Endothelial cell SMAD6 balances Alk1 function to regulate adherens junctions and hepatic vascular development.

BMP signaling is crucial to blood vessel formation and function, but how pathway components regulate vascular development is not well-understood. Here, we find that inhibitory SMAD6 functions in endothelial cells to negatively regulate ALK1-mediated responses, and it is required to prevent vessel dysmorphogenesis and hemorrhage in the embryonic liver vasculature. Reduced Alk1 gene dosage rescued embryonic hepatic hemorrhage and microvascular capillarization induced by Smad6 deletion in endothelial cells in vivo. At the cellular level, co-depletion of Smad6 and Alk1 rescued the destabilized junctions and impaired barrier function of endothelial cells depleted for SMAD6 alone. Mechanistically, blockade of actomyosin contractility or increased PI3K signaling rescued endothelial junction defects induced by SMAD6 loss. Thus, SMAD6 normally modulates ALK1 function in endothelial cells to regulate PI3K signaling and contractility, and SMAD6 loss increases signaling through ALK1 that disrupts endothelial cell junctions. ALK1 loss-of-function also disrupts vascular development and function, indicating that balanced ALK1 signaling is crucial for proper vascular development and identifying ALK1 as a 'Goldilocks' pathway in vascular biology that requires a certain signaling amplitude, regulated by SMAD6, to function properly.

Endothelial Cell Flow-Mediated Quiescence Is Temporally Regulated and Utilizes the Cell Cycle Inhibitor p27.

BACKGROUND: Endothelial cells regulate their cell cycle as blood vessels remodel and transition to quiescence downstream of blood flow-induced mechanotransduction. Laminar blood flow leads to quiescence, but how flow-mediated quiescence is established and maintained is poorly understood. METHODS: Primary human endothelial cells were exposed to laminar flow regimens and gene expression manipulations, and quiescence depth was analyzed via time-to-cell cycle reentry after flow cessation. Mouse and zebrafish endothelial expression patterns were examined via scRNA-seq (single-cell RNA sequencing) analysis, and mutant or morphant fish lacking p27 were analyzed for endothelial cell cycle regulation and in vivo cellular behaviors. RESULTS: Arterial flow-exposed endothelial cells had a distinct transcriptome, and they first entered a deep quiescence, then transitioned to shallow quiescence under homeostatic maintenance conditions. In contrast, venous flow-exposed endothelial cells entered deep quiescence early that did not change with homeostasis. The cell cycle inhibitor p27 (CDKN1B) was required to establish endothelial flow-mediated quiescence, and expression levels positively correlated with quiescence depth. p27 loss in vivo led to endothelial cell cycle upregulation and ectopic sprouting, consistent with loss of quiescence. HES1 and ID3, transcriptional repressors of p27 upregulated by arterial flow, were required for quiescence depth changes and the reduced p27 levels associated with shallow quiescence. CONCLUSIONS: Endothelial cell flow-mediated quiescence has unique properties and temporal regulation of quiescence depth that depends on the flow stimulus. These findings are consistent with a model whereby flow-mediated endothelial cell quiescence depth is temporally regulated downstream of p27 transcriptional regulation by HES1 and ID3. The findings are important in understanding endothelial cell quiescence misregulation that leads to vascular dysfunction and disease.

Differential endothelial cell cycle status in postnatal retinal vessels revealed using a novel PIP-FUCCI reporter and zonation analysis.

Cell cycle regulation is critical to blood vessel formation and function, but how the endothelial cell cycle integrates with vascular regulation is not well-understood, and available dynamic cell cycle reporters do not precisely distinguish all cell cycle stage transitions in vivo. Here we characterized a recently developed improved cell cycle reporter (PIP-FUCCI) that precisely delineates S phase and the S/G2 transition. Live image analysis of primary endothelial cells revealed predicted temporal changes and well-defined stage transitions. A new inducible mouse cell cycle reporter allele was selectively expressed in postnatal retinal endothelial cells upon Cre-mediated activation and predicted endothelial cell cycle status. We developed a semi-automated zonation program to define endothelial cell cycle status in spatially defined and developmentally distinct retinal areas and found predicted cell cycle stage differences in arteries, veins, and remodeled and angiogenic capillaries. Surprisingly, the predicted dearth of S-phase proliferative tip cells relative to stalk cells at the vascular front was accompanied by an unexpected enrichment for endothelial tip and stalk cells in G2, suggesting G2 stalling as a contribution to tip-cell arrest and dynamics at the front. Thus, this improved reporter precisely defines endothelial cell cycle status in vivo and reveals novel G2 regulation that may contribute to unique aspects of blood vessel network expansion.

Development of a mammalian cell-based ZZ display system for IgG quantification.

BACKGROUND: Biological laboratories and companies involved in antibody development need convenient and versatile methods to detect highly active antibodies. METHODS: To develop a mammalian cell-based ZZ display system for antibody quantification, the eukaryotic ZZ-displayed plasmid was constructed and transfected into CHO cells. After screening by flow cytometric sorting, the stable ZZ display cells were incubated with reference IgG and samples with unknown IgG content for 40 min at 4℃, the relative fluorescence intensity of cells was analyzed and the concentration of IgG was calculated. RESULTS: By investigating the effects of different display-associated genetic elements, a eukaryotic ZZ-displaying plasmid with the highest display efficiency were constructed. After transfection and screening, almost 100% of the cells were able to display the ZZ peptide (designated CHO-ZZ cells). These stable CHO-ZZ cells were able to capture a variety of IgG, including human, rabbit, donkey and even mouse and goat. CHO-ZZ cells could be used to quantify human IgG in the range of approximately 12.5-1000 ng/mL, and to identify high-yielding engineered monoclonal cell lines. CONCLUSIONS: We have established a highly efficient CHO-ZZ display system in this study, which enables the quantification of IgG from various species under physiological conditions. This system offers the advantage of eliminating the need for antibody purification and will contribute to antibody development.

Functional coordination of non-myocytes plays a key role in adult zebrafish heart regeneration.

Cardiac regeneration occurs primarily through proliferation of existing cardiomyocytes, but also involves complex interactions between distinct cardiac cell types including non-cardiomyocytes (non-CMs). However, the subpopulations, distinguishing molecular features, cellular functions, and intercellular interactions of non-CMs in heart regeneration remain largely unexplored. Using the LIGER algorithm, we assemble an atlas of cell states from 61,977 individual non-CM scRNA-seq profiles isolated at multiple time points during regeneration. This analysis reveals extensive non-CM cell diversity, including multiple macrophage (MC), fibroblast (FB), and endothelial cell (EC) subpopulations with unique spatiotemporal distributions, and suggests an important role for MC in inducing the activated FB and EC subpopulations. Indeed, pharmacological perturbation of MC function compromises the induction of the unique FB and EC subpopulations. Furthermore, we developed computational algorithm Topologizer to map the topological relationships and dynamic transitions between functional states. We uncover dynamic transitions between MC functional states and identify factors involved in mRNA processing and transcriptional regulation associated with the transition. Together, our single-cell transcriptomic analysis of non-CMs during cardiac regeneration provides a blueprint for interrogating the molecular and cellular basis of this process.

Discovery of novel resorcinol biphenyl ether-based macrocyclic small molecules as PD-1/PD-L1 inhibitors with favorable pharmacokinetics for cancer immunotherapy.

Programmed death protein 1 (PD-1)/programmed death protein ligand 1 (PD-L1) is one of the most promising immune checkpoints (ICs) in tumor immunology and has been actively pursued as a target for anticancer drug discovery. Based on our previous research in small molecule PD-1/PD-L1 modulators, we designed and synthesized a series of resorcinol biphenyl ether-bearing macrocyclic compounds and evaluated their anti-PD-1/PD-L1 activities. Among them, compound 8d exhibited the highest inhibitory activity against PD-1/PD-L1 interaction with IC50 of 259.7 nM in the homogenous time-resolved fluorescence (HTRF) assay. In addition, 8d displayed in vitro immunomodulatory effects by promoting HepG2 cell death in a HepG2/Jurkat cell co-culture model. Furthermore, 8d effectively inhibited tumor growth (TGI = 74.6% at 40 mg/kg) in a melanoma tumor model in mice without causing obvious toxicity. Moreover, 8d exhibited favorable pharmacokinetics [e.g. high stability, reasonable half-life, and good oral bioavailability (F = 21.5%)]. Finally, molecular modeling studies showed that 8d bound to PD-L1 with high affinity. These results suggest that 8d may serve as a starting point for further development of macrocyclic small molecule-based PD-1/PD-L1 inhibitors for cancer treatment.

Single-cell transcriptomics reconstructs fate conversion from fibroblast to cardiomyocyte.

Direct lineage conversion offers a new strategy for tissue regeneration and disease modelling. Despite recent success in directly reprogramming fibroblasts into various cell types, the precise changes that occur as fibroblasts progressively convert to the target cell fates remain unclear. The inherent heterogeneity and asynchronous nature of the reprogramming process renders it difficult to study this process using bulk genomic techniques. Here we used single-cell RNA sequencing to overcome this limitation and analysed global transcriptome changes at early stages during the reprogramming of mouse fibroblasts into induced cardiomyocytes (iCMs). Using unsupervised dimensionality reduction and clustering algorithms, we identified molecularly distinct subpopulations of cells during reprogramming. We also constructed routes of iCM formation, and delineated the relationship between cell proliferation and iCM induction. Further analysis of global gene expression changes during reprogramming revealed unexpected downregulation of factors involved in mRNA processing and splicing. Detailed functional analysis of the top candidate splicing factor, Ptbp1, revealed that it is a critical barrier for the acquisition of cardiomyocyte-specific splicing patterns in fibroblasts. Concomitantly, Ptbp1 depletion promoted cardiac transcriptome acquisition and increased iCM reprogramming efficiency. Additional quantitative analysis of our dataset revealed a strong correlation between the expression of each reprogramming factor and the progress of individual cells through the reprogramming process, and led to the discovery of new surface markers for the enrichment of iCMs. In summary, our single-cell transcriptomics approaches enabled us to reconstruct the reprogramming trajectory and to uncover intermediate cell populations, gene pathways and regulators involved in iCM induction.

Association of mitochondrial DNA copy number with chronic kidney disease in older adults.

BACKGROUND: Mitochondrial dysfunction in kidney cells has been implicated in the pathogenesis of chronic kidney disease (CKD). Estimation of mitochondrial DNA copy number (mtDNA-CN) is considered a convenient method for representing mitochondrial function in large samples. However, no study has investigated the association between mtDNA-CN and CKD in older adults with the highest prevalence. The objective is to examine cross-sectional and prospective associations between mtDNA-CN values and CKD risk in older adults to determine whether mtDNA-CN represents a novel potential biomarker for the recognition of CKD risk. PATIENTS AND METHODS: In a Chinese community-based cohort of over 65-year-olds, we included 14,467 participants (52.6% females). CKD was defined by eGFR < 60 mL/min/1.73 m2 or ICD-10 codes (patients = 3831 (26.5%)). Participants had peripheral blood levels of mtDNA-CN calculated from probe intensities of the Axiom CAS Array. RESULTS: The risk of CKD prevalence decreased with mtDNA-CN per 1-SD increment, independent of established risk factors for older CKD (odds ratio [OR] per SD 0.90, 95% confidence interval [CI] 0.86, 0.93, P < 0.001), and has comparable strength of association with these established risk factors. Furthermore, the progression of kidney function was stratified according to the worsening of eGFR categories. The risk of kidney function progression to a more severe stage gradually decreased as the mtDNA-CN increased (P trend < 0.001). Non-CKD participants in the highest quartile of mtDNA-CN had a lower risk of developing CKD compared to the lowest quartile within 2 years of follow-up, reducing the risk of CKD by 36% (95% CI 0.42, 0.97; P = 0.037). CONCLUSIONS: Based on the analysis of the largest sample to date investigating the association between mtDNA-CN and CKD in older adults, higher levels of mtDNA-CN were found to be associated with a lower risk of CKD, suggesting that a reduced level of mtDNA-CN is a potential risk factor for CKD.

Effects of regular aerobic exercise on vascular function in overweight or obese older adults: A systematic review and meta-analysis.

Background: Overweight and obese older adults have a high risk for developing cardiovascular disease. Aerobic exercise is a valuable strategy to improve vascular health, but the effects of aerobic exercise on vascular endothelial function in obese and overweight older adults remain controversial. The purpose of this meta-analysis was to investigate the effects of aerobic exercise on vascular function in obese and overweight older adults with or without comorbidity. Methods: A systematic literature search for related studies published in English was conducted between January 1989 and October 30, 2022, in the PubMed, Embase, and Cochrane Library databases. A random effects model was chosen for meta-analysis, which calculated the effect sizes of control and intervention groups after exercise intervention using standardized mean differences (SMDs) corrected for Hedges' g bias and 95% confidence intervals (95% CIs). Results: Twenty-six studies containing 1418 participants were included in the study. After excluding three studies contributing to higher heterogeneity by sensitivity analysis, there are small effects of regular aerobic exercise on vascular function of obese and overweight older adults, including flow-mediated dilation (FMD) [SMD = 0.21, 95% CI (0.02, 0.41), z = 2.16, df = 19, I2 = 52.2%, P = 0.031] and pulse wave velocity (PWV) [SMD = -0.24, 95% CI (-0.46, -0.02), z = 2.17, df = 10, I2 = 8.6%, P = 0.030], and no significant effect was observed on augmentation index (Aix). Subgroup analysis showed small effects of regular aerobic exercise on FMD [SMD = 0.37, 95% CI (0.13, 0.61), z = 3.05, df = 9, I2 = 52.6%, P = 0.002] in the overweight not obese subgroup (25 = BMI <30 kg/m2), but no significant effect on the obese subgroup (BMI ≥30 kg/m2). Regular aerobic exercise for more than 24 weeks improved FMD by small effect sizes [SMD = 0.48, 95% CI (0.04, 0.93), z = 2.12, df = 5, I2 = 56.4%, P = 0.034] and for more than three times per week improved FMD by moderate effect sizes [SMD = 0.55, 95% CI (0.12, 0.98), z = 2.50, df = 3, I2 = 31.1%, P = 0.012] in obese and overweight older adults with or without CVD. Conclusion: In obese and overweight older adults with or without comorbidity, regular aerobic exercise for more than 24 weeks improved FMD by small effect sizes and exercise for more than three times per week improved FMD by moderate effect sizes and regular aerobic exercise reduced PWV by small effect sizes and had no influence on Aix. Taken together, it was recommended that obese and overweight older adults should adhere to regular aerobic exercise, training at least 3 times per week for better results.

Controlled Deformation of Soft Nanogel Particles Generates Artificial Biominerals with Ordered Internal Structure.

Biominerals can exhibit exceptional mechanical properties owing to their hierarchically-ordered organic/inorganic nanocomposite structure. However, synthetic routes to oriented artificial biominerals of comparable complexity remain a formidable technical challenge. Herein we design a series of soft, deformable nanogels that are employed as particulate additives to prepare nanogel@calcite nanocomposite crystals. Remarkably, such nanogels undergo a significant morphological change-from spherical to pseudo-hemispherical-depending on their degree of cross-linking. This deformation occurs normal to the growth direction of the (104) face of the calcite and the underlying occlusion mechanism is revealed by in situ atomic force microscopy studies. This model system provides new mechanistic insights regarding the formation of oriented structures during biomineralization and offers new avenues for the design of synthetic nanocomposites comprising aligned anisotropic nanoparticles.

Single-Cell RNA Sequencing Reveals Endothelial Cell Transcriptome Heterogeneity Under Homeostatic Laminar Flow.

Objective: Endothelial cells (ECs) that form the innermost layer of all vessels exhibit heterogeneous cell behaviors and responses to pro-angiogenic signals that are critical for vascular sprouting and angiogenesis. Once vessels form, remodeling and blood flow lead to EC quiescence, and homogeneity in cell behaviors and signaling responses. These changes are important for the function of mature vessels, but whether and at what level ECs regulate overall expression heterogeneity during this transition is poorly understood. Here, we profiled EC transcriptomic heterogeneity, and expression heterogeneity of selected proteins, under homeostatic laminar flow. Approach and Results: Single-cell RNA sequencing and fluorescence microscopy were used to characterize heterogeneity in RNA and protein gene expression levels of human ECs under homeostatic laminar flow compared to nonflow conditions. Analysis of transcriptome variance, Gini coefficient, and coefficient of variation showed that more genes increased RNA heterogeneity under laminar flow relative to genes whose expression became more homogeneous, although small subsets of cells did not follow this pattern. Analysis of a subset of genes for relative protein expression revealed little congruence between RNA and protein heterogeneity changes under flow. In contrast, the magnitude of expression level changes in RNA and protein was more coordinated among ECs in flow versus nonflow conditions. Conclusions: ECs exposed to homeostatic laminar flow showed overall increased heterogeneity in RNA expression levels, while expression heterogeneity of selected cognate proteins did not follow RNA heterogeneity changes closely. These findings suggest that EC homeostasis is imposed post-transcriptionally in response to laminar flow.

Metal-Organic Frameworks@Calcite Composite Crystals.

The direct incorporation of guest crystals into another type of host crystals during the formation of the latter is technically challenging due to the large difference in surface energy for different crystalline components. Nevertheless, we herein demonstrate that metal-organic frameworks (MOFs, UiO-66-NH2 as a model guest crystal) after postsynthetic modification with poly(methacrylic acid) can be efficiently incorporated into calcite single crystals, forming a unique composite structure where the MOF crystals are uniformly distributed throughout the whole calcite host crystals. Remarkably, such MOF@calcite composite crystals exhibit superior performance in fluoride removal compared with the MOF or calcite alone. Moreover, this incorporation strategy is general as it can be extended to other guest particles. In principle, this study opens up a versatile avenue for the rational design and preparation of a wide range of hybrid functional materials with controllable compositions and enhanced physicochemical properties.

SMAD6 transduces endothelial cell flow responses required for blood vessel homeostasis.

Fluid shear stress provided by blood flow instigates a transition from active blood vessel network expansion during development, to vascular homeostasis and quiescence that is important for mature blood vessel function. Here we show that SMAD6 is required for endothelial cell flow-mediated responses leading to maintenance of vascular homeostasis. Concomitant manipulation of the mechanosensor Notch1 pathway and SMAD6 expression levels revealed that SMAD6 functions downstream of ligand-induced Notch signaling and transcription regulation. Mechanistically, full-length SMAD6 protein was needed to rescue Notch loss-induced flow misalignment. Endothelial cells depleted for SMAD6 had defective barrier function accompanied by upregulation of proliferation-associated genes and down regulation of junction-associated genes. The vascular protocadherin PCDH12 was upregulated by SMAD6 and required for proper flow-mediated endothelial cell alignment, placing it downstream of SMAD6. Thus, SMAD6 is a required transducer of flow-mediated signaling inputs downstream of Notch1 and upstream of PCDH12, as vessels transition from an angiogenic phenotype to maintenance of a homeostatic phenotype.

Lin28a Regulates Pathological Cardiac Hypertrophic Growth Through Pck2-Mediated Enhancement of Anabolic Synthesis.

BACKGROUND: Hypertrophic response to pathological stimuli is a complex biological process that involves transcriptional and epigenetic regulation of the cardiac transcriptome. Although previous studies have implicated transcriptional factors and signaling molecules in pathological hypertrophy, the role of RNA-binding protein in this process has received little attention. METHODS: Here we used transverse aortic constriction and in vitro cardiac hypertrophy models to characterize the role of an evolutionary conserved RNA-binding protein Lin28a in pathological cardiac hypertrophy. Next-generation sequencing, RNA immunoprecipitation, and gene expression analyses were applied to identify the downstream targets of Lin28a. Epistatic analysis, metabolic assays, and flux analysis were further used to characterize the effects of Lin28a and its downstream mediator in cardiomyocyte hypertrophic growth and metabolic remodeling. RESULTS: Cardiac-specific deletion of Lin28a attenuated pressure overload-induced hypertrophic growth, cardiac dysfunction, and alterations in cardiac transcriptome. Mechanistically, Lin28a directly bound to mitochondrial phosphoenolpyruvate carboxykinase 2 ( Pck2) mRNA and increased its transcript level. Increasing Pck2 was sufficient to promote hypertrophic growth similar to that caused by increasing Lin28a, whereas knocking down Pck2 attenuated norepinephrine-induced cardiac hypertrophy. Epistatic analysis demonstrated that Pck2 mediated, at least in part, the role of Lin28a in cardiac hypertrophic growth. Furthermore, metabolomic analyses highlighted the role for Lin28a and Pck2 in promoting cardiac biosynthesis required for cell growth. CONCLUSIONS: Our study demonstrates that Lin28a promotes pathological cardiac hypertrophy and glycolytic reprograming, at least in part, by binding to and stabilizing Pck2 mRNA.

Real time low-complexity adaptive channel equalization for coherent optical transmission systems.

In this paper, a novel low-complexity adaptive channel equalization (ACE) algorithm for digital coherent optical systems is proposed and experimentally demonstrated. We divide the conventional N-tap butterfly ACE into two N-tap polarization independent filters and a 1-tap butterfly adaptive equalization filter. The computational complexity can reduce about 40% of multiplier operations in the digital signal processing (DSP). We evaluate the effectiveness of our proposed ACE algorithm in a 10-Gb/s real-time coherent transmission platform. It is shown that our proposed ACE algorithm has similar performance as conventional ACE algorithm and better polarization tracking ability.

A comparative quantitative study of utilizing artificial intelligence on electronic health records in the USA and China during 2008-2017.

BACKGROUND: The application of artificial intelligence techniques for processing electronic health records data plays increasingly significant role in advancing clinical decision support. This study conducts a quantitative comparison on the research of utilizing artificial intelligence on electronic health records between the USA and China to discovery their research similarities and differences. METHODS: Publications from both Web of Science and PubMed are retrieved to explore the research status and academic performances of the two countries quantitatively. Bibliometrics, geographic visualization, collaboration degree calculation, social network analysis, latent dirichlet allocation, and affinity propagation clustering are applied to analyze research quantity, collaboration relations, and hot research topics. RESULTS: There are 1031 publications from the USA and 173 publications from China during 2008-2017 period. The annual numbers of publications from the USA and China increase polynomially. JAMIA with 135 publications and JBI with 13 publications are the top prolific journals for the USA and China, respectively. Harvard University with 101 publications and Zhejiang University with 12 publications are the top prolific affiliations for the USA and China, respectively. Massachusetts is the most prolific region with 211 publications for the USA, while for China, Taiwan is the top 1 with 47 publications. China has relatively higher institutional and international collaborations. Nine main research areas for the USA are identified, differentiating 7 for China. CONCLUSIONS: There is a steadily growing presence and increasing visibility of utilizing artificial intelligence on electronic health records for the USA and China over the years. The results of the study demonstrate the research similarities and differences, as well as strengths and weaknesses of the two countries.

A bibliometric analysis of natural language processing in medical research.

BACKGROUND: Natural language processing (NLP) has become an increasingly significant role in advancing medicine. Rich research achievements of NLP methods and applications for medical information processing are available. It is of great significance to conduct a deep analysis to understand the recent development of NLP-empowered medical research field. However, limited study examining the research status of this field could be found. Therefore, this study aims to quantitatively assess the academic output of NLP in medical research field. METHODS: We conducted a bibliometric analysis on NLP-empowered medical research publications retrieved from PubMed in the period 2007-2016. The analysis focused on three aspects. Firstly, the literature distribution characteristics were obtained with a statistics analysis method. Secondly, a network analysis method was used to reveal scientific collaboration relations. Finally, thematic discovery and evolution was reflected using an affinity propagation clustering method. RESULTS: There were 1405 NLP-empowered medical research publications published during the 10 years with an average annual growth rate of 18.39%. 10 most productive publication sources together contributed more than 50% of the total publications. The USA had the highest number of publications. A moderately significant correlation between country's publications and GDP per capita was revealed. Denny, Joshua C was the most productive author. Mayo Clinic was the most productive affiliation. The annual co-affiliation and co-country rates reached 64.04% and 15.79% in 2016, respectively. 10 main great thematic areas were identified including Computational biology, Terminology mining, Information extraction, Text classification, Social medium as data source, Information retrieval, etc. CONCLUSIONS: A bibliometric analysis of NLP-empowered medical research publications for uncovering the recent research status is presented. The results can assist relevant researchers, especially newcomers in understanding the research development systematically, seeking scientific cooperation partners, optimizing research topic choices and monitoring new scientific or technological activities.

Stoichiometry of Gata4, Mef2c, and Tbx5 influences the efficiency and quality of induced cardiac myocyte reprogramming.

RATIONALE: Generation of induced cardiac myocytes (iCMs) directly from fibroblasts offers great opportunities for cardiac disease modeling and cardiac regeneration. A major challenge of iCM generation is the low conversion rate of fibroblasts to fully reprogrammed iCMs, which could in part be attributed to unbalanced expression of reprogramming factors Gata4 (G), Mef2c (M), and Tbx5 (T) using the current gene delivery approach. OBJECTIVE: We aimed to establish a system to express distinct ratios of G, M, T proteins in fibroblasts and determine the effect of G, M, T stoichiometry on iCM reprogramming. METHODS AND RESULTS: We took advantage of the inherent feature of the polycistronic system and generated all possible combinations of G, M, T with identical 2A sequences in a single transgene. We demonstrated that each splicing order of G, M, T gave rise to distinct G, M, T protein expression levels. Combinations that resulted in higher protein level of Mef2c with lower levels of Gata4 and Tbx5 significantly enhanced reprogramming efficiency compared with separate G, M, T transduction. Importantly, after further optimization, the MGT vector resulted in more than 10-fold increase in the number of mature beating iCM loci. Molecular characterization revealed that more optimal G, M, T stoichiometry correlated with higher expression of mature cardiac myocyte markers. CONCLUSIONS: Our results demonstrate that stoichiometry of G, M, T protein expression influences the efficiency and quality of iCM reprogramming. The established optimal G, M, T expression condition will provide a valuable platform for future iCM studies.

Real-time experimental demonstration of PS-QPSK transmission with manipulated rotating and novel correlated CMA.

In this paper, we propose a novel manipulated rotating polarization switched quadrature phase shift keying (MR-PS-QPSK) technique, and corresponding correlated constant modulus algorithm (CMA) for signal recovery. The latter utilizes the correlation between the PS-QPSK symbols in the two polarizations to lock the phase of output signals. Then the signals in the two polarizations are merged according to the recovered switching bit, which suppresses the noise and simplifies the subsequent process. A field programmable gate array (FPGA) based real-time platform is built for experimental demonstration. The experimental results show that the proposed MR-PS-QPSK modulation format with correlated CMA can provide 3.2 dB optical signal-to-noise ratio (OSNR) improvement over dual-polarization QPSK (DP-QPSK) at back-to-back case and 3.8 dB OSNR improvement after fiber transmission at the same symbol rate, which corresponds to be about 2 dB OSNR improvement at the same bit rate. The resource consumption analysis in FPGA digital signal processing blocks and logic utilizations shows that the MR-PS-QPSK with correlated CMA only requires a small additional computational effort.

Exosome-associated hepatitis C virus in cell cultures and patient plasma.

Hepatitis C virus (HCV) infects its target cells in the form of cell-free viruses and through cell-cell contact. Here we report that HCV is associated with exosomes. Using highly purified exosomes and transmission electron microscopic imaging, we demonstrated that HCV occurred in both exosome-free and exosome-associated forms. Exosome-associated HCV was infectious and resistant to neutralization by an anti-HCV neutralizing antibody. There were more exosome-associated HCV than exosome-free HCV detected in the plasma of HCV-infected patients. These results suggest exosome-associated HCV as an alternative form for HCV infection and transmission.