Highly Efficient Production of Soluble Proteins from Insoluble Inclusion Bodies by a Two-Step-Denaturing and Refolding Method

The production of recombinant proteins in a large scale is important for protein functional and structural studies, particularly by using Escherichia coli over-expression systems; however, approximate 70% of recombinant proteins are over-expressed as insoluble inclusion bodies. Here we presented an efficient method for generating soluble proteins from inclusion bodies by using two steps of denaturation and one step of refolding. We first demonstrated the advantages of this method over a conventional procedure with one denaturation step and one refolding step using three proteins with different folding properties. The refolded proteins were found to be active using in vitro tests and a bioassay. We then tested the general applicability of this method by analyzing 88 proteins from human and other organisms, all of which were expressed as inclusion bodies. We found that about 76% of these proteins were refolded with an average of >75% yield of soluble proteins. This “two-step-denaturing and refolding” (2DR) method is simple, highly efficient and generally applicable; it can be utilized to obtain active recombinant proteins for both basic research and industrial purposes

Electroacupuncture Ameliorates Learning and Memory via Activation of the CREB Signaling Pathway in the Hippocampus to Attenuate Apoptosis after Cerebral Hypoperfusion

Studies have shown that electroacupuncture (EA) ameliorates learning and memory after ischemic injury. However, there have been few studies elucidating the mechanisms of EA on learning and memory in cerebral hypoperfusion. In this study, we explored the cAMP response element-binding protein (CREB) signaling pathway-mediated antiapoptotic action involved in EA-induced improvement of learning and memory. EA at GV20 and GV14 acupoints was applied in cerebral hypoperfusion rats. A Morris water maze task was performed, and the immunoreactivities of pCREB, Bcl-2, and Bax in the hippocampal CA1 area were evaluated by the Western blotting technique. Our findings indicated that (1) EA ameliorated spatial learning and memory impairment in cerebral hypoperfusion rats; (2) EA increased the immunoreactivities of pCREB and Bcl-2 and decreased the immunoreactivity of Bax; (3) intracerebroventricular administration of H89 (the inhibitor of protein kinase A) blocked EA-induced, pCREB-mediated antiapoptotic action and improved learning and memory. These results suggest that EA can ameliorate learning and memory via activation of the CREB signaling pathway in the hippocampus to attenuate apoptosis after cerebral hypoperfusion

Research Progress in Components, Structure and Functions of Human Milk and Bovine Milk Fat Globules

As an important nutrient in milk, fat exists in the form of milk fat globules. Cow’s milk is an important substitute for breast milk, but the difference between its milk fat globules and those of human milk remains to be clarified. In this paper, the main differences in protein composition, lipid composition, structure and function between cow’s and human milk fat globules are reviewed. The types of milk fat globule membrane proteins (MFGMPs) in human milk are significantly more than those in cow’s milk, and there are also obvious differences in the abundance of some special proteins. The composition and distribution of unsaturated and saturated fatty acids in cow’s milk and human milk are also different. Sphingomyelin is more abundant in human milk phospholipids, but in cow’s milk, lecithin is the main phospholipid. In all mammalian milk, the core of the lipid structure is triglyceride, encapsulated by a complex three-layer membrane. In terms of composition and structure, there is heterogeneity in milk fat globule membrane (MFGM) between the same species and different species. By summarizing the differences between human milk and cow’s milk fat globules, this review aims to increase the utilization rate and value of milk MFGM, to improve the structure of simulated milk fat globules, and to further optimize infant formula

High-throughput Sequencing to Analyze Changes in the Structural Diversity of the Flora of Cheddar Cheese during Processing

In order to clarify the microflora structure in Cheddar cheese processing, MiSeq high-throughput sequencing technology was used to analyze the community structure of Cheddar cheese at three stages of processing (post-pasteurization, curdling, and ripening 0, 30, 60 and 90 d) in this study. The results showed that the community structure varies widely of cheddar cheese during processing. The highest microbial community diversity and abundance were found after pasteurization (Chao1 index and Shannon index mean values were 6.09 and 1415.78, respectively). The dominant microflora in the pasteurization stage at the genus level was Stenotrophomonas (21.04%). The community structure was relatively similar in the curd and ripening stages, Lactococcus were the dominant flora in both stages, with abundance averaging more than 85%. During the ripening period, the relative abundance of Lactococcus increased first and then decreased. The community structure in the pasteurized cheeses was different compared to the other groups, and there was less change in the community structure of the groups during the ripening period. This study provides a basis for clarifying the community structure of Cheddar cheese, and has a certain reference value for the expansion of Cheddar cheese microbiome information

Ultra-low-dose spectral-detector computed tomography for the accurate quantification of pulmonary nodules: an anthropomorphic chest phantom study

PURPOSETo assess the quantification accuracy of pulmonary nodules using virtual monoenergetic images (VMIs) derived from spectral-detector computed tomography (CT) under an ultra-low-dose scan protocol.METHODSA chest phantom consisting of 12 pulmonary nodules was scanned using spectral-detector CT at 100 kVp/10 mAs, 100 kVp/20 mAs, 120 kVp/10 mAs, and 120 kVp/30 mAs. Each scanning protocol was repeated three times. Each CT scan was reconstructed utilizing filtered back projection, hybrid iterative reconstruction, iterative model reconstruction (IMR), and VMIs of 40–100 keV. The signal-to-noise ratio and air noise of images, absolute differences, and absolute percentage measurement errors (APEs) of the diameter, density, and volume of the four scan protocols and ten reconstruction images were compared.RESULTSWith each fixed reconstruction image, the four scanning protocols exhibited no significant differences in APEs for diameter and density (all P > 0.05). Of the four scan protocols and ten reconstruction images, APEs for nodule volume had no significant differences (all P > 0.05). At 100 kVp/10 mAs, APEs for density using IMR were the lowest (APE-mean: 6.69), but no significant difference was detected between VMIs at 50 keV (APE-mean: 11.69) and IMR (P = 0.666). In the subgroup analysis, at 100 kVp/10 mAs, there were no significant differences between VMIs at 50 keV and IMR in diameter and density (all P > 0.05). The radiation dose at 100 kVp/10 mAs was reduced by 77.8% compared with that at 120 kVp/30 mAs.CONCLUSIONCompared with IMR, reconstruction at 100 kVp/10 mAs and 50 keV provides a more accurate quantification of pulmonary nodules, and the radiation dose is reduced by 77.8% compared with that at 120 kVp/30 mAs, demonstrating great potential for ultra-low-dose spectral-detector CT

Pre-Treatment with Melatonin Enhances Therapeutic Efficacy of Cardiac Progenitor Cells for Myocardial Infarction

Background/Aims: Melatonin possesses many biological activities such as antioxidant and anti-aging. Cardiac progenitor cells (CPCs) have emerged as a promising therapeutic strategy for myocardial infarction (MI). However, the low survival of transplanted CPCs in infarcted myocardium limits the successful use in treating MI. In the present study, we aimed to investigate if melatonin protects against oxidative stress-induced CPCs damage and enhances its therapeutic efficacy for MI. Methods: TUNEL assay and EdU assay were used to detect the effects of melatonin and miR-98 on H2O2-induced apoptosis and proliferation. MI model was used to evaluate the potential cardioprotective effects of melatonin and miR-98. Results: Melatonin attenuated H2O2-induced the proliferation reduction and apoptosis of c-kit+ CPCs in vitro, and CPCs which pretreated with melatonin significantly improved the functions of post-infarct hearts compared with CPCs alone in vivo. Melatonin was capable to inhibit the increase of miR-98 level by H2O2 in CPCs. The proliferation reduction and apoptosis of CPCs induced by H2O2 was aggravated by miR-98. In vivo, transplantation of CPCs with miR-98 silencing caused the more significant improvement of cardiac functions in MI than CPCs. MiR-98 targets at the signal transducer and activator of the transcription 3 (STAT3), and thus aggravated H2O2-induced the reduction of Bcl-2 protein. Conclusions: Pre-treatment with melatonin protects c-kit+ CPCs against oxidative stress-induced damage via downregulation of miR-98 and thereby increasing STAT3, representing a potentially new strategy to improve CPC-based therapy for MI

Do Animations Impair Executive Function in Young Children? Effects of Animation Types on the Executive Function of Children Aged Four to Seven Years

This study used a three (animation types: educational, entertainment, and control groups) × four (age group: four-, five-, six-, and seven-year-olds) between-group experimental design to investigate the short-term effects of animation type and age on each component of children’s executive function (EF) (inhibitory control [IC], cognitive flexibility [CF], and working memory [WM]). One hundred twenty-six kindergarten and first-grade elementary school students in a city in Henan Province of China were selected for the experimental study. The results showed that briefly watching animation affected children’s EF. Specifically, watching entertainment cartoons weakened children’s IC and CF, while cartoons did not affect children’s WM. The moderating effect of age in the relationship between animation type and EFs was non-significant. This study suggests that researchers should focus on the uniqueness of each component of EF in children aged four to seven years, and parents should try to limit children’s viewing of animation, especially entertainment animation

Detection of Type, Blended Ratio, and Mixed Ratio of Pu’er Tea by Using Electronic Nose and Visible/Near Infrared Spectrometer

The objective of this study was to find an intelligent and fast method to detect the type, blended ratio, and mixed ratio of ancient Pu’er tea, which is significant in maintaining order in the Pu’er tea industry. An electronic nose (E-nose) and a visible near infrared spectrometer (VIS/NIR spectrometer) were applied for tea sampling. Feature extraction was conducted using both the traditional method and a convolutional neural network (CNN) technique. Linear discriminant analysis (LDA) and partial least square regression (PLSR) were applied for pattern recognition. After sampling while using the traditional method, the analysis of variance (ANOVA) results showed that the mean differential value of each sensor should be selected as the optimal feature extraction method for E-nose data, and raw data comparison results showed that 19 peak/valley values and two slope values were extracted. While the format of E-nose data was in accord with the input format for CNN, the VIS/NIR spectrometer data required matrixing to meet the format requirements. The LDA and PLSR analysis results showed that CNN has superior detection ability, being able to acquire more local features than the traditional method, but it has the risk of mixing in redundant information, which can act to reduce the detection ability. Multi-source information fusion (E-nose and VIS/NIR spectrometer fusion) can collect more features from different angles to improve the detection ability, but it also contains the risk of adding redundant information, which reduces the detection ability. For practical detection, the type of Pu’er tea should be recognizable using a VIS/NIR spectrometer and the traditional feature extraction method. The blended ratio of Pu’er tea should also be identifiable by using a VIS/NIR spectrometer with traditional feature extraction. Multi-source information fusion with traditional feature extraction should be used if the accuracy requirement is extremely high; otherwise, a VIS/NIR spectrometer with traditional feature extraction is preferred

The trafficking machinery of lytic and protein storage vacuoles: how much is shared and how much is distinct?

Plant cells contain two types of vacuoles, the lytic vacuole (LV) and protein storage vacuole (PSV). LVs are present in vegetative cells, whereas PSVs are found in seed cells. The physiological functions of the two types of vacuole differ. Newly synthesized proteins must be transported to these vacuoles via protein trafficking through the endomembrane system for them to function. Recently, significant advances have been made in elucidating the molecular mechanisms of protein trafficking to these organelles. Despite these advances, the relationship between the trafficking mechanisms to the LV and PSV remains unclear. Some aspects of the trafficking mechanisms are common to both types of vacuole, but certain aspects are specific to trafficking to either the LV or PSV. In this review, we summarize recent findings on the components involved in protein trafficking to both the LV and PSV and compare them to examine the extent of overlap in the trafficking mechanisms. In addition, we discuss the interconnection between the LV and PSV provided by the protein trafficking machinery and the implications for the identity of these organelles.11Nsciescopu