq). The robustness of the strategy was revealed by parallel testing on different MS systems including quadrupole orbitrap mass spectrometer (Q-Exactive), hybrid Q-TOF (Triple-TOF 5600), and dual pressure linear ion-trap orbitrap mass spectrometer (LTQ-Orbitrap Velos), indicating the inherent strength of the approach as to merely taking advantage of the better MS instruments. This strategy increases the efficiency of MS sequencing in unit time for the identification of proteins. We achieved identification of 2200 proteins/30 mins on LTQ-Orbitrap Velos, 2800 proteins/30 mins on Q-Exactive and Triple-TOF 5600 respectively. We further optimized Fast-seq and worked out a quantitative-version of the Fast-seq workflow: Fast-quantification (Fast-quan) and applied it for protein abundance quantification in HUVEC cell that was treated with a drug candidate MLN4924 (a drug in phase III clinical trial). We were able to quantify > 6700 GPs in 1 day of MS running time and found 99 proteins were up-regulated with high confidence. We expect this efficient alternative approach for in-depth proteome analysis will make the application of MS-based proteomics more accessible to biological applications.     

Mining Proteins with Non-Experimental Annotations Based on an Active Sample Selection Strategy for Predicting Protein Subcellular Localization

Subcellular localization of a protein is important to understand proteins’ functions and interactions. There are many techniques based on computational methods to predict protein subcellular locations, but it has been shown that many prediction tasks have a training data shortage problem. This paper introduces a new method to mine proteins with non-experimental annotations, which are labeled by non-experimental evidences of protein databases to overcome the training data shortage problem. A novel active sample selection strategy is designed, taking advantage of active learning technology, to actively find useful samples from the entire data pool of candidate proteins with non-experimental annotations. This approach can adequately estimate the “value” of each sample, automatically select the most valuable samples and add them into the original training set, to help to retrain the classifiers. Numerical experiments with for four popular multi-label classifiers on three benchmark datasets show that the proposed method can effectively select the valuable samples to supplement the original training set and significantly improve the performances of predicting classifiers.     

Community Based Promotion on VCT Acceptance among Rural Migrants in Shanghai,China

Objective

Voluntary counseling and testing (VCT) plays an important integral role in response to the HIV/AIDS epidemic. However, VCT service has not been effectively utilized among rural migrants, a high risk group in China. In this study, we developed a community based intervention to examine if community mobilization with comprehensive VCT is more effective than current HIV preventions with routine VCT service in promoting VCT acceptability among rural migrants in Shanghai, China.

Methods

A comprehensive intervention with community mobilization and comprehensive VCT services including community-based VCT and mobile VCT was implemented during 2007–2009. Three communities in Minhang District of Shanghai were randomly selected and were designed to receive community mobilization and comprehensive VCT, traditional VCT and none intervention, respectively. After 24 months intervention, effects were evaluated by comparing outcome indicators between the baseline (2,690 participants) and follow-up surveys (1,850 participants).

Findings

A substantial increase in VCT acceptance was observed among community mobilization group (94.9% vs. 88.5%, P<0.001), whereas the reverse effect was seen in the traditional VCT group (86.1% vs. 94.6%, P<0.001) and control group (69.0% vs. 91.7%, P<0.001). Rural migrants from community mobilization group were more likely to accept VCT (OR = 2.91, 95% CI 1.69–4.97). Rural migrants from community mobilization group also showed significant increase in HIV/AIDS knowledge, positive attitude towards HIV positive individuals and condom use.

Conclusion

Community mobilization with comprehensive VCT has significant impact on promotion of VCT acceptance and utilization among rural migrants in Shanghai. These findings provide evidence to support community mobilization as a suitable strategy for VCT promotion among rural migrants in Shanghai, China.     

Msb1 Interacts with Cdc42, Boi1, and Boi2 and May Coordinate Cdc42 and Rho1 Functions during Early Stage of Bud Development in Budding Yeast

Msb1 is not essential for growth in the budding yeast Saccharomyces cerevisiae since msb1Δ cells do not display obvious phenotypes. Genetic studies suggest that Msb1 positively regulates Cdc42 function during bud development, since high-copy MSB1 suppressed the growth defect of temperature-sensitive cdc24 and cdc42 mutants at restrictive temperature, while deletion of MSB1 showed synthetic lethality with cdc24, bem1, and bem2 mutations. However, the mechanism of how Msb1 regulates Cdc42 function remains poorly understood. Here, we show that Msb1 localizes to sites of polarized growth during bud development and interacts with Cdc42 in the cells. In addition, Msb1 interacts with Boi1 and Boi2, two scaffold proteins that also interact with Cdc42 and Bem1. These findings suggest that Msb1 may positively regulate Cdc42 function by interacting with Cdc42, Boi1, and Boi2, which may promote the efficient assembly of Cdc42, Cdc24, and other proteins into a functional complex. We also show that Msb1 interacts with Rho1 in the cells and Msb1 overproduction inhibits the growth of rho1-104 and rho1-3 but not rho1-2 cells. The growth inhibition appears to result from the down-regulation of Rho1 function in glucan synthesis, specifically during early stage of bud development. These results suggest that Msb1 may coordinate Cdc42 and Rho1 functions during early stage of bud development by promoting Cdc42 function and inhibiting Rho1 function. Msb1 overproduction also affects cell morphology, septin organization, and causes increased, aberrant deposition of 1,3-β-glucan and chitin at the mother-bud neck. However, the stimulation of glucan synthesis mainly occurs during late, but not early, stage of bud development.     

Compromised NK Cell-Mediated Antibody-Dependent Cellular Cytotoxicity in Chronic SIV/SHIV Infection

Increasing evidence indicates that antibody-dependent cellular cytotoxicity (ADCC) contributes to the control of HIV/SIV infection. However, little is known about the ADCC function of natural killer (NK) cells in non-human primate model. Here we demonstrated that ADCC function of NK cells was significantly compromised in chronic SIV/SHIV infection, correlating closely with the expression of FcγRIIIa receptor (CD16) on NK cells. CD32, another class of IgG Fc receptors, was identified on NK cells with higher expression in the infected macaques and the blockade of CD32 impacted the ability of NK cells to respond to antibody-coated target cells. The inhibition of matrix metalloproteases (MMPs), a group of enzymes normally involved in tissue/receptor remodeling, could restore NK cell-mediated ADCC with increased CD16 expression on macaque NK cells. These data offer a clearer understanding of NK cell-mediated ADCC in rhesus macaques, which will allow us to evaluate the ADCC repertoire arising from preclinical vaccination studies in non-human primates and inform us in the future design of effective HIV vaccination strategies.     

Cyanine 5.5 Conjugated Nanobubbles as a Tumor Selective Contrast Agent for Dual Ultrasound-Fluorescence Imaging in a Mouse Model

Nanobubbles and microbubbles are non-invasive ultrasound imaging contrast agents that may potentially enhance diagnosis of tumors. However, to date, both nanobubbles and microbubbles display poor in vivo tumor-selectivity over non-targeted organs such as liver. We report here cyanine 5.5 conjugated nanobubbles (cy5.5-nanobubbles) of a biocompatible chitosan–vitamin C lipid system as a dual ultrasound-fluorescence contrast agent that achieved tumor-selective imaging in a mouse tumor model. Cy5.5-nanobubble suspension contained single bubble spheres and clusters of bubble spheres with the size ranging between 400–800 nm. In the in vivo mouse study, enhancement of ultrasound signals at tumor site was found to persist over 2 h while tumor-selective fluorescence emission was persistently observed over 24 h with intravenous injection of cy5.5-nanobubbles. In vitro cell study indicated that cy5.5-flurescence dye was able to accumulate in cancer cells due to the unique conjugated nanobubble structure. Further in vivo fluorescence study suggested that cy5.5-nanobubbles were mainly located at tumor site and in the bladder of mice. Subsequent analysis confirmed that accumulation of high fluorescence was present at the intact subcutaneous tumor site and in isolated tumor tissue but not in liver tissue post intravenous injection of cy5.5-nanobubbles. All these results led to the conclusion that cy5.5-nanobubbles with unique crosslinked chitosan–vitamin C lipid system have achieved tumor-selective imaging in vivo.     

δ-Opioid Receptor Activation Modified MicroRNA Expression in the Rat Kidney under Prolonged Hypoxia

Hypoxic/ischemic injury to kidney is a frequently encountered clinical problem with limited therapeutic options. Since microRNAs are differentially involved in hypoxic/ischemic events and δ-opioid receptor (DOR) activation is known to protect against hypoxic/ischemic injury, we speculated on the involvement of DOR activation in altering the microRNA (miRNA) expression in kidney under hypoxic condition. We selected 31 miRNAs based on microarray data for quantitative PCR analysis. Among them, 14 miRNAs were significantly altered after prolonged hypoxia, DOR activation or a combination of both. We found that 1) DOR activation alters miRNA expression profiles in normoxic conditions; 2) hypoxia differentially alters miRNA expression depending on the duration of hypoxia; and 3) DOR activation can modify hypoxia-induced changes in miRNA expression. For example, 10-day hypoxia reduced the level of miR-212 by over 70%, while DOR activation could mimic such reduction even in normoxic kidney. In contrast, the same stress increased miR-29a by >100%, which was reversed following DOR activation. These first data suggest that hypoxia comprehensively modifies the miRNA profile within the kidney, which can be mimicked or modified by DOR activation. Ascertaining the targeted pathways that regulate the diverse cellular and molecular functions of miRNA may provide new insights into potential therapies for hypoxic/ischemic injury of the kidney.