Co-expression of Dsb proteins enables soluble expression of a single-chain variable fragment (scFv) against human type 1 insulin-like growth factor receptor (IGF-1R) in E. coli

Type 1 insulin-like growth factor receptor (IGF-1R) is a promising therapeutic target for cancer treatment. A single-chain variable fragment (scFv) against human IGF-1R forms inclusion body when expressed in periplasmic space of E. coli routinely. Here, we described that co-expression of appropriate disulfide bonds (Dsb) proteins known to catalyze the formation and isomerization of Dsb can markedly recover the soluble expression of target scFv in E. coli. A 50 % recovery in solubility of the scFv was observed upon co-expression of DsbC alone, and a maximum solubility (80 %) was obtained when DsbA and DsbC were co-expressed in combination. Furthermore, the soluble scFv present full antigen-binding activity with IGF-1R, suggesting its correct folding. This study also suggested that the selection of Dsb proteins should be tested case-by-case if the approach of co-expression of Dsb system is adopted to address the problem of insoluble expression of proteins carrying Dsb.     

MiR-133a Is Functionally Involved in Doxorubicin-Resistance in Breast Cancer Cells MCF-7 via Its Regulation of the Expression of Uncoupling Protein 2

The development of novel targeted therapies holds promise for conquering chemotherapy resistance, which is one of the major hurdles in current breast cancer treatment. Previous studies indicate that mitochondria uncoupling protein 2 (UCP-2) is involved in the development of chemotherapy resistance in colon cancer and lung cancer cells. In the present study we found that lower level of miR133a is accompanied by increased expression of UCP-2 in Doxorubicin-resistant breast cancer cell cline MCF-7/Dox as compared with its parental cell line MCF-7. We postulated that miR133a might play a functional role in the development of Doxorubicin-resistant in breast cancer cells. In this study we showed that: 1) exogenous expression of miR133a in MCF-7/Dox cells can sensitize their reaction to the treatment of Doxorubicin, which is coincided with reduced expression of UCP-2; 2) knockdown of UCP-2 in MCF-7/Dox cells can also sensitize their reaction to the treatment of Doxorubicin; 3) intratumoral delivering of miR133a can restore Doxorubicin treatment response in Doxorubicin-resistant xenografts in vivo, which is concomitant with the decreased expression of UCP-2. These findings provided direct evidences that the miR133a/UCP-2 axis might play an essential role in the development of Doxorubicin-resistance in breast cancer cells, suggesting that the miR133a/UCP-2 signaling cohort could be served as a novel therapeutic target for the treatment of chemotherapy resistant in breast cancer.     

A novel thermophilic endo-β-1,4-mannanase from Aspergillus nidulans XZ3: functional roles of carbohydrate-binding module and Thr/Ser-rich linker region

The gene man5XZ3 from Aspergillus nidulans XZ3 encodes a multimodular β-mannanase of glycoside hydrolase family 5 that consists of a family 1 carbohydrate-binding module (CBM1), a Thr/Ser-rich linker region, and a catalytic domain. Recombinant Man5XZ3 and its two truncated derivatives, Man5ΔCBM (removing the CBM1) and Man5ΔCL (removing both the CBM1 and linker region), were produced in Pichia pastoris and showed significant variance in the secondary structure. The three enzymes had similar biochemical properties, such as optimal pH and temperature (pH 5.0 and 80 °C) and excellent pH stability at pH 4.0–10.0. Removal of the CBM1 alone could improve the thermostability of Man5XZ3, but further removal of the linker region resulted in worse thermostability. Man5XZ3 retained greater enzyme activity in the presence of an organic solvent (acetone), two detergents (SDS and Triton X-100), and a chaotropic agent (urea) compared with Man5ΔCBM and Man5ΔCL. This study provides an excellent β-mannanase candidate favorable for various industries and primarily demonstrates the relationship between enzyme structure and function.     

Dynamic modification of cortical orientation tuning mediated by recurrent connections

Receptive field properties of visual cortical neurons depend on the spatiotemporal context within which the stimuli are presented. We have examined the temporal context dependence of cortical orientation tuning using dynamic visual stimuli with rapidly changing orientations. We found that tuning to the orientation of the test stimulus depended on a briefly presented preceding stimulus, with the preferred orientation shifting away from the preceding orientation. Analyses of the spatial-phase dependence of the shift showed that the effect cannot be explained by purely feedforward mechanisms, but can be accounted for by activity-dependent changes in the recurrent interactions between different orientation columns. Thus, short-term plasticity of the intracortical circuit can mediate dynamic modification of orientation tuning, which may be important for efficient visual coding.     

Mesenchymal Stem Cell Transplantation Enhancement in Myocardial Infarction Rat Model under Ultrasound Combined with Nitric Oxide Microbubbles

Objective

This study evaluated the effects of ultrasound combined with the homemade nitric oxide (NO) micro-bubble destruction on the in vitro proliferation, apoptosis, and migration of mesenchymal stem cells (MSCs). Furthermore, we studied whether or not irradiation of the NO micro-bubble combined with bone-marrow derived MSC infusion had a better effect on treating myocardial infarction. The possible mechanism of MSC delivery into the infarcted myocardium was also investigated.

Methods

The murine bone marrow-derived MSCs were isolated, cultured, irradiated, and combined with different concentrations of NO microbubbles. MTT proliferation assay, annexin V-FITC apoptosis detection, migration assay, and RT-PCR were performed 24 h after the irradiation. The NO micro-bubbles was a intravenously injected, followed by the infusion of MSCs, which were labeled by CM-Dil. Myocardium was harvested 48 h later and the distribution of MSCs was observed by laser scanning confocal microscope after frozen sectioning. Echocardiography, histological examination, RT-PCR, and western blotting were performed four weeks after the cell transplantation.

Results

Ultrasound combined with 1:70 NO micro-bubbles had no significant impact on the proliferation or apoptosis of MSCs. Transwell chamber findings demonstrated that MSCs migrated more efficiently in group that underwent ultrasound combined with 1:70 NO micro-bubbles. The Real-time PCR results indicated that the expression of CXCR4 was much higher in the group undergoing ultrasound combined with 1:70 NO micro-bubbles. The normalized fluorescence intensity greatly increased in the group of US+NO micro-bubbles and the cardiac function was also markedly improved. Immunohistochemical staining showed that the capillary density was much greater in the group of US+NO micro-bubbles as compared to that of the other groups. RT-PCR and western blotting also revealed a higher SDF-1 and VEGF expression in the group of US+NO micro-bubbles.

Conclusions

NO micro-bubbles could be used in the cell transplantation, which efficiently promoted the MSC homing into the infarcted myocardium.     

Development and Evaluation of a Pseudovirus-Luciferase Assay for Rapid and Quantitative Detection of Neutralizing Antibodies against Enterovirus 71

The level of neutralizing antibodies (NtAb) induced by vaccine inoculation is an important endpoint to evaluate the efficacy of EV71 vaccine. In order to evaluate the efficacy of EV71 vaccine, here, we reported the development of a novel pseudovirus system expression firefly luciferase (PVLA) for the quantitative measurement of NtAb. We first evaluated and validated the sensitivity and specificity of the PVLA method. A total of 326 serum samples from an epidemiological survey and 144 serum specimens from 3 clinical trials of EV71 vaccines were used, and the level of each specimen''s neutralizing antibodies (NtAb) was measured in parallel using both the conventional CPE-based and PVLA-based assay. Against the standard neutralization assay based on the inhibition of the cytopathic effect (CPE), the sensitivity and specificity of the PVLA method are 98% and 96%, respectively. Then, we tested the potential interference of NtAb against hepatitis A virus, Polio-I, Polio-II, and Polio-III standard antisera (WHO) and goat anti-G10/CA16 serum, the PVLA based assay showed no cross-reactivity with NtAb against other specific sera. Importantly, unlike CPE based method, no live replication-competent EV71 is used during the measurement. Taken together, PVLA is a rapid and specific assay with higher sensitivity and accuracy. It could serve as a valuable tool in assessing the efficacy of EV71 vaccines in clinical trials and disease surveillance in epidemiology studies.     

Biocontrol of Postharvest Rhizopus Decay of Peaches with Pichia caribbica

A new yeast antagonist, Pichia caribbica, isolated in our laboratory from the soil collected from unsprayed orchards, was evaluated for its biocontrol capability against Rhizopus stolonifer on peaches and the possible mechanisms involved. The decay incidence and lesion diameter of Rhizopus decay of peaches treated by P. caribbica were significantly reduced compared with the control fruits, and the higher the concentration of P. caribbica, the better the efficacy of the biocontrol. Rapid colonization of the yeast in peach wounds stored at 25 °C was observed. In peaches, the activities of peroxidase (POD), catalase (CAT), and phenylalanine ammonia-lyase (PAL) were significantly induced by P. caribbica treatment compared to those of the control fruits. All these results indicated that P. caribbica has a great potential for the development of commercial formulations to control postharvest Rhizopus decay of peaches. Its modes of action were based on competition for space and nutrients with pathogens, inducement of activities of defense-related enzymes such as POD, CAT, and PAL of peaches.