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Abstract Background Exosomes, a special subtype of extracellular vesicles derived from human cells, serve as vital mediators of intercellular communication by transporting diverse bioactive cargos, including proteins and enzymes. However, the underlying mechanisms governing exosome secretion and regulation remain poorly understood. In this study, we employed a dual-reporter system consisting of bioluminescent Gaussia luciferase and fluorescent proteins to investigate the dynamics and regulation of exosome secretion in cultured human cells. Results Our results demonstrated that the engineered dual-reporters effectively monitored both exosome-mediated and ER-Golgi-mediated secretory pathways in a specific and quantitative manner. Notably, we observed distinct characteristics of exosome-mediated protein secretion, including significantly lower capacity and different dynamics compared to the ER-Golgi pathway. This phenomenon was observed in human kidney 293T cells and liver HepG2 cells, emphasizing the conserved nature of exosome-mediated secretion across cell types. Furthermore, we investigated the impact of brefeldin A (BFA), an inhibitor of ER-to-Golgi membrane trafficking, on protein secretion. Interestingly, BFA inhibited protein secretion via the ER-Golgi pathway while stimulating exosome-mediated protein secretion under same experimental conditions. Conclusions Collectively, our study highlights the utility of the dual-reporter system for real-time monitoring and quantitative analysis of protein secretion through conventional ER-Golgi and unconventional exosome pathways. Moreover, our findings unveil distinct features of exosome-mediated protein secretion, shedding light on its differential capacity, dynamics, and regulatory mechanisms compared to ER-Golgi-mediated proteins in human cells.

To enable unmanned aerial vehicles to generate coverage paths that balance inspection quality and efficiency when performing three-dimensional inspection tasks, we propose a quality and efficiency coupled iterative coverage path planning (QECI-CPP) method. First, starting from a cleaned and refined mesh model, this was segmented into narrow and normal spaces, each with distinct constraint settings. During the initialization phase of viewpoint generation, factors such as image resolution and orthogonality degree were considered to enhance the inspection quality along the path. Then, the optimization objective was designed to simultaneously consider inspection quality and efficiency, with the relative importance of these factors adjustable according to specific task requirements. Through iterative adjustments and optimizations, the coverage path was continuously refined. In numerical simulations, the proposed method was compared with three other classic methods, evaluated across five aspects: image resolution, orthogonality degree, path distance, computation time, and total path cost. The comparative simulation results show that the QECI-CPP achieves maximum image resolution and orthogonality degree while maintaining inspection efficiency within a moderate computation time, demonstrating the effectiveness of the proposed method. Additionally, the flexibility of the planned path is validated by adjusting the weight coefficient in the optimized objective function.

Exosomes, nanoscale vesicles derived from human cells, offer great promise for targeted drug delivery. However, their inherent diversity and genetic modifications present challenges in terms of ensuring quality in clinical use. To explore solutions, we employed advanced gene fusion and transfection techniques in human 293T cells to generate two distinct sets of genetically engineered samples. We used dual-omics analysis, combining transcriptomics and proteomics, to comprehensively assess exosome quality by comparing with controls. Transcriptomic profiling showed increased levels of engineering scaffolds in the modified groups, confirming the success of genetic manipulation. Through transcriptomic analysis, we identified 15 RNA species, including 2008 miRNAs and 13,897 mRNAs, loaded onto exosomes, with no significant differences in miRNA or mRNA levels between the control and engineered exosomes. Proteomics analysis identified changes introduced through genetic engineering and over 1330 endogenous exosome-associated proteins, indicating the complex nature of the samples. Further pathway analysis showed enrichment in a small subset of cellular signaling pathways, aiding in our understanding of the potential biological impacts on recipient cells. Detection of over 100 cow proteins highlighted the effectiveness of LC-MS for identifying potential contaminants. Our findings establish a dual-omics framework for the quality control of engineered exosome products, facilitating their clinical translation and therapeutic applications in nanomedicine.

Methanol–gasoline blends have emerged as a promising and environmentally friendly bio-fuel option, garnering widespread attention and promotion globally. The methanol content within these blends significantly influences their quality and combustion performance. This study explores the qualitative and qualitative analysis of methanol–gasoline blends using Raman spectroscopy coupled with machine learning methods. Experimentally, methanol–gasoline blends with varying methanol concentrations were artificially configured, commencing with initial market samples. For qualitative analysis, the partial least squares discriminant analysis (PLS-DA) model was employed to classify the categories of blends, demonstrating high prediction performance with an accuracy of nearly 100% classification. For the quantitative analysis, a consensus model was proposed to accurately predict the methanol content. It integrates member models developed on clustered variables, using the unsupervised clustering method of the self-organizing mapping neural network (SOM) to accomplish the regression prediction. The performance of this consensus model was systemically compared to that of the PLS model and uninformative variable elimination (UVE)–PLS model. Results revealed that the unsupervised consensus model outperformed other models in predicting the methanol content across various types of methanol gasoline blends. The correlation coefficients for prediction sets consistently exceeded 0.98. Consequently, Raman spectroscopy emerges as a suitable choice for both qualitative and quantitative analysis of methanol–gasoline blend quality. This study anticipates an increasing role for Raman spectroscopy in analysis of fuel composition.

Satellite gravimetry missions have enabled the calculation of high-accuracy and high-resolution Earth gravity field models from satellite-to-satellite tracking data and gravitational gradients. However, calculating high maximum degree/order (e.g., 240 or even higher) gravity field models using the least squares method is time-consuming due to the vast amount of gravimetry observations. To improve calculation efficiency, a parallel algorithm has been developed by combining Message Passing Interface (MPI) and Open Multi-Processing (OpenMP) programming models to calculate and invert normal equations for the Earth gravity field recovery. The symmetrical feature of normal equations has been implemented to speed up the calculation progress and reduce computation time. For example, the computation time to generate the normal equation of an IGGT_R1 test version of degree/order 240 was reduced from 88 h to 27 h by considering the symmetrical feature. Here, the calculation was based on the high-performance computing cluster with 108 cores in the School of Geodesy and Geomatics, at Wuhan University. Additionally, the MPI parallel Gaussian-Jordan elimination method was modified to invert normal equation matrices and scaled up to 100 processor cores in this study while the traditional method was limited in a certain number of processors. Furthermore, the Cholesky decomposition from the ScaLAPACK library was used to compare with the parallel Gauss-Jordan elimination method. The numerical algorithm has effectively reduced the amount of calculation and sped up the calculation progress, and has been successfully implemented in applications such as building the gravity field models IGGT_R1 and IGGT_R1C.

Comprehensive radiant heat transfer coefficient, convective heat transfer coefficient and furnace temperature are the key parameters constituting the boundary conditions of the billet heat transfer model. This paper presents a novel method to obtain these parameters by combining ''black box'' test with mathematical model. Average relative errors of the calculated surface temperature and centre temperature of the case reheating furnace compared with the measured values are 2.34% and 3.51%, which verifies the method's feasibility. Influence law of furnace temperature characteristic parameters on billet temperature is analyzed. When the intercept, slope and amplitude change, the regions where the billet temperature field changes significantly is in or near the HEⅠsection, which are the key section to adjust the billet temperature field through single characteristic parameter. The longer the period is, the smoother the change of billet temperature in the same spatial region is, which is conducive to the uniform heating of billet. However, if only increasing the period, there may be a situation that the billet discharging temperature exceeds the target. Change of single characteristic parameter has its limitations. In order to achieve the accurate control of billet temperature, the collaborative change of each characteristic parameter is required.

Electron transport layer (ETL), one of the important layers for high-performing perovskite solar cells (PSCs), also has great potential in bioengineering applications. It could be used for biological sensors, biological imaging, and biomedical treatments with high resolution or efficiency. Seldom research focused on the development of biological material for ETL and their application in biological uses. This review will introduce commercial and biological materials used in ETL to help readers understand the working mechanism of ETL. And the ways to prepare ETL at low temperatures will also be introduced to improve the performance of ETL. Then this review summarizes the latest research on material doping, material modification, and bilayer ETL structures to improve the electronic transmission capacity of ETLs. Finally, the application of ETLs in bioengineering will be also shown to demonstrate that ETLs and their used material have a high potential for biological applications.

Surface electromyographic (sEMG) signals are weak physiological electrical signals, which are highly susceptible to coupling external noise and cause major difficulties in signal acquisition and processing. The study of using sEMG signals to analyze human motion intention mainly involves data preprocessing, feature extraction, and model classification. Feature extraction is an extremely critical part; however, this often involves many manually designed features with specialized domain knowledge, so the experimenter will spend time and effort on feature extraction. To address this problem, deep learning methods that can automatically extract features are applied to the sEMG-based gesture recognition problem, drawing on the success of deep learning for image classification. In this paper, sEMG is captured using a wearable, flexible bionic device, which is simple to operate and highly secure. A multi-stream convolutional neural network algorithm is proposed to enhance the ability of sEMG to characterize hand actions in gesture recognition. The algorithm virtually augments the signal channels by reconstructing the sample structure of the sEMG to provide richer input information for gesture recognition. The methods for noise processing, active segment detection, and feature extraction are investigated, and a basic method for gesture recognition based on the combination of multichannel sEMG signals and inertial signals is proposed. Suitable filters are designed for the common noise in the signal. An improved moving average method based on the valve domain is used to reduce the segmentation error rate caused by the short resting signal time in continuous gesture signals. In this paper, three machine learning algorithms, K-nearest neighbor, linear discriminant method, and multi-stream convolutional neural network, are used for hand action classification experiments, and the effectiveness of the multi-stream convolutional neural network algorithm is demonstrated by comparison of the results. To improve the accuracy of hand action recognition, a final 10 gesture classification accuracy of up to 93.69% was obtained. The separability analysis showed significant differences in the signals of the two cognitive-behavioral tasks when the optimal electrode combination was used. A cross-subject analysis of the test set subjects illustrated that the average correct classification rate using the pervasive electrode combination could reach 93.18%.

With the improper use of antibiotics, an increasing number of multidrug-resistant bacteria have been reported worldwide, posing challenges for disease treatment. Klebsiella pneumoniae is an important zoonotic pathogen that colonises the respiratory tract. Endolysin therapy has emerged with the development of phages. In this study, a lytic phage vB_KpnS_MK54 was isolated from the drinking water of a forest musk deer (FMD) farm in Sichuan Province. It was the first reported phage obtained from FMD. The primary biological characteristics were determined, and whole-genome sequencing analysis was performed. The phage which belongs to the family Siphoviridae is highly specific for lytic host bacteria and is moderately adaptable to different environments. Whole-genome sequencing results showed that the phage genome size was 46,218 bp. There were 80 coding DNA sequences (CDSs) in total, 32 of which had known functions. The last CDS is the phage endolysin LysG24. A new peptide-modified endolysin (LysCA) was constituted by connecting the cecropin A peptide residues with LysG24 to investigate the antibacterial activities of both LysG24 and LysCA. The results showed that the lytic profile of LysG24 and LysCA was wider than that of phage MK54. For in vitro tests, both endolysins destroyed 99% of the host bacteria within 6 h. The lysing ability and environmental adaptability of LysCA were significantly stronger than those of LysG24. For in vivo tests, LysG24 and LysCA exhibited therapeutic effects in a mouse model of pneumonia wherewith the mice were infected with K. pneumoniae (LPKP), wherein both LysG24 and LysCA can effectively reduce the pulmonary inflammatory response. The LPKP bacterial load in the treatment group was significantly lower than that in the bacterial group, among which LysCA displayed a more obvious therapeutic effect. Furthermore, the safety test showed that the endolysins had no toxic effects on mice. In general, both LysG24 and LysCA showed excellent antibacterial activity in vivo and in vitro, with high safety and strong adaptability to the environment, manifesting their latent potential as new antimicrobial agents.

Bovine mastitis is a common disease occurring in dairy farms and can be caused by more than 150 species of pathogenic bacteria. One of the most common causative organisms is Streptococcus agalactiae, which is also potentially harmful to humans and aquatic animals. At present, research on S. agalactiae in China is mostly concentrated in the northern region, with limited research in the southeastern and southwestern regions. In this study, a total of 313 clinical mastitis samples from large-scale dairy farms in five regions of Sichuan were collected for isolation of S. agalactiae. The epidemiological distribution of S. agalactiae was inferred by serotyping isolates with multiplex polymerase chain reaction. Susceptibility testing and drug resistance genes were detected to guide the clinical use of antibiotics. Virulence genes were also detected to deduce the pathogenicity of S. agalactiae in Sichuan Province. One hundred and five strains of S. agalactiae (33.6%) were isolated according to phenotypic features, biochemical characteristics, and 16S rRNA sequencing. Serotype multiplex polymerase chain reaction analysis showed that all isolates were of type Ia. The isolates were up to 100% sensitive to aminoglycosides (kanamycin, gentamicin, neomycin, and tobramycin), and the resistance rate to β-lactams (penicillin, amoxicillin, ceftazidime, and piperacillin) was up to 98.1%. The TEM gene (β-lactam-resistant) was detected in all isolates, which was in accordance with a drug-resistant phenotype. Analysis of virulence genes showed that all isolates harbored the cfb, cylE, fbsA, fbsB, hylB, and α-enolase genes and none harbored bac or lmb. These data could aid in the prevention and control of mastitis and improve our understanding of epidemiological trends in dairy cows infected with S. agalactiae in Sichuan Province.

ContextLong-acting recombinant human growth hormone (rhGH) has transformed growth hormone deficiency (GHD) treatment. However, the possibility and rationality for flexible time regimen are pending.ObjectiveWe studied the efficacy of biweekly versus weekly PEGylated rhGH (PEG-rhGH) therapy in GHD children.Design, Setting, and PatientsThis multicenter, phase IV trial with a non-inferiority threshold ≥20% enrolled 585 Tanner stage I GHD children.InterventionSubjects randomly received 0.20 mg/kg once-weekly or biweekly PEG-rhGH, or 0.25 mg/kg.w rhGH once daily for 26 weeks.Main Outcome MeasureThe primary outcome was height SD scores for chronological age (HtSDSCA) at week 26 and safety measurements including adverse events (AEs), IGF-2, and IGFBP-2 changes.ResultsAt week 26, the median HtSDSCA changed from −2.75, −2.82, and −2.78 to −2.31, −2.43, and −2.28 with weekly and biweekly PEG-rhGH, and daily rhGH, respectively. The difference in HtSDSCA was 0.17 ± 0.28 between weekly and biweekly PEG-rhGH, and 0.17 ± 0.27 between daily rhGH and biweekly PEG-rhGH, failing the non-inferiority threshold. Nevertheless, the height velocity of children receiving biweekly PEG-rhGH reached 76.42%–90.34% and 76.08%–90.60% that of children receiving weekly PEG-rhGH and daily rhGH, respectively. The rate of AEs was comparable among the groups. No statistical difference was observed in IGF-2 and IGFBP-2 levels among the groups. IGFBP-2 levels decreased over time in all groups, with no notable difference in IGF-2 and IGFBP-2 changes among the three treatment groups.ConclusionsAlthough notably promoted height velocity, biweekly PEG-rhGH failed the non-inferiority threshold as compared with either weekly PEG-rhGH or daily rhGH. Compared with short-term rhGH, long-acting PEG-rhGH did not significantly increase tumor-associated IGF-2 and IGFBP-2 expressions.Clinical Trial Registrationclinicaltrials.gov, identifier NCT02976675.

Klebsiella pneumoniae is an important pathogen that can infect both humans and cattle. The widespread K. pneumoniae and its high drug resistance make it difficult to treat Klebsiella infections/diseases. In this study, a lytic K. pneumoniae bacteriophage vB_Kpn_B01 was isolated from a dairy farm trough in Sichuan Province, and its biological properties were studied, and the entire genome of vB_Kpn_B01 was sequenced. The therapeutic effects of the phage on disease-causing mice were preliminarily tested. Phages found in this study are double-stranded DNA bacterial viruses belonging to the family Siphoviridae, Sugarlandvirus. The results suggest that vB_Kpn_B01 has strong specificity and low adaptability to different adverse conditions. Meanwhile, the predicted gene products of phage vB_Kpn_B01 comprised 149 coding sequences (CDS) and 25 tRNAs, of which 34 CDS had known functions. Of course, vB_Kpn_B01 did not contain any known antibiotic-resistant or virulent genes. The pathological sections of the liver and lungs of mice showed that the inflammatory scores of the treatment group were lower than in the bacterial group. Phage vB_Kpn_B01 alleviated the inflammatory response in the organs of the infected mice, and the organ tissue bacterial load of the treatment group was significantly lower than that of the bacterial group. Therefore, vB_Kpn_B01 can inhibit the proliferation of K. pneumoniae 18 in vivo and can alleviate the inflammation of target organs caused by infectious bacteria, which preliminarily indicates that vB_Kpn_B01 has a certain therapeutic effect on laboratory-infected mice.

Background: Human cell-secreted extracellular vesicles (EVs) are versatile nanomaterials suitable for disease-targeted drug delivery and therapy. Native EVs, however, usually do not interact specifically with target cells or harbor therapeutic drugs, which limits their potential for clinical applications. These functions can be introduced to EVs by genetic manipulation of membrane protein scaffolds, although the efficiency of these manipulations and the impacts they have on the properties of EVs are for the most part unknown. In this study, we quantify the effects of genetic manipulations of different membrane scaffolds on the physicochemical properties, molecular profiles, and cell uptake of the EVs. Methods: Using a combination of gene fusion, molecular imaging, and immuno-based on-chip analysis, we examined the effects of various protein scaffolds, including endogenous tetraspanins (CD9, CD63, and CD81) and exogenous vesicular stomatitis virus glycoprotein (VSVG), on the efficiency of integration in EV membranes, the physicochemical properties of EVs, and EV uptake by recipient cells. Results: Fluorescence imaging and live cell monitoring showed each scaffold type was integrated into EVs either in membranes of the endocytic compartment, the plasma membrane, or both. Analysis of vesicle size revealed that the incorporation of each scaffold increased the average diameter of vesicles compared to unmodified EVs. Molecular profiling of surface markers in engineered EVs using on-chip assays showed the CD63-GFP scaffold decreased expression of CD81 on the membrane surface compared to control EVs, whereas its expression was mostly unchanged in EVs bearing CD9-, CD81-, or VSVG-GFP. The results from cell uptake studies demonstrated that VSVG-engineered EVs were taken up by recipient cells to a greater degree than control EVs. Conclusion: We found that the incorporation of different molecular scaffolds in EVs altered their physicochemical properties, surface protein profiles, and cell-uptake functions. Scaffold-induced changes in the physical and functional properties of engineered EVs should therefore be considered in engineering EVs for the targeted delivery and uptake of therapeutics to diseased cells.

Since its discovery in 1909, the Moho was routinely studied by seismological methods. However, from the 1950s, a possible alternative was introduced by gravimetric inversion. Thanks to satellite gravity missions launched from the beginning of the 21st century, a global inversion became feasible, e.g., leading to the computation of the GEMMA model in 2012. This model was computed inverting the GOCE second radial derivatives of the anomalous potential by a Wiener filter, which was applied in the spherical harmonic domain, considering a two-layer model with lateral and vertical density variations. Moreover, seismic information was introduced in the inversion to deal with the joint estimation/correction of both density and geometry of the crustal model. This study aims at revising the GEMMA algorithm from the theoretical point of view, introducing a cleaner formalization and studying the used approximations more thoroughly. The updates are on: (1) the management of the approximations due to the forward operator linearization required for the inversion; (2) the regularization of spherical harmonic coefficients in the inversion by proper modelling the Moho signal and the gravity error covariances; (3) the inclusion of additional parameters and their regularization in the Least Squares adjustment to correct the density model by exploiting seismic information. Thanks to these updates, a significant improvement from the computational point of view is achieved too, thus the convergence of the iterative solution and the differences with respect to the previous algorithm can be assessed by closed-loop tests, showing the algorithm performance in retrieving the simulated “true” Moho.

To systematically and quantitatively analyze the influence factors on billet gas consumption (BGC) in reheating furnace, a novelty data mining approach for multi-influence factors BGC analysis was proposed in this paper. This multi-influence factors data mining model mainly includes four steps: Firstly, the BGC apportionment model was established based on energy apportionment model in reheating furnace; Secondly, the BGC data set could be achieved according to the division of billet sample space (BSS); Thirdly, the data interpolation calculation method of various BSS subsets (BSSSs) was put forward; Lastly, the influence degree analysis method of various factors on BGC was described in detail. Especially, contribution degree model, which could quantitatively describe the influence degree of each factor on BGC, was established. Case study showed that working groups (WGs) should be eliminated because of weak influence on BGC. Then the order of contribution degree on BGC from weak to strong was working shifts (WSs) (1.61%), residence time (9.7%), loading temperature (88.68%). Therefore, residence time and loading temperature should be highlighted in all factors. Finally, some measures and suggestions, which could improve the residence time and loading temperature, were put forward.

Background: Recent technological advancements have enabled live-cell imaging of intracellular organelles to monitor their biogenesis in mammalian cells. However, applying this method to gain insight into extracellular organelles, such as extracellular vesicles (EVs), presents unique challenges that require special considerations in design and engineering.Results: We have developed a dual-reporter system that combines genetic fusion, fluorescence microcopy and magnetic beads capture of EVs to study the biogenesis of EVs in mammalian cell cultures. First, we genetically produced a series of reporters by fusing a green fluorescent protein (GFP) and an affinity peptide (6xHis), with either the endogenous transmembrane protein, CD63, or EVs targeting vesicular stomatitis viral glycoprotein (VSVG). Transfection of these reporters into human 293T cells resulted in expression and integration of these reporters into pre-exosome compartments, which were subsequently released into the culture medium. Confocal imaging and nano-particle tracking analysis demonstrated that EVs were appropriately labeled and exhibited a single dominant peak in the 80–110 nm size range, indicating that isolated EVs were comprised of micro-vesicles and/or exosome subpopulations. Incubation of isolated EVs with nickel-coated magnetic beads resulted in successful capture of GFP-positive EVs. Finally, addition of EVs into culture medium was able to reveal the cellular uptake of GFP-labeled EVs by recipient cells. Taken together, our dual-reporter system provides a powerful method for both monitoring and capturing of EVs in mammalian cell culture systems.Conclusion: A dual-reporter system provides a robust tool to study the life cycle of EVs in mammalian cells from biogenesis and excretion to cellular uptake.

Marine gravimetry provides high-quality gravity measurements, particularly in coastal areas. After the update of new sensors in GFZ’s air-marine gravimeter Chekan-AM, gravimetry measurements showed a significant improvement from the first new campaign DENEB2017 with an accuracy of 0.3/2=0.21 mGal @ 1 km along the tracks, which is at the highest accuracy level of marine gravimetry. Then, these measurements were used to assess gravity data derived from satellite altimetry (about 3 mGal) and a new finding is that a bias of −1.5 mGal exists in the study area. Additionally, ship soundings were used to assess existing seafloor topography models. We found that the accuracy of SRTM model and SIO model is at a level of 2 m, while the accuracy of the regional model EMODnet reaches the lever of sub-meters. Furthermore, a bias of 0.7 m exists and jumps above 5 m in the SRTM model near the coast of Sweden. Finally, new combined gravity anomalies with sounding data are used to reveal the fine structure of ocean topography. Our estimated seafloor topography model is more accurate than existing digital elevation data sets such as EMODnet, SRTM and SIO models and, furthermore, shows some more detailed structure of seafloor topography. The marine gravimetry and sounding measurements as well as the estimated seafloor topography are crucial for future geoid determination, 3D-navigation and resource exploration in the Baltic Sea.

Abstract Background Targeted nucleases have transformed genome editing technology, providing more efficient methods to make targeted changes in mammalian genome. In parallel, there is an increasing demand of Cre-LoxP technology for complex genome manipulation such as large deletion, addition, gene fusion and conditional removal of gene sequences at the target site. However, an efficient and easy-to-use Cre-recombinase delivery system remains lacking. Results We designed and constructed two sets of expression vectors for Cre-recombinase using two highly efficient viral systems, the integrative lentivirus and non-integrative adeno associated virus. We demonstrate the effectiveness of those methods in Cre-delivery into stably-engineered HEK293 cells harboring LoxP-floxed red fluorescent protein (RFP) and puromycin (Puro) resistant reporters. The delivered Cre recombinase effectively excised the floxed RFP-Puro either directly or conditionally, therefore validating the function of these molecular tools. Given the convenient options of two selections markers, these viral-based systems offer a robust and easy-to-use tool for advanced genome editing, expanding complicated genome engineering to a variety of cell types and conditions. Conclusions We have developed and functionally validated two viral-based Cre-recombinase delivery systems for efficient genome manipulation in various mammalian cells. The ease of gene delivery with the built-in reporters and inducible element enables live cell monitoring, drug selection and temporal knockout, broadening applications of genome editing.

Abstract Background High-throughput methods based on molecular reporters have greatly advanced our knowledge of cell signaling in mammalian cells. However, their ability to monitor various types of cells is markedly limited by the inefficiency of reporter gene delivery. Recombinant adeno-associated virus (AAV) vectors are efficient tools widely used for delivering and expressing transgenes in diverse animal cells in vitro and in vivo. Here we present the design, construction and validation of a novel AAV-based dual-reporter circuit that can be used to monitor and quantify cell signaling in living human cells. Results We first design and construct the AAV-based reporter system. We then validate the versatility and specificity of this system in monitoring and quantifying two important cell signaling pathways, inflammation (NFκB) and cell growth and differentiation (AP-1), in cultured HEK293 and MCF-7 cells. Our results demonstrate that the AAV reporter system is both specific and versatile, and it can be used in two common experimental protocols including transfection with plasmid DNA and transduction with packaged viruses. Importantly, this system is efficient, with a high signal-to-background noise ratio, and can be easily adapted to monitor other common signaling pathways. Conclusions The AAV-based system extends the dual-reporter technology to more cell types, allowing for cost-effective and high throughput applications.

Electrocaloric properties of [110] and [111] oriented 0.73Pb(Mg1/3Nb2/3)O3-0.27PbTiO3 single crystals were studied in the temperature range of 293–423 K. The Maxwell relations and the Landau–Ginsburg–Devonshire (LGD) phenomenological theory were employed as the indirect method to calculate the electrocaloric properties, while a high-resolution calorimeter was used to measure the adiabatic temperature change of the electrocaloric effect (ECE) directly. The results indicate that the directly measured temperature changes of ΔT > 2.5 K at room temperature were procured when the applied electric field was reversed from 1 MV/m to −1 MV/m, which are larger than those deduced pursuant to the Maxwell relation, and even larger than those calculated using the LGD theory in the temperature range of 293–~380 K.

The electrocaloric effect (ECE) in lead-free BaSnxTi1−xO3 (BSnT, x=0.05, 0.10 and 0.15) ceramics were investigated using a direct ECE measurement. Results indicate that the maximum ECE occurs near the Curie temperature and it slightly shifts towards high temperatures. The directly measured ECE is larger than that calculated using the Maxwell relation and P-E loops. A maximum electrocaloric efficiency ΔT∕ΔE=0.49×10−6KmV−1 was procured for the composition with x=0.15 at T=299.2K. And it also shows a broad peak in the temperature range of 250–320K.

Conventional relative kinematic positioning is difficult to be applied in the polar region of Earth since there is a very sparse distribution of reference stations, while precise point positioning (PPP), using data of a stand-alone receiver, is recognized as a promising tool for obtaining reliable and accurate trajectories of moving platforms. However, PPP and its integer ambiguity fixing performance could be much degraded by satellite orbits and clocks of poor quality, such as those of the geostationary Earth orbit (GEO) satellites of the BeiDou navigation satellite system (BDS), because temporal variation of orbit errors cannot be fully absorbed by ambiguities. To overcome such problems, a network-based processing, referred to as precise orbit positioning (POP), in which the satellite clock offsets are estimated with fixed precise orbits, is implemented in this study. The POP approach is validated in comparison with PPP in terms of integer ambiguity fixing and trajectory accuracy. In a simulation test, multi-GNSS (global navigation satellite system) observations over 14 days from 136 globally distributed MGEX (the multi-GNSS Experiment) receivers are used and four of them on the coast of Antarctica are processed in kinematic mode as moving stations. The results show that POP can improve the ambiguity fixing of all system combinations and significant improvement is found in the solution with BDS, since its large orbit errors are reduced in an integrated adjustment with satellite clock offsets. The four-system GPS+GLONASS+Galileo+BDS (GREC) fixed solution enables the highest 3D position accuracy of about 3.0 cm compared to 4.3 cm of the GPS-only solution. Through a real flight experiment over Antarctica, it is also confirmed that POP ambiguity fixing performs better and thus can considerably speed up (re-)convergence and reduce most of the fluctuations in PPP solutions, since the continuous tracking time is short compared to that in other regions.

Phospholipids are required for epidermal lamellar body formation. Glycerol 3-phosphate acyltransferases (GPATs) catalyze the initial step in the biosynthesis of glycerolipids. Little is known about the expression and regulation of GPATs in epidermis/keratinocytes. Here, we demonstrate that GPAT 1, 3, and 4 are expressed in epidermis/keratinocytes, whereas GPAT2 is not detected. In mouse epidermis, GPAT 3 and 4 are mainly localized to the upper layers whereas GPAT1 is found in both the upper and lower layers. GPAT1 and 3 mRNA increase during fetal rat epidermal development. No change in GPAT expression was observed in adult mice following acute permeability barrier disruption. Calcium-induced human keratinocyte differentiation increased GPAT3 mRNA whereas both GPAT1 and 4 mRNA levels decreased. In parallel, total GPAT activity increased 2-fold in differentiated keratinocytes attributable to an increase in N-ethylmaleimide (NEM) sensitive GPAT activity localized to microsomes with little change in NEM resistant activity, consistent with an increase in GPAT3. Furthermore, PPARγ or PPARδ activators increased GPAT3 mRNA, microsomal GPAT activity, and glycerol lipid synthesis without affecting the expression of GPAT1 or 4. Finally, both PPARγ and PPARδ activators increased GPAT3 mRNA via increasing its transcription. Thus, multiple isoforms of GPAT are expressed and differentially regulated in epidermis/keratinocytes.

ABCG1, a member of the ATP binding cassette superfamily, facilitates the efflux of cholesterol from cells to HDL. In this study, we demonstrate that ABCG1 is expressed in cultured human keratinocytes and murine epidermis, and induced during keratinocyte differentiation, with increased levels in the outer epidermis. ABCG1 is regulated by liver X receptor (LXR) and peroxisome proliferator-activated receptor-δ (PPAR-δ) activators, cellular sterol levels, and acute barrier disruption. Both LXR and PPAR-δ activators markedly stimulate ABCG1 expression in a dose- and time-dependent fashion. PPAR-γ activators also increase ABCG1 expression, but to a lesser degree. In contrast, activators of PPAR-α, retinoic acid receptor, retinoid X receptor, and vitamin D receptor do not alter ABCG1 expression. In response to increased intracellular sterol levels, ABCG1 expression increases, whereas inhibition of cholesterol biosynthesis decreases ABCG1 expression. In vivo, ABCG1 is stimulated 3–6 h after acute barrier disruption by either tape stripping or acetone treatment, an increase that can be inhibited by occlusion, suggesting a potential role of ABCG1 in permeability barrier homeostasis. Although Abcg1-null mice display normal epidermal permeability barrier function and gross morphology, abnormal lamellar body (LB) contents and secretion leading to impaired lamellar bilayer formation could be demonstrated by electron microscopy, indicating a potential role of ABCG1 in normal LB formation and secretion.

OBJECTIVES/SPECIFIC AIMS: Exosomes are living nanoscale vesicles that can shuttle large amounts of bioactive cargo for intercellular communication. The potential of these nanovesicles to serve as both biomarkers for disease diagnosis and vehicles for delivery of therapeutics has only begun to be explored. To realize these potentials, molecular tools for effective exosome tracking and capturing must be invented in order to advance basic research and clinical translation. METHODS/STUDY POPULATION: We utilize a surface display strategy that enables exosome modification in living mammalian systems. By reconfiguring the surface protein CD63 or viral envelope glycoprotein VSV-G, we generate 3 topologically distinctive protein chimeras for exosome imaging and capture in mammalian systems. RESULTS/ANTICIPATED RESULTS: We have shown that these genetically encoded protein chimeras have the ability to correctly target and integrate into exosomes in cultured human cells. Furthermore, we have demonstrated that the secreted exosomes could be successfully captured by an affinity peptide intentionally displayed on the outer surface of exosomes. DISCUSSION/SIGNIFICANCE OF IMPACT: Our study highlights the potential of these fusion proteins for exosome tracking and provides novel genetic tools for exosome research and translation, one of which is loading protein therapeutics for targeted delivery.

Keratinocytes require abundant cholesterol for cutaneous permeability barrier function; hence, the regulation of cholesterol homeostasis is of great importance. ABCA1 is a membrane transporter responsible for cholesterol efflux and plays a pivotal role in regulating cellular cholesterol levels. We demonstrate that ABCA1 is expressed in cultured human keratinocytes (CHKs) and murine epidermis. Liver X receptor (LXR) activation markedly stimulates ABCA1 mRNA and protein levels in CHKs and mouse epidermis. In addition to LXR, activators of peroxisome proliferator-activated receptor (PPAR)α, PPARβ/δ, and retinoid X receptor (RXR), but neither PPARγ nor retinoic acid receptor, also increase ABCA1 expression in CHKs. Increases in cholesterol supply induced by LDL or mevalonate stimulate ABCA1 expression, whereas inhibiting cholesterol synthesis with statins or cholesterol sulfate decreases ABCA1 expression in CHKs. After acute permeability barrier disruption by either tape-stripping or acetone treatment, ABCA1 expression declines, and this attenuates cellular cholesterol efflux, making more cholesterol available for regeneration of the barrier. In addition, during fetal epidermal development, ABCA1 expression decreases at days 18–22 of gestation (term = 22 days), leaving more cholesterol available during the critical period of barrier formation. Together, our results show that ABCA1 is expressed in keratinocytes, where it is negatively regulated by a decrease in cellular cholesterol levels or altered permeability barrier requirements and positively regulated by activators of LXR, PPARs, and RXR or increases in cellular cholesterol levels.

The acute-phase response (APR) leads to alterations in lipid metabolism and type II nuclear hormone receptors, which regulate lipid metabolism, are suppressed, in liver, heart, and kidney. Here, we examine the effect of the APR in adipose tissue. In mice, lipopolysaccharide produces a rapid, marked decrease in mRNA levels of nuclear hormone receptors [peroxisome proliferator-activated receptor γ (PPARγ), liver X receptor α (LXRα) and LXRβ, thyroid receptor α (TRα) and TRβ, and retinoid X receptor α (RXRα) and RXRβ] and receptor coactivators [cAMP response element binding protein, steroid receptor coactivator 1 (SRC1) and SRC2, thyroid hormone receptor-associated protein, and peroxisome proliferator-activated receptor γ co-activator 1α (PGC1α) and PGC1β] along with decreased expression of target genes (adipocyte P2, phosphoenolpyruvate carboxykinase, glycerol-3-phosphate acyltransferase, ABCA1, apolipoprotein E, sterol-regulatory element binding protein-1c, glucose transport protein 4 (GLUT4), malic enzyme, and Spot14) involved in triglyceride (TG) and carbohydrate metabolism. We show that key TG synthetic enzymes, 1-acyl-sn-glycerol-3-phosphate acyltransferase-2, monoacylglycerol acyltransferase 1, and diacylglycerol acyltransferase 1, are PPARγ-regulated genes and that they also decrease in the APR. In 3T3-L1 adipocytes, tumor necrosis factor-α (TNF-α) significantly decreases PPARγ, LXRα and LXRβ, RXRα and RXRβ, SRC1 and SRC2, and PGC1α and PGC1β mRNA levels, which are associated with a marked reduction in receptor-regulated genes. Moreover, TNF-α significantly reduces PPAR and LXR response element-driven transcription. Thus, the APR suppresses the expression of many nuclear hormone receptors and their coactivators in adipose tissue, which could be a mechanism to coordinately downregulate TG biosynthesis and thereby redirect lipids to other critical organs during the APR.

Cardiolipin (CL) is a phospholipid localized to the mitochondria, and its biosynthesis is essential for mitochondrial structure and function. We report here the identification and characterization of a cDNA encoding the first mammalian cardiolipin synthase (CLS1) in humans and mice. This cDNA exhibits sequence homology with members of a CLS gene family that share similar domain structure and chemical properties. Expression of the human CLS (hCLS1) cDNA in reticulocyte lysates or insect cells led to a marked increase in CLS activity. The enzyme is specific for CL synthesis, because no significant increase in phosphatidylglycerol phosphate synthase activity was observed. In addition, CL pool size was increased in hCLS1-overexpressing cells compared with controls. Furthermore, the hCLS1 gene was highly expressed in tissues such as heart, skeletal muscle, and liver, which have been shown to have high CLS activities. These results demonstrate that hCLS1 encodes an enzyme that synthesizes CL.

ABSTRACT Invasive pathogens interface with the host and its resident microbiota through interkingdom signaling. The bacterial receptor QseC, which is a membrane-bound histidine sensor kinase, responds to the host stress hormones epinephrine and norepinephrine and the bacterial signal AI-3, integrating interkingdom signaling at the biochemical level. Importantly, the QseC signaling cascade is exploited by many bacterial pathogens to promote virulence. Here, we translated this basic science information into development of a potent small molecule inhibitor of QseC, LED209. Extensive structure activity relationship (SAR) studies revealed that LED209 is a potent prodrug that is highly selective for QseC. Its warhead allosterically modifies lysines in QseC, impairing its function and preventing the activation of the virulence program of several Gram-negative pathogens both in vitro and during murine infection. LED209 does not interfere with pathogen growth, possibly leading to a milder evolutionary pressure toward drug resistance. LED209 has desirable pharmacokinetics and does not present toxicity in vitro and in rodents. This is a unique antivirulence approach, with a proven broad-spectrum activity against multiple Gram-negative pathogens that cause mammalian infections. IMPORTANCE There is an imminent need for development of novel treatments for infectious diseases, given that one of the biggest challenges to medicine in the foreseeable future is the emergence of microbial antibiotic resistance. Here, we devised a broad-spectrum antivirulence approach targeting a conserved histidine kinase, QseC, in several Gram-negative pathogens that promotes their virulence expression. The LED209 QseC inhibitor has a unique mode of action by acting as a prodrug scaffold to deliver a warhead that allosterically modifies QseC, impeding virulence in several Gram-negative pathogens.

Phospholipids are a major class of lipids in epidermis, where they serve as a source of free fatty acids that are important for the maintenance of epidermal permeability barrier function. The phospholipid biosynthetic enzyme, 1-acyl-sn-glycerol-3-phosphate acyltransferase (AGPAT), catalyzes the acylation of lysophosphatidic acid to form phosphatidic acid, the major precursor of all glycerolipids. We identified an expression pattern of AGPAT isoforms that is unique to epidermis, with relatively high constitutive expression of mouse AGPAT (mAGPAT) 3, 4, and 5 but low constitutive expression of mAGPAT 1 and 2. Localization studies indicate that all five isoforms of AGPAT were expressed in all nucleated layers of epidermis. Furthermore, rat AGPAT 2 and 5 mRNAs increased in parallel with both an increase in enzyme activity and permeability barrier formation late in rat epidermal development. Moreover, after two methods of acute permeability barrier disruption, mAGPAT 1, 2, and 3 mRNA levels increased rapidly and were sustained for at least 24 h. In parallel with the increase in mRNA levels, an increase in AGPAT activity also occurred.Because upregulation of mAGPAT mRNAs after tape-stripping could be partially reversed by artificial barrier restoration by occlusion, these studies suggest that an increase in the expression of AGPATs is linked to barrier requirements.

The role of peroxisome proliferator-activated receptor α (PPARα)-stimulated phospholipase A2 (PLA2) in cardiac mitochondrial cardiolipin (CL) biosynthesis was examined in both in vivo and in vitro models. Treatment of rat heart H9c2 cells with clofibrate increased the expression and activity of 14 kDa PLA2 but did not affect the pool size of CL. Clofibrate treatment stimulated de novo CL biosynthesis via an increase in phosphatidylglycerolphosphate (PGP) synthase activity, accounting for the unaltered CL content. Cardiac PLA2, PGP synthase, and CDP-1,2-diacyl-sn-glycerol synthase (CDS-2) activities and CDS-2 mRNA levels were elevated in mice fed clofibrate for 14 days compared with controls. In PPARα-null mice, clofibrate feeding did not alter cardiac PLA2, PGP synthase activities, or CDS-2 activity and mRNA level, confirming that these enzymes are regulated by PPARα activation. In contrast to mouse heart, clofibrate treatment did not affect the activity or mRNA levels of CDS-2 in H9c2 cells, indicating that CDS-2 is regulated differently in rat heart H9c2 cells in vitro and in mouse heart in vivo.These results clearly indicate that cardiac CL de novo biosynthesis is stimulated by PPARα activation in responsive rodent models and that CDS-2 is an example of an enzyme that exhibits alternative regulation in vivo and in cultured cell lines. This study is the first to demonstrate that CL de novo biosynthesis is regulated by PPARα activation.