Antimicrobial activity of the recombinant peptide Melittin-Thanatin with three glycine to tryptophan mutations.

The antimicrobial peptide was considered an important target for developing novel antibacterial drugs. However, the unstable biological activity and the low antibacterial activity are challenges for the application of recombinant proteins. In this study, the fusion peptide of Melittin-Thanatin (MT) was designed and produced, and its derivative sequence (MT-W) was obtained by replacing three glycines (Gly, G) with tryptophan (Trp, W). The MT-W peptide were synthesized in Bacillus subtilis WB700 by EDDIE self-cleavage protein fusion. Compared with MT, MT-W exhibited 2-4 times higher antibacterial rate against Escherichia coli K88. In addition, MT-W showed lower cytotoxicity (IC50 > 300 mg·L-1) to the red blood cell, and more stable biological activities under the conditions of different temperatures (20, 30, 40, 50, 60, 70, 80, and 90 °C), pH values (2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0) and different proteases. Especially, MT-W showed a broader antibacterial effect on three drug-resistant strains than florfenicol and oxytetracycline calcium. In conclusion, compared with MT, the MT-W showed increased antibacterial activity, stability, lower cytotoxicity, and broader antimicrobial effect. Therefore, it would become a promising alternative to conventional antibiotics.

Improving the Performance of Time-Relative GNSS Precise Positioning in Remote Areas.

Global navigation satellite systems (GNSS) can attain centimeter level positioning accuracy, which is conventionally provided by real-time precise point positioning (PPP) and real-time kinematic (RTK) techniques. Corrections from the data center or the reference stations are required in these techniques to reduce various GNSS errors. The time-relative positioning approach differs from the traditional PPP and RTK in the sense that it does not require external real-time corrections. It computes the differences in positions of a single receiver at different epochs using phase observations. As the code observations are not used in this approach, its performance is not affected by the noise and multipath of code observations. High reliability is another advantage of time-relative precise positioning because the ambiguity resolution is not needed in this approach. Since the data link is not required in the method, this approach has been widely used in remote areas where wireless data link is not available. The main limitation of time-relative positioning is that its accuracy degrades over time between epochs because of the temporal variation of various errors. The application of the approach is usually limited to be within a time interval of less than 20 min. The purpose of this study was to increase the time interval of time-relative positioning and to extend the use of this method to applications with a longer time requirement, especially in remote areas without wireless communication. In this paper, the main error sources of the time-relative method are first analyzed in detail, and then the approach to improve the accumulated time relative positioning method is proposed. The performance of the proposed method is assessed using both static and dynamic observations with a duration as long as several hours. The experiments presented in this paper show that, among the four scenarios tested (i.e., GPS, GPS/Galileo, GPS/Galileo/BeiDou, and GPS/Galileo/BeiDou/GLONASS), GPS/Galileo/BeiDou performed best and GPS/Galileo/BeiDou/GLONASS performed worst. The maximum positioning errors were mostly within 0.5 m in the horizontal direction, even after three hours with GPS/Galileo/BeiDou. It is expected that the method could be used for positioning and navigation for as long as several hours with decimeter level horizontal accuracy in remote areas without wireless communication.

An Improved Long-Period Precise Time-Relative Positioning Method Based on RTS Data.

The high precision positioning can be easily achieved by using real-time kinematic (RTK) and precise point positioning (PPP) or their augmented techniques, such as network RTK (NRTK) and PPP-RTK, even if they also have their own shortfalls. A reference station and datalink are required for RTK or NRTK. Though the PPP technique can provide high accuracy position data, it needs an initialisation time of 10-30 min. The time-relative positioning method estimates the difference between positions at two epochs by means of a single receiver, which can overcome these issues within short period to some degree. The positioning error significantly increases for long-period precise positioning as consequence of the variation of various errors in GNSS (Global Navigation Satellite System) measurements over time. Furthermore, the accuracy of traditional time-relative positioning is very sensitive to the initial positioning error. In order to overcome these issues, an improved time-relative positioning algorithm is proposed in this paper. The improved time-relative positioning method employs PPP model to estimate the parameters of current epoch including position vector, float ionosphere-free (IF) ambiguities, so that these estimated float IF ambiguities are used as a constraint of the base epoch. Thus, the position of the base epoch can be estimated by means of a robust Kalman filter, so that the position of the current epoch with reference to the base epoch can be obtained by differencing the position vectors between the base epoch and the current one. The numerical results obtained during static and dynamic tests show that the proposed positioning algorithm can achieve a positioning accuracy of a few centimetres in one hour. As expected, the positioning accuracy is highly improved by combining GPS, BeiDou and Galileo as a consequence of a higher amount of used satellites and a more uniform geometrical distribution of the satellites themselves. Furthermore, the positioning accuracy achieved by using the positioning algorithm here described is not affected by the initial positioning error, because there is no approximation similar to that of the traditional time-relative positioning. The improved time-relative positioning method can be used to provide long-period high precision positioning by using a single dual-frequency (L1/L2) satellite receiver.

A Regulatory Circuit Orchestrated by Novel-miR-3880 Modulates Mammary Gland Development.

Milk casein and triglyceride content are important production traits in goats. Studies on mechanisms in milk casein secretion and mammary gland development is essential for milk goat breeding. miRNAs play an important role in goat lactation. While novel-miR-3880 is highly expressed at goat peak lactation stage, its molecular mechanism has not been studied. The purpose of the present study was to explore the relationship between novel-miR-3880 and lactation, as well as to construct a network among novel-miR-3880, ciRNA13761, and E74 like ETS transcription factor 2 (ELF2), thus further exploring their potential roles in milk components and mammary gland development. ELF2 was previously proven to be important in cell survival and proliferation, and 3'-UTR of ELF2 was predicted to have binding sites of novel-miR-3880. Our study found that the overexpression of novel-miR-3880 exerted anti-apoptotic and proliferative roles in GMEC, induced a boost in triglyceride synthesis, and caused a decrease in α s1-, α s2-, and ß-casein, but an increase in κ-casein secretion. Furthermore, treatment in mice indicated that novel-miR-3880 could promote mammary gland development and extend the lactation period, while novel-miR-3880 expression was found to be suppressed by ciRNA13761 as a miRNA sponge. The present study explores a mechanism of triglyceride synthesis and casein secretion, and reveals a crosstalk between ciRNA13761/novel-miR-3880/ELF2 axis and PI3K/AKT/mTOR/S6K1 pathway, to gain a better understanding of lactation traits in dairy goats.

A New DGNSS Positioning Infrastructure for Android Smartphones.

One's position has become an important piece of information for our everyday lives in a smart city. Currently, a position can be obtained easily using smartphones that is equipped with low-cost Global Navigation Satellite System (GNSS) chipsets with accuracy varying from 5 m to 10 m. Differential GNSS (DGNSS) is an efficient technology that removes the majority of GNSS errors with the aid of reference stations installed at known locations. The sub-meter accuracy can be achieved when applying the DGNSS technology on the advanced receivers. In 2016, Android has opened the accesses of raw GNSS measurements to developers. However, most of the mid and low-end smartphones only provide the data using the National Marine Electronics Association (NMEA) protocol. They do not provide the raw measurements, and thus do not support the DGNSS operation either. We proposed a DGNSS infrastructure that correct the standalone GNSS position of smartphones using the corrections from the reference station. In the infrastructure, the position correction is generated considering the GNSS satellite IDs that contribute to the standalone solution in smartphones, and the position obtained is equivalent to the solution of using the range-domain correction directly. To serve a large number of smartphone users, a Client/Server architecture is developed to cope with a mass of DGNSS positioning requests efficiently. The comparison of the proposed infrastructure against the ground truth, for all field tests in open areas, showed that the infrastructure achieves the horizontal positioning accuracy better than 2 m. The improvement in accuracy can reach more than 50% for the test in the afternoon. The infrastructure brings benefits to applications that require more accuracy without requiring any hardware modifications.

Characteristics of BeiDou Navigation Satellite System (BDS) Code Observations for Different Receiver Types and Their Influence on Wide-Lane Ambiguity Resolution.

The Chinese BeiDou Navigation Satellite System (BDS) has been an important constitute of the Global Navigation Satellite System (GNSS), and the combination of GPS and BDS shows significant improvements when compared with single GPS system for real-time kinematic (RTK) positioning, and improves on availability and fixing rates, especially in the East Asian area. While network RTK might have different types of receivers, both for global and regional networks, different types of receiver may adopt different internal multipath mitigation methods and other techniques that result in different pseudorange characteristics, especially for a multipath. Then, the performance of wide-lane ambiguity resolution (WL AR) is affected. In this study, we first analyze and compare the characteristics of BDS dual-frequency observations for different types of receivers, including Trimble, Leica, Javad, and Septentrio, based on multipath (MP) observables, and then we assess their influence on double-differenced (DD) WL AR. The numerical results show that an obvious low-frequency component exists in MP observables of BDS geostationary earth-orbit satellites (GEOs) for Leica receivers, while its high-frequency measurement noise is very small. For geosynchronous orbit satellites (IGSOs) and medium earth-orbit satellites (MEOs), a slight fluctuation can also be observed that is similar to that of GPS satellites, except for the satellite-included code bias. In Trimble, Javad, and Septentrio receivers, the MP series are dominated by high-frequency measurement noise, both for GEOs and non-GEOs, except for satellite-included code bias. Furthermore, the characteristic of Leica receivers for BDS GEOs seriously affects WL AR and, even for a short baseline, it takes a long time for WL ambiguities to converge, or not converge for many GEO-related DD WL ambiguities, while Trimble, Javad, and Septentrio receivers perform well for short and medium baselines. Then, a time-difference method is proposed to mitigate the multipath of BDS GEOs for a Leica receiver. After applying the proposed method, WL ambiguity fixing rates of GEO-related satellite pairs are improved significantly and the convergence time is shortened from several hours to ten minutes.

Improvement Activity of 1-Deoxynojirimycin in the Growth of Dairy Goat Primary Mammary Epithelial Cell through Upregulating LEF-1 Expression.

LEF-1/wnt10b is one of the most important signaling pathways regulating mammary gland growth and development and is also a potential target for molecular breeding. In this work, 1-deoxynojirimycin (DNJ), a natural alkaloid extracted from plant mulberry or microorganism, was found to have a positive activity in primary breast epithelial cell growth of dairy goats. The findings showed that, compared to the control, 6 µM DNJ in the DMEM/F12 medium in vitro greatly improved the density of dairy goat breast epithelial cell and significantly increased the LEF-1 mRNA level (P < 0.01) and thus enhanced cell growth. In addition, DNJ displayed a similar function in alleviating the growth suppression of epithelial cell and the decrease of LEF-1 mRNA level resulting from lentiviral-mediated LEF-1 knockdown. Simultaneously, no significant change of the mRNA levels of IGF-1 and Fgf10, the other two key regulators in mammary gland growth and development, could be detected. Furthermore, the mammary duct of DNJ-fed mouse illustrated a better development accompanied with a higher LET-1 mRNA level than that of the control. In conclusion, DNJ could improve breast epithelial cell growth through upregulating LEF-1 expression, which supplied a new means in studying mammary gland growth and development.

LncRNA H19 promotes the differentiation of bovine skeletal muscle satellite cells by suppressing Sirt1/FoxO1.

BACKGROUND: H19 is a well-characterized Long noncoding RNA (lncRNA) that has been proven to promote myoblast differentiation in humans and mice. However, its mechanism of action is still not fully interpreted. METHODS: Using RT-qPCR, we examined H19 RNA levels in various tissues from 1-week, 1-month, 6-month and 36-month old male cattle (i.e., newborn, infant, young and adult). The protein and mRNA levels of MyoG, MyHC, Sirt1 and FoxO1 in the satellite and C2C12 cells with an H19 silencing or overexpression vector were respectively detected using western blot and real-time qPCR. RESULTS: H19 was highly expressed in skeletal muscle at all the studied ages. High expression of H19 was required for the differentiation of bovine satellite cells. Knockdown of H19 caused a remarkable increase in the myoblast-inhibitory genes Sirt1/FoxO1, suggesting that H19 suppresses Sirt1/FoxO1 expression during myogenesis. Western blotting analysis of co-transfection of Sirt1 or FoxO1 expression vectors with pcDNA-H19 indicated that Sirt1/FoxO1 overexpression neutralized the promotion of myoblast differentiation through transfection of pcDNA-H19. CONCLUSION: H19 promoted the differentiation of bovine skeletal muscle satellite cells by suppressing Sirt1/FoxO1.

Isolation and characterisation of a novel antibacterial peptide from a native swine intestinal tract-derived bacterium.

Antimicrobial peptides (AMPs) are highly associated with antipathogenic activity, without generating drug resistance in targeted bacteria. In this study, the existence of AMPs in the Tibetan swine, a China-native, cold-resistant and seldom-sick breed of pig, was investigated. A peptide secreted by a Tibetan swine intestinal tract-derived Bacillus strain was isolated using reversed-phase chromatography (RPC), ultrafiltration and reversed-phase high-performance liquid chromatography (RP-HPLC). The peptide was identified by mass spectrometry and was characterised for activity against Escherichia coli and Staphylococcus aureus. The 16-amino acid peptide (ASVVNKLTGGVAGLLK), named TP, had a molecular mass of 1568.919 Da and exhibited inhibitory activity against Gram-positive and Gram-negative bacteria [minimum inhibitory concentrations (MICs) of 2.5-5 µM and 10-20 µM for E. coli and S. aureus, respectively] as well as human MKN-45 and NB4 tumour cell lines [50% inhibitory concentration (IC50) = 4.686 µM and 11.479 µM, respectively]. TP also exhibited weak haemolytic activity. Furthermore, TP enhanced cell membrane permeability and K+ outflow, bound with E. coli genomic DNA in vitro and inhibited E. coli growth. Thus, TP represents a strong candidate as an antibacterial peptide.

Efficient biosynthesis of a Cecropin A-melittin mutant in Bacillus subtilis WB700.

The efficient production of antimicrobial peptides (AMPs) for clinical applications has attracted the attention of the scientific community. To develop a novel microbial cell factory for the efficient biosynthesis of a cecropin A-melittin mutant (CAM-W), a recombinant Bacillus subtilis WB700 expression system was genetically modified with a novel vector, including a fusion gene encoding CAM-W, the autoprotease EDDIE and the signal peptide SacB under the control of the maltose-inducible promoter Pglv. A total of 159 mg of CAM-W was obtained from 1 L of fermentation supernatant. The purified CAM-W showed a consistent size with the expected molecular weight of 3.2 kDa. Our findings suggest that this novel expression system can be used as a powerful tool for the efficient production of CAM-W.

Improved Production of Sublancin 168 Biosynthesized by Bacillus subtilis 168 Using Chemometric Methodology and Statistical Experimental Designs.

Sublancin 168, as a distinct S-linked antimicrobial glycopeptide produced by Bacillus subtilis 168, is effective in killing specific microorganisms. However, the reported yield of sublancin 168 is at a low level of no more than 60 mg from 1 L fermentation culture of B. subtilis 168 by using the method in the literature. Thus optimization of fermentation condition for efficiently producing sublancin 168 is required. Here, Box-Behnken design was used to determine the optimal combination of three fermentation parameters, namely, corn powder, soybean meal, and temperature that were identified previously by Plackett-Burman design and the steepest ascent experiment. Subsequently, based on the response surface methodology, the quadratic regression model for optimally producing sublancin 168 was developed, and the optimal combination of culture parameters for maximum sublancin 168 production of 129.72 mg/L was determined as corn powder 28.49 g/L, soybean meal 22.99 g/L, and incubation temperature 30.8°C. The results showed that sublancin 168 production obtained experimentally was coincident with predicted value of 125.88 mg/L, and the developed model was proved to be adequate, and the aim of efficiently producing sublancin 168 was achieved.

Expression and regulative function of tissue inhibitor of metalloproteinase 3 in the goat ovary and its role in cultured granulosa cells.

Tissue inhibitor of metalloproteinase 3 (TIMP3) played a key role in female reproduction. However, its expression and function in goat are still unclear. In the present study, the full-length cDNA of goat TIMP3 was cloned from adult goat ovary; meanwhile, we demonstrated that putative TIMP3 protein shared a highly conserved amino acid sequence with known mammalian homologs. Real-time PCR results showed that TIMP3 was widely expressed in the tissues of adult goat. In the ovary, increasing expression of TIMP3 mRNA was discovered during the growth process of follicle and corpus luteum. Immunohistochemistry results suggested that TIMP3 protein existed in oocytes of all types of follicles, corpus luteum and granulosa and theca cells of primary, secondary, and antral but not primordial follicles. In vitro, human chorionic gonadotropin (hCG) stimulated the expression of TIMP3 in goat granulosa cells. hCG-induced TIMP3 mRNA expression was reduced by the inhibitors of protein kinase A, protein kinase C, MAPK kinase, or p38 kinase. Functionally, over-expression of TIMP3 significantly increased apoptosis and decreased the viability of cultured granulosa cells. Knockdown of TIMP3 could decrease hCG-induced progesterone secretion and the mRNA abundance of key steroidogenic enzymes (StAR, p450scc and HSD3B) as well as ECM proteins (DCN and FN). These findings provided evidence that the hCG induced expression of TIMP3 may play an important role in regulating goat granulosa cell survival and steroidogenesis.

A New Indoor Positioning System Architecture Using GPS Signals.

The pseudolite system is a good alternative for indoor positioning systems due to its large coverage area and accurate positioning solution. However, for common Global Positioning System (GPS) receivers, the pseudolite system requires some modifications of the user terminals. To solve the problem, this paper proposes a new pseudolite-based indoor positioning system architecture. The main idea is to receive real-world GPS signals, repeat each satellite signal and transmit those using indoor transmitting antennas. The transmitted GPS-like signal can be processed (signal acquisition and tracking, navigation data decoding) by the general receiver and thus no hardware-level modification on the receiver is required. In addition, all Tx can be synchronized with each other since one single clock is used in Rx/Tx. The proposed system is simulated using a software GPS receiver. The simulation results show the indoor positioning system is able to provide high accurate horizontal positioning in both static and dynamic situations.

Improved production of sublancin via introduction of three characteristic promoters into operon clusters responsible for this novel distinct glycopeptide biosynthesis.

BACKGROUND: Sublancin is a novel and distinct antimicrobial glycopeptide that can be used as an alternative to conventional antibiotics. The reported production of sublancin by Bacillus subtilis 168 is poor because transcriptional regulatory circuit of sunA, a gene that encodes presublancin, is complex and difficult to control. RESULTS: A strong inducible and easy to control vegetative σA promoter of Pglv was introduced to replace that of sunA in situ in B. subtilis 1A747 [SPßc, prototroph, the derivative of B. subtilis 168 (trpC2)]. Meanwhile, other two strong promoters of P43 and PluxS were respectively placed before sunI and sunT-bdbA-sunS-bdbB, encoding five functional proteins that involved in the biosynthesis of mature sublancin. 642 mg sublancin was obtained from 1 L culture supernatant of recombinant B. subtilis 1A747 strains. Analysises of electrospray ionization mass spectrometry and circular dichroism spectrum showed that mature sublancin had a molecular weight of 3877.642 Da and displayed a α-helical conformation that are consistent with reported results. In addition, the mature sublancin was proved to be a potent antimicrobial glycopeptide with broad activity spectrum, moderate cytotoxicity and good conditional stability under high temperature, extreme pH and protease-rich environments, thus showing its potential for clinical applications. CONCLUSIONS: Our present findings suggest that recombinant B. subtilis 1A747 strains can effectively and efficiently biosynthesize mature sublancin. The replacement of native promoters provides an extra method for production improvement of some other complicated peptides such as nisin and subtilin.

Cecropin A-melittin mutant with improved proteolytic stability and enhanced antimicrobial activity against bacteria and fungi associated with gastroenteritis in vitro.

Cecropin A-melittin (CAM), a chimeric antimicrobial peptide with potent antimicrobial activity, is threatened by some special extracellular proteases when used to deal with certain drug-resistant pathogenic microbes in the gastrointestinal tract. Thus, a four-tryptophan-substitution mutant (CAM-W) from CAM was developed via the replacement of special amino acid residues to enhance the antimicrobial potency and to improve the proteolytic stability of this agent. The pharmaceutical index of CAM-W was investigated, with a focus on biological potency, cytotoxicity, and proteolytic stability, as well as pH and thermal resistance. CAM-W exhibited potent antimicrobial activity and was approximately 3-12 times higher than that of CAM. CAM-W also exhibited a strong antifungal activity against a series of common pathogenic fungi, in a lower IC50 range between 2.1mg/L and 3.3mg/L than that of its reference CAM ranging from 9.8mg/L to 14.2mg/L. Besides, CAM-W showed moderate cytotoxicity (IC50>300mg/L) in erythrocyte lysis test. In addition, CAM-W overcame challenges under various conditions, including specific temperatures (20, 30, 40, 50, 60, 70, 80, and 90°C), pH values (2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, and 9.0), and proteases (trypsin, pepsin, human neutrophil elastase, Pseudomonas aeruginosa elastase, and Staphylococcus aureus V8 protease) that are commonly present in human gastrointestinal tract. These results suggest that the four-tryptophan-substitution can confer CAM-W with a high pharmaceutical index, which is important for CAM-W to become a potential alternative to conventional antibiotics against bacteria and fungi associated with gastroenteritis.

PU.1 promotes miR-191 to inhibit adipogenesis in 3T3-L1 preadipocytes.

PU.1, an Ets family transcription factor, was previously demonstrated expressed in 3T3-L1 preadipocytes and had an negative effect on adiopogenesis. However, the underlying mechanism remains elusive. Here, miR-191 was identified as an inhibitor of adipocyte differentiation through targeting the 3' untranslated regions of C/EBPß, the initial factor in the C/EBPα/ß-PPARγ terminal pathway of adipogenic differentiation. MiR-191 suppressed the lipid accumulation by Oil Red O staining and downregulated the levels of adipogenic marker genes PPARγ (P<0.01), aP2 (P<0.01) and FAS (P<0.05). Then, we found that PU.1 overexpression resulted in upregulation of miR-191 and adipogenic inhibition. Likewise, PU.1 overexpression rescued the miR-191 decrease and resisted the adipogenic promotion caused by miR-191 oligonucleotide inhibitor. Collectively, these results revealed that PU.1 promoted miR-191 to suppress adipogenesis 3T3-L1 preadipocyte and indicated a new mechanism of PU.1 inhibiting adipogenesis.

Generation of an artificial double promoter for protein expression in Bacillus subtilis through a promoter trap system.

Bacillus subtilis is an attractive host for production of recombinant proteins. Promoters and expression plasmid backbones have direct impacts on the efficiency of gene expression. To screen and isolate strong promoters, a promoter trap vector pShuttleF was developed in this study. Using the vector, approximately 1000 colonies containing likely promoters from Bacillus licheniformis genomic DNA were obtained. Amongst them, pShuttle-09 exhibited the highest ß-Gal activities in both Escherichia coli and B. subtilis. The activity of pShuttle-09 in B. subtilis was eight times of that of the P43 promoter, a commonly used strong promoter for B. subtilis. A sequence analysis showed that pShuttle-09 contained P(luxS) and truncated luxS in-frame fused with the reporter gene as well as another fragment upstream of P(luxS) containing a putative promoter. This putative promoter was a hybrid promoter and its ß-Gal activity was higher than P(luxS). Reconstructing the hybrid promoter from pShuttle-09 to P(lapS) further improved the ß-Gal production by 60%. The usefulness of our promoter trap system is likely due to random shuffling and recombination of DNA fragments and adoption of a rapid and high-throughput screening. Thus, our data provide additional evidence to support the concept of using a promoter trap system to create new promoters.

Interactive and fuzzy search: a dynamic way to explore MEDLINE.

MOTIVATION: The MEDLINE database, consisting of over 19 million publication records, is the primary source of information for biomedicine and health questions. Although the database itself has been growing rapidly, the search paradigm of MEDLINE has remained largely unchanged. RESULTS: Here, we propose a new system for exploring the entire MEDLINE collection, represented by two unique features: (i) interactive: providing instant feedback to users' query letter by letter, and (ii) fuzzy: allowing approximate search. We develop novel index structures and search algorithms to make such a search model possible. We also develop incremental-update techniques to keep the data up to date. AVAILABILITY: Interactive and fuzzy searching algorithms for exploring MEDLINE are implemented in a system called iPubMed, freely accessible over the web at http://ipubmed.ics.uci.edu/ and http://tastier.cs.tsinghua.edu.cn/ipubmed.