Virus-like particles (VLPs) derived from viral nucleocapsids are an important class of nanoparticles. The structure, uniformity, stability, and function of these VLPs have attracted scientists in utilizing them as a unique tool in various applications in biomedical fields. Their interaction with the innate immune system is of major importance for the adaptive immune response they induce. The innate immune cells and molecules recognize and interact with VLPs on the basis of two major characteristics: size and surface geometry. This review discusses the interaction of viral capsid-derived VLPs with the innate immune system. Virus-like particles (VLPs) are supramolecular complexes formed by viral proteins that self-assemble into capsids resembling real viruses, albeit non-infectious due to the lack of viral genome packaged within the particle. Such non-infectious particles can be naturally formed during infection or genetically engineered and produced in large scales in laboratories. Most VLPs are characterized by stability, together with uniform and repetitive structure that allow diverse applications in the field of biomedical science. Since their early discovery five decades ago, VLPs led to enormous progress in the field of vaccine development with seven vaccines approved for humans and several others in pre-clinical phase or in clinical trials ,2. Most The virus-like property of VLPs is a potent geometric pathogen-associated structural pattern (PASP) facilitating their engagement with innate and adaptive immune cells. The exterior and interior facets of VLPs can be functionalized and modified to enhance their immunogenicity and stability or to present heterologous antigens . The virsize and surface geometry [VLPs—especially those packaging nucleic acids—are capable of inducing a comparable humoral immune response to viruses. This is considered a major advantage of VLPs over attenuated virus-derived vaccines as it guarantees safety of the vaccines whilst retaining their immunogenicity . The immgeometry  as discuThe lymphatic system regulates the balance of fluid in the body as well as the trafficking of immune cells and antigens to lymphoid organs, in particular lymph nodes (LNs) . The effThe diameter of the blind-ended structure of the initial lymphatic vessels is about 10–60 μm. The vessels are lined up with single flattened endothelial cells forming valves that ensure the unidirectional movement of fluids through and along the lymphatic vessels . FurtherhiCD40hiCD8− and CD11cintCD40hiCD8− DCs—can successfully uptake VLPs after intradermal injection. Both identified subsets of skin-derived DCs have also been shown to effectively cross-prime cytotoxic T-cells following VLPs uptake [+ DCs are usually much more potent in cross-presenting VLP-derived antigens [The kinetics of free drainage of viral capsid-derived VLPs has been studied extensively and previous data has shown that VLPs with average size of 30 nm can be detected in mice footpads for at least 40 min post injection and at the draining LN 2 h post injection . Recentls uptake . Even thantigens .In summary, the size of antigen influences the immunogenicity largely by two mechanisms: first, by allowing the appropriate draining from periphery to secondary lymphoid organs and second by facilitating the interaction with APCs due to their repetitive surface. VLPs size distribution falls within the aforementioned optimal range, which explains in part its remarkable immunogenicity.In general, particles ranging between 20–200 nm upon reaching the LN are distributed throughout the different areas of the draining LN. Previous studies have shown that VLPs are detected at the sub-capsular, medullar and cortical regions of LNs as fast as 2 h post subcutaneous injection. At a later stage, VLPs are also detected deeper in B-cell follicles bound to follicular dendritic cells (fDCs) . At 48 h+F4/80+, different subsets of conventional dendritic cells (cDCs) including CD8+CD11c+, CD8−CD11b+, and CD8+CD11c+. B-cells characterized by CD45R/B220+ were less efficient in up taking VLPs [+ DCs for effective CTL priming [The predominant subsets of APCs actively up taking viral capsid-derived VLPs in the popliteal LN post subcutaneous injection have been recently classified. These cells include the sub-capsular sinus (SCS) macrophages CD11bing VLPs  but are ing VLPs . Medullaing VLPs . Therefo priming .As described above, the draining and trafficking of VLPs within the immune system and subsequent interaction with immune cells such as DCs, macrophages, and B cells allow the initiation of both humoral and cellular immune responses. Details of the immune response to VLPs used as vaccines will be discussed in the following sections.The highly repetitive surface structure of viral capsid-derived VLPs facilitates their interaction with components of the innate humoral immune system that in turn mediates opsonization and phagocytosis by APCs . The natThe pentraxin protein family including the short pentraxin C-reactive protein (CRP), serum amyloid P (SAP) and long pentraxin (PTX3) may also recognize the repetitive structure of VLPs due to high avidity interactions. SAP, for example, has been shown to bind to influenza A viral particles . This reThe hallmark of the immune response induced by VLPs is the induction of high levels of long lasting humoral responses. In contrast to soluble antigens, the highly organized and repetitive structure of VLPs promotes crosslinking of BCRs that surpasses the activation threshold and bypasses the initial need of T-cell help. As a consequence, any antigen exposed on the surface of VLPs in this same organized and repetitive array will benefit from this viral fingerprint and induce high levels of humoral responses.Another important feature of the humoral response elicited by VLPs is the promotion of isotype switching dependent on direct TLR7 or TLR9 signaling on B-cells ,28. UponFurthermore, intranasal administration of VLPs in murine models resulted in strong B-cell responses and germinal center formation in the spleen. Roaming B-cells in the lungs have been shown to bind VLPs through their BCR and shuttle them to B-cell follicles within the spleen .h CD4+ cells following endosomal processing [+ CTLs priming. The classical source of MHC-I antigens is newly synthesized proteins which are misfolded and degraded in the proteasomes. Nevertheless, it has been found that both presentation pathways are not strictly separated [h cells has been previously documented to be highly efficient and biologically relevant. Cross-presentation on MHC-I molecules has also been studied extensively and has been documented for tumor cells, necrotic cells, apoptotic cells, viruses, and VLPs [+ DCs only while CD8− DCs present them on MHC-II molecules [Following the uptake by professional APCs, processed VLPs will be subsequently presented on MHC molecules. In general, exogenous antigens (endocytosed or phagocytosed) are presented on MHC-II molecules to trigger the activation of Tocessing . Endogeneparated  as exogeeparated ,33,34. Ceparated ,36. The and VLPs ,38,39. Tolecules .+ and CD8− DCs can efficiently present VLP-derived antigens on MHC-II molecules, however only CD8+ DCs were able to cross-present VLPs derived peptides. This implies that VLPs presentation on MHC-II molecules to CD4+ T-cells is not restricted to a specific DCs subset but cross-presentation to CD8+ T-cells is usually restricted to a CD8+ DCs subset [However, this dichotomy hypothesis was not supported in other studies which have shown that both CD8s subset .h1 and CTL response [Many VLPs, including Norovirus, Papaya Mosaic Virus, Human Papilloma Virus and Qβ-derived VLPs activate innate cells through various means ,36,41. Aresponse . Non-metresponse . Class Aresponse . In contresponse . The homresponse . Free RNresponse . It has response .In general, recognition of ssRNA, dsRNAm or CpGs packaged into VLPs by endosomal TLRs will result in the activation of signaling pathways that are essential for the expression of different genes required to initiate the inflammatory responses . The proApart from TLRs, other PRRs and nucleic acid sensors have also been reported to play a role in the immune response elicited by VLPs. RIG-I and the stimulator of IFN genes protein (STING) induces significant levels of type-I IFN upon viral infection mediating autocrine and paracrine signaling . ActivatVLPs had been consistently successful at inducing protective immune responses in various pre-clinical models and clinical trials both for infectious and non-infectious diseases. The field had started employing naturally occurring VLPs during infection as vaccines, as exemplified by the Hepatitis B vaccine  and has The success of this approach relies on presenting heterologous antigens to the surface of model VLPs and in this manner, conferring the highly immunogenic viral fingerprint to those antigens. There are two main methods for presentation of heterologous antigens: chemical crosslinking or genetic fusion. The choice of method will impact the quality of the immune response as it will affect the valency of decoration of the particle with the antigen and the overall stability of the vaccine. For that reason, small changes in the crosslinking method must be closely evaluated as it can directly affect immunogenicity.A second important consideration for VLPs is the expression system of VLPs. During the assembly phase VLPs will randomly pack host derived components that can have an unbeknownst impact in the immune response. Thus, the same VLP can exert different immune responses depending on the manufacturing process. This is an important consideration when comparing results across different groups and when scaling up the manufacturing process.As is common to other vaccine development platforms, there are several challenges that should be considered for successful development of VLP-based vaccines. There are some risks that are specific to the VLP-platform technologies and some risks that are related to the specific vaccine candidates.Challenges specific to VLP-based vaccine platformsEven though several VLP-based vaccines are on the market, some more recent candidates struggle with stability. In addition, no vaccine that displays foreign epitopes has made it to the market so far. Hence, real-life, market PoC for such vaccines is missing. While there is no a priori reason that this should not be possible, it may still be perceived as a potential risk.Most if not all nucleocapsid VLPs derived from RNA viruses package RNA from the production host cells. This may need an additional Quality Control effort.If epitopes are to be fused into VLPs, this can create substantial problems, as VLPs may not assemble anymore.Challenges for individual vaccinesThe selected epitope may not be protectiveInduced immune responses may be too lowThe selected indication may sound interesting but does not attract interest from industry and/or the end-customerThe highly immunogenic properties of VLPs are a direct consequence of the viral fingerprint retained from the parental viruses. Such properties can be summarized by size, surface geometry, and ability to package nucleic acids. VLPs immunogenicity can also be extended by presenting heterologous antigens on their surface.


Given the increased attention to sepsis at the population level there is a need to assess hospital performance in the care of sepsis patients using widely-available administrative data. The goal of this study was to develop an administrative risk-adjustment model suitable for profiling hospitals on their 30-day mortality rates for patients with sepsis.We conducted a retrospective cohort study using hospital discharge data from general acute care hospitals in Pennsylvania in 2012 and 2013. We identified adult patients with sepsis as determined by validated diagnosis and procedure codes. We developed an administrative risk-adjustment model in 2012 data. We then validated this model in two ways: by examining the stability of performance assessments over time between 2012 and 2013, and by examining the stability of performance assessments in 2012 after the addition of laboratory variables measured on day one of hospital admission.In 2012 there were 115,213 sepsis encounters in 152 hospitals. The overall unadjusted mortality rate was 18.5%. The final risk-adjustment model had good discrimination (C-statistic = 0.78) and calibration . Based on this model, hospital-specific risk-standardized mortality rates ranged from 12.2 to 24.5%. Comparing performance assessments between years, correlation in risk-adjusted mortality rates was good (Pearson’s correlation = 0.53) and only 19.7% of hospitals changed by more than one quintile in performance rankings. Comparing performance assessments after the addition of laboratory variables, correlation in risk-adjusted mortality rates was excellent (Pearson’s correlation = 0.93) and only 2.6% of hospitals changed by more than one quintile in performance rankings.A novel claims-based risk-adjustment model demonstrated wide variation in risk-standardized 30-day sepsis mortality rates across hospitals. Individual hospitals’ performance rankings were stable across years and after the addition of laboratory data. This model provides a robust way to rank hospitals on sepsis mortality while adjusting for patient risk.The online version of this article (10.1186/s12913-019-4037-x) contains supplementary material, which is available to authorized users. Sepsis is a leading cause of in-hospital mortality and a major driver of health care spending in developed nations . SeveralUnderstanding the impact of these efforts and providing hospitals with feedback on their quality of care in patients with sepsis requires a robust method for assessing hospital-specific mortality rates. Such a method would ideally use widely available data that is readily accessible across hospital systems and must effectively account for individual patients’ variation in risk of mortality. At the same time, mortality-based performance measures should not adjust for variation in treatment practices that may modify the risk of mortality, which are reflective of hospital quality.To address this need, we used a state-wide Pennsylvania discharge database that captures administrative claims data along with a selection of laboratory data to create a novel method to adjust for individual patients’ severity of illness on presentation in order to meaningfully compare sepsis outcomes across hospitals.We conducted a retrospective cohort study of patients with sepsis admitted to non-federal general acute care hospitals in the Commonwealth of Pennsylvania in the United States during calendar years 2012 and 2013. First, we developed a de novo risk-adjustment model using 2012 administrative data. Next, we examined the construct validity of our model by examining the stability of hospital rankings over time (comparing the 2012 administrative model to the 2013 administrative model) and after addition of clinical laboratory variables . In this context, a valid administrative model would produce relatively stable performance estimates over time . A valid administrative model would also yield performance estimates that are similar to those estimated from a more granular clinical model which better accounts for variation in risk.International Classification of Diseases—version 9.0—Clinical Modification (ICD-9-CM) diagnosis and procedure codes. Unlike most administrative claims-based data sets, these data also contain a selection of laboratory values obtained on the day of admission, enabling us to create a clinical model in addition to the standard administrative model [We used the Pennsylvania Health Care Cost Containment Council (PHC4) database. PHC4 collects administrative data on all hospital admissions in Pennsylvania and makes them available for research, including both demographic information and ve model . We augmAll encounters for patients meeting the “Angus” definition of sepsis—either an explicit ICD-9-CM code for sepsis or co-documentation of ICD-9-CM codes for an infection and an organ dysfunction—were eligible for the study . We chosWe first created a base logistic regression model for risk-adjusted mortality using exclusively risk-adjustment variables that are available in administrative data. The primary outcome variable for this model was all-cause mortality within 30 days of the admission date, as determined using the Pennsylvania vital status records. The model was based on five categories of risk-adjustment variables hypothesized to be associated with sepsis outcomes based on prior work , 11, 12:Demographic variables were obtained directly from the claims and included age and gender. Gender was modeled as an indicator covariate, and age was modeled as a linear spline by age quintile. Admission source was obtained directly from the claims and modelled as an indicator covariate defined as admission through the emergency department versus admission from another source. Comorbidities were defined using ICD-9-CM codes in the manner of Elixhauser  and modeInfection source was modeled as hierarchical infection categories in which we assigned each patient an infectious source category identified using ICD-9-CM diagnosis codes -based stepwise logistic regression to identify the laboratory value covariates to be included in the model. This regression included all the covariates in the claims-based model. Laboratory values that did not contribute to a maximal BIC were excluded from the final model. Each laboratory value’s categories were assessed in the BIC regression as a group and ultimately either included in or excluded from the model as a group, so as not to partially remove categories for a given laboratory value. Laboratory values deemed contributory by the BIC regression entered the final model as categorical variables with the normal category as the reference group.Based on these models we use mixed-effects logistic regression to create risk-standardized hospital-specific 30-day mortality rates. These rates account for variation in both risk and reliability across hospitals: they account for variation in risk in that they control for the different baseline characteristics of sepsis patients across hospitals; they account for reliability in that the rates for small hospitals, which are more susceptible to random variation than rates for large hospitals, are adjusted toward the state-wide mean .We calculated hospital-specific risk adjusted mortality rates by dividing each hospital’s predicted mortality  by each hospital’s expected mortality , generating a risk-standardized mortality ratio. Multiplying the risk-standardized mortality ratio by the mean 30-day mortality of the state-wide sample yielded a hospital-specific risk-standardized mortality rate.We performed this process separately for 2012 and 2013 without laboratory data and then again for 2012 with laboratory data, resulting in three sets of hospital-specific mortality rates: 2012 administrative rates, 2013 administrative rates, and 2012 clinical rates.For all models we assessed discrimination, using the C-statistic, and calibration, using the slope and intercept of regression lines fit to the calibration plots. We assessed the validity of our administrative model by examining the consistency of hospital rankings over time and with the addition of laboratory data. As noted above, we assumed that a valid model would yield hospital rankings that did not markedly change between years or after the addition of laboratory values. We generated scatter plots to compare the hospital-specific risk-standardized mortality rates between the 2012 and 2013 administrative rates; and between the 2012 administrative and clinical rates, calculating a coefficient of determination. Additionally, for each of the three sets of hospital-specific mortality rates, we calculated performance quintiles, with the outer quintiles representing the highest and lowest performing 20% of hospitals, respectively. We compared the composition of the quintiles between the 2012 and 2013 administrative rates and then between the 2012 administrative and clinical rates. We considered hospital movement of one quintile or less between comparison groups to be a marker of stability.Data management and analysis was performed using Stata version 14.0 . All aspects of this work were reviewed and approved by the University of Pittsburgh institutional review board.A total of 236,154 patients met our final inclusion criteria: 115,213 in 2012 and 120,941 in 2013 . Comparing the 2012 administrative model to the 2012 clinical model, 113 (74%) hospitals stayed in the same quintile, and only 1 hospital (1%) moved by more than one quintile


Significant progress has been made toward understanding the impact of chromatin states on gene expression. Nevertheless, the mechanism of transcription factors binding combinatorially in different chromatin states to enable selective regulation of gene expression remains an interesting research area. We introduce a nonparametric Bayesian clustering method for inhomogeneous Poisson processes to detect heterogeneous binding patterns of multiple proteins including transcription factors to form regulatory modules in different chromatin states. We applied this approach on ChIP-seq data for mouse neural stem cells containing 21 proteins and observed different groups or modules of proteins clustered within different chromatin states. These chromatin-state-specific regulatory modules were found to have significant influence on gene expression. We also observed different motif preferences for certain TFs between different chromatin states. Our results reveal a degree of interdependency between chromatin states and combinatorial binding of proteins in the complex transcriptional regulatory process. The software package is available on Github at -  Transcription factors (TFs) and other proteins that bind to specific DNA sequences play key roles in the regulation of gene expression. Binding locations of a protein of interest can be determined with chromatin immunoprecipitation followed by sequencing (ChIP-seq). This produces millions of short reads covering the protein-DNA binding sites across the genome. Several computational tools have been developed to identify these binding locations from ChIP-seq data. Widely used among these is MACS2 , a nonparametric Bayesian clustering method DPM-LGCP is applied to identify transcriptional regulatory modules within each chromatin state. In downstream analyses, proximal . We chose this modeling strategy for the following reasons: (i) the event of each binding site falling into a minuscule interval is a rare event, independent of the events in other non-overlapping intervals, and (ii) the non-uniform distribution of the peaks at different genomic locations can be well characterized by the intensity function of the inhomogeneous Poisson process. For a protein with n binding site locations, we map these locations to points in a closed interval D on the real line, denoted by S = {s1, …, sn}. Following the inhomogeneous Poisson process model setting, the likelihood of observing S can be written as Simpson et al. f, s ∈ D is the intensity function. The Poisson process likelihood (1) provides the basis for nonparametric clustering of proteins based on their binding patterns, resulting in identification of modules of co-binding proteins that share similar regulatory functions. For a given ChIP-seq dataset of N proteins coming from K clusters (with K unknown), we assume that proteins in the same cluster share a common intensity function, distinct from those in other clusters. Under this assumption, we implement a Dirichlet process mixture of log Gaussian Cox process (DPM-LGCP) model that employs a Dirichlet process (DP) prior to the latent log intensity functions to facilitate clustering of the intensity functions. Let Si denote the binding site locations of the ith protein, the DPM-LGCP model can be described as follows:where |G is a random distribution with a DP prior. The DP prior is characterized by two parameters m and G0, where m is the precision parameter, and G0 is the base measure. The base measure G0 is assumed to be a Gaussian process with mean 0 and covariance kernel Cθ, and θ contains parameters that control the shape of the covariance kernel. The introduction of this DP prior to the latent log intensity functions naturally facilitates clustering of the N point processes based on their intensity functions. With this model, neither the number of clusters nor ad-hoc distance measure between two point processes needs to be specified.where Si, we employed an approximate but efficient posterior inference using the Integrated Nested Laplace Approximations (INLA) package  into a discrete precision matrix of the B-spline basis coefficients on a regular grid, which enables very fast covariance computation . For the nucleosome level states, annotation of the chromatin states to functionally relevant categories was performed by using information from the emission probabilities of the nucleosome states Figure , fractioTo analyze the distribution of protein-DNA binding sites in each chromatin state, we integrated ChIP-seq data with the chromatin state map of mouse neural stem cells (NSCs) Figure . For mosNext, to study the regulatory effect of histone marks on proximal genes, we compared the expression levels of genes (Transcripts Per Kilobase Million or TPM) with promoters located in different chromatin states. We observed that proximal genes in the Broad Promoter state had a higher median expression than proximal genes in the Polycomb Repressed or Low Coverage states Figure . To undeThe distributions of ChIP-seq peaks across distinct chromatin states indicate that functionally relevant proteins may have similar binding patterns  regulated by the predicted clusters in each state to understand transcriptional regulation by combinatorial binding of proteins in different chromatin states. We observed that the median expression level of the genes regulated by distinct clusters are close to each other in the Broad Promoter state Figure . On the We compared the clustering results of the proposed algorithm with K-means and CLARANS  are often enriched in genes targeted by β-catenin/TCF/LEF  De-novo sequences resembling the secondary motifs such the SMAD family. For SMAD4, we observed that sequences with ‘GCCGC' pattern were highly enriched in both active and repressed chromatin states, as reported previously in Hu et al.  1 Figure , MAX, NFe Figure . Howeveru et al.  where thu et al. . Interesu et al. , the autDevelopment of the semi-automated genome annotation tools has enabled genome segmentation and identification of distinct chromatin states at fine resolutions. In this study, we designed a two-step process to identify transcriptional regulatory modules within distinct chromatin states. First, we segmented the genome using the diHMM software. Second, we designed a novel nonparametric Bayesian clustering algorithm to identify clusters of co-binding proteins on the segmented genome. Existing work have adopted distance thresholds and empirical tests to define pairwise co-bound regions and context-dependent co-regulators (Ji et al., We applied the approach on ChIP-seq data for neural stem cells obtained from ChIP-Atlas, an integrated and comprehensive database rapidly gaining importance in cell replacement therapy. Despite the methodological advantages, this approach may have limitations in practical use. First, ChIP-seq can produce millions of short reads, which may result in varying strengths of signal intensities along the genome. In the current study, we did not consider the peak-height for different proteins but treated the center of each peak as a binary binding event along the genome. The overlook of the signal intensity effects may impact the modeling of protein binding patterns. Another possible limitation of our approach lies in handling the three dimensional structural information of the histone marks. This restricted our downstream gene expression analysis to gene promoters present in the Enhancer states. While not in scope of the current study, including such information may improve the accuracy of the model and enable the prediction of long distance Enhancer activity.Nevertheless, we were able to establish several interesting findings. It has been known that protein-DNA binding sites are not randomly distributed but rather clustered together at enhancer or promoter regions. Hence, some specific proteins may team up to have a significant epigenetic impact on gene expression. In our study, transcriptional regulatory modules identified in different chromatin states revealed several known protein-protein interactions in neural stem cells, for example, SOX family and NF1 in the Enhancer states (Webb et al., de-novo sequences predicted for the some proteins resembled the consensus PWM across distinct chromatin states whereas for certain proteins such as SMAD family the sequences resembled secondary motifs in specific chromatin states. Further, we also noticed that the prediction of binding preferences might help the identification of indirect protein bindings when the de-novo sequences do not match the consensus PWM (Yin et al., An important issue in transcription regulation is to understand the binding specificity and affinity of a protein. A TF may have several thousands of DNA binding sites along the genome, which collectively can be represented as a motif—a consensus sequence demonstrating the nucleotide preferences at each position of the binding site. In this study, we observed that chromatin state can have an impact on the binding preferences of transcription factors and their co-activators (Jolma et al., HX conceived and designed the study. HZ and XW designed and implemented the clustering model. SB and MT designed computational experiments and performed data analyses. HX, WF, XW, and SB wrote the original draft. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


A three-dimensional (3D) tissue model has significant advantages over the conventional two-dimensional (2D) model. A 3D model mimics the relevant in-vivo physiological conditions, allowing a cell culture to serve as an effective tool for drug discovery, tissue engineering, and the investigation of disease pathology. The present reviews highlight the recent advances and the development of microfluidics based methods for the generation of cell spheroids. The paper emphasizes on the application of microfluidic technology for tissue engineering including the formation of multicellular spheroids (MCS). Further, the paper discusses the recent technical advances in the integration of microfluidic devices for MCS-based high-throughput drug screening. The review compares the various microfluidic techniques and finally provides a perspective for the future opportunities in this research area. In the past decade, cell-based assays have undergone a noticeable transition from two-dimensional 2D) to three-dimensional 3D) cell culture. A cell culture is the basic tool for drug discovery, investigation of the mechanism of diseases, and tissue engineering. A 3D cell culture maintains the significant physiological relevance of cell-based assays . A 3D ceD to threD cell cuThe limitations of 2D culture systems motivate the development of 3D culture. In contrast to the flat 2D culture, a 3D culture consists of multi-cellular layers, which are critical for both biochemical and mechanical characteristics of a tissue. Thus, a 3D construct allows for the optimal transport of nutrient, gas, growth factors and cellular waste similar to in-vivo processes. To date, countless efforts have been reported on the production of more biologically relevant 3D tissue models using both scaffold-based and scaffold-free strategies. Microtissues constructed with scaffold rely on supporting materials, which raises issues of biocompatibility and cell–material biorecognition. Biodegradable scaffold substitutes a large amount ECM, resulting in tissue that is composed of less densely packed cells . FurtherHoltfreter and Moscona demonstrated the first formation of MCS using self-assembled cells suspension without external forced interaction with a biomaterial . With thSince the 1990s, the cutting-edge technology of microfluidics has been adopted for cell culture and producing reliable 3D tissue models that are highly complex, reproducible and tuneable ; (ii) coThe present review highlights the recent advances in the development of microfluidics-based 3D spheroid culture. The review focuses on: (i) the formation of MCS in a microfluidic system; (ii) optimization of a microfluidic system for 3D culture; and (iii) integrated systems for high-throughput drug screening. Furthermore, the review also compares and discusses the advantages and limitations of various microfluidic techniques and proposes the future research opportunities, especially to address the current challenges in the field of health care.Tissue compaction and cohesion are essential for the spontaneous formation of cell constructs. An engineered spherical tissue should possess viable cells, organized matrix and biomechanical properties to achieve the ultimate biomimicry. Scaffold-free approaches produce tissues by mimicking natural processes that occur during embryogenesis, morphogenesis and organogenesis. If cells are seeded on a planar surface without exogenous material, they inhibit surface adhesion and form clumps in suspension. The two distinct assembly categories are the organization of extracellular matrix and the self-assembly of cells aggregation. The self-assembly process is spontaneous and more pronounced to intrinsic sorting. The process initiates the self-arrangement and cell–cell interactions to organize aggregates in the form of the spheroids or other shapes. Initially, cells aggregate and undergo self-sorting in response to the signal they generate. The cells subsequently form loose clusters. In addition, the self-sorting mechanism allows cells to selectively form a discrete population and segregate from other populations. A number of intercellular adhesion models have been formulated to describe the formation and compaction of MCS. These models mainly consider the three-step process shown in The interaction between ECM and integrin plays an important role as physical linkers to mediate the cells merging process. A long-chain polymerized fibronectin matrix is the major ECM component that links the cells . Mono-diE-cadherin expression is established to initiate the cell compaction and it is auto regulated until a specific threshold . Further2-sensitive microelectrodes . F. F64]. F−9 to 10−18 litters); in a size scale that matches the size of cells and microtissue. Microfluidics is categorized as continuous-flow and digital microfluidics  the formation of biocompatible, monodisperse (<1%–3% dispersity) and stable droplets; and (ii) the real-time observation of cell activity. The size and stability of droplet is critical to sustain long term culture of MCSs as well as drug testing. The hydrodynamic properties such as type of flow, laminar or turbulent, are crucial for scaling of the size of the droplets during emulsification process . The emuThe outer oil layer of a droplet serves as a selectively permeable barrier and allows the transport of small molecule across this barrier, which is essential to provide a discrete microenvironment for cell culture. To further enhance cell growth the droplets can be formulated by encapsulating cells with biological additives. Tumarkin et al. demonstrated the possibility of using microgel-based biomaterials, which potentially promoted the cell functionality in terms of enhanced proliferation and adhesiveness for the formation of MCS . For insd,l-lactide) porous microspheres [Biomimetic or biodegradable material based microcarriers are useful for cell culture and drug delivery. Shi et al. modified the method of double emulsion solvent evaporation to fabricate a biodegradable Poly is a branch of microfluidics, which integrates the microfluidic devices and electrical forces to manipulate discrete droplets ,120. TheHowever, common fluid operation using DMF is restricted to a 2D platform, and therefore it has limitations such as cross contamination, solute adsorption and degradation of soluble factors . InteresOur recent approach for multiple spheroids of olfactory ensheathing cells was carried out using floating liquid marble (LM) . A drop The combination of microfluidics and optics results in a unique technology called optofluidics. The initial optical applications in microfluidic domain were optical tweezers  and optiOrgan printing is promising to transform tissue engineering into customized organ biofabrication. Spheroids make an excellent candidate for organ bioassembly. A spheroid has an ideal geometry and may serve as “bioink” for bioprinting. Printing the spheroids layer-by-layer for tissue constructions is a common approach in biopriniting. With the advance computer-aided robotic bioprinting technology, the predefined structure can be precisely printed to achieve desired organ/tissue assembly. The positioning and the placement of the dispersed spheroids are critical factors to achieve a controllable fusion in a 3D tissue. Most recently, Moldovan et al. reported a latest invention called the “Kenzan” method, which utilises microneedles for the spheroids assembly . This teMany other technologies can be considered to improve the use of spheroids for bioprinting as well as tissue fabrication. For instance, a bioprintable scaffold such as electrospun matrix can accurately pattern printed spheroids into desired tissue construct . FurtherTo date, the microfluidics-based lab-on-a-chip technologies are facing challenges to reduce the costs and increase the efficiency for drug screening and development. However, with further development it may potentially serve as a tool for preclinical models for human efficacy and safety. Prominently, this technology contributes towards an alternative step to reduce the extensive use of animal testing. Further, the technology allows for mimicking the complexity of animal-based testing models. The combination of fluid physics with 3D cell compartmentalization has gained popularity as organ-on-chip devices. Interestingly, the organ-on-chip concept simplifies clinical bioanalysis by integrating realistic organ models in a single device ,153.Initially, the organ-on-a-chip concept was established by combining the cultures of the liver spheroid and neurospheres in a separate chamber and connecting them by a microfluidic circuit . SubsequMany biological processes involve the interaction between multiple organs. Thus, a futuristic organ-on-a-chip model will incorporate multiple organ interactions in physiologically relevant orders. This technology is likely to have a higher accuracy for testing drug metabolism and toxicity for translating basic bench research to clinical practice. This device can be fabricated by using spheroids to further mimic the in-vivo conations of cells and enhance the results . This apBoth spheroids based “organ-on-a-chip” and even a “body-on-a-chip” is still in infancy to accurately reproduce and mimic the in vivo niche. Although spheroids consist of organized tissues, the quality of tissues function is limited. Thus, the developments towards the use of organoid cultures have gained attention in the recent years. Organoid can be grown onto a microfluidic platform to model organ features such as development, homeostasis and diseases. These cultures form a higher order tissue organization such as hollow spherical tissue. The organoids are generated using stems cells or embryonic stem. The organoids exclusively represent development system of the embryonic tissue. The stem cells potentially aggregate and assemble into a spatially patterned structure that supports organogenesis. Moreover, organoids also can be developed using induced pluripotent stem (IPS) derived from a patient who represent a personalized cell system that functions as a disease model for an individual.+ stem cells can be differentiated and develop organoids of gastrointestinal system (GI) including hepatic system. The organoids of the hepatic system enable the acquisition of biotransformation drugs and toxins to predict drug safety. Recent advances in a microfluidic system can potentially help to miniaturize the hepatic organoids in a single chip for high-throughput screening. For instance, digital microfluidic system was developed for drug testing using arrays of liver organoids [The organoids are “near physiological” models for drug metabolism and toxicity testing. The development of organoids of the gastrointestinal system also represents an important resource for drug development programs. Initially, intestinal organoids were developed by using stem cells expressing leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5) . Furtherrganoids . The plarganoids .Organoids are promising candidates for cellular therapeutics. Microfluidics-assisted encapsulation of organoids provides a prominent role in therapeutic delivery systems. For instance, LSFM4LIFE project is an ongoing EU Horizon 2020 program seeks to achieve cellular therapy for type 1 diabetes by using Human Pancreas Organoids (hPOs). Further, alginate-poly-L-lysine is used to encapsulate cells for secreting antibody and therapeutic protein. These microcapsules are called immunotherapeutic organoids. These organoids can be implanted in vivo to deliver therapeutics. They are alternative to drug based treatment for cancer  and immuThe integration of biomaterial supports the further development of organoid-based platforms. For instance, patterning of hyaluronic acid (HA) as substrate was used to fabricate 3D contractile cardiac organoids . RecentlIn line with the current trend, 3D cell cultures are cost effective and easy-to-use solutions for various applications. The microfluidic technology evidently demonstrated its suitability for the translation of 3D cell spheroid technology into commercial products. In fact, advances in microfluidics-based spheroid culturing techniques made substantial progress in biomedical ventures including drug screening, tissue engineering, disease modelling and cellular therapeutics. However, further optimization is needed to accurately mimic the in-vivo environment. Ideally, physiologically relevant models will help in overcoming the animal testing and animal based assays. Nonetheless, heed should be given to ensure that these microfluidic tools do not interfere or manipulate the ideal cellular behaviour. Ultimately, the usefulness of iPSC, organoids technology and microfluidic system may serve as a breakthrough for featuring next generation human models. Organoids potentially enhance the similarity of 3D tissue organization to that of real organs. Another interesting direction is the utility of 3D-bioprinted organoids for tissue construct. It is also critical to improve efficacy of drug testing by using this technology. In that regard, an advance look on coupling digitalized organ-on-a-chip systems with mass spectrometry analysis will widen the pipeline of future of drug development. Furthermore, it is crucial to integrate new biosensors technology into multi-organ systems. This is particularly important for feedback and molecular diagnostics. Collectively, the future challenges will be scaling up this technology towards the target for personalized medicine. By this, individualized therapeutic methods can be established as more precise medical approaches for intracellular delivery, cell transplantation and personalized tissue engineering.


This allows more precise fine-tuning of the circuit output in contrast to the sigmoidal, steep input–output response of the autorepressor alone. In the second circuit, the output is a transcription factor that induces expression of an sRNA, which inhibits translation of the mRNA encoding the output, creating direct, closed-loop, negative feedback. Analysis of the noise profiles of both circuits showed that the use of sRNAs did not result in large increases in noise. Stochastic and deterministic modelling of both circuits agreed well with experimental data. Finally, simulations using fitted parameters allowed dynamic attributes of each circuit such as response time and disturbance rejection to be investigated.Negative feedback is known to enable biological and man-made systems to perform reliably in the face of uncertainties and disturbances. To date, synthetic biological feedback circuits have primarily relied upon protein-based, transcriptional regulation to control circuit output. Small RNAs (sRNAs) are non-coding RNA molecules that can inhibit translation of target messenger RNAs (mRNAs). In this work, we modelled, built and validated two synthetic negative feedback circuits that use rationally-designed sRNAs for the first time. The first circuit builds upon the well characterised  Natural biological systems function through the regulation of a large number of genes and their products, which interact with each other dynamically in response to a wide variety of signals. The ability to engineer biological systems to perform such complex tasks is a key goal of synthetic biology , with soEmploying negative feedback control is known to endow systems with robustness and improved performance. This is particularly valuable when prediction of a circuit component's behaviour in a particular context is impossible, and when disturbances to the system are applied ,13–17. IIn vivo feedback has been employed in many synthetic gene circuits, including circuits such as the toggle switch  with improved understanding leading to more successful synthetic feedback circuits  cells were transformed with appropriate plasmid(s) and isolated transformants used to inoculate 5 ml of EZ rich defined media (rich media hereafter) or M9 minimal media  supplemented with 0.5% (v/v) glycerol, and ampicillin or chloramphenicol. These cultures were incubated with shaking at 240 rpm at 37°C for 16 h. For flow cytometry experiments, these cultures were then subcultured into 300 μl of fresh media in deep 96-well multiplates, to an optical density  of 0.05 and the plate incubated at 37°C with rapid shaking (700 rpm). For circuits using the tet promoter, the tetracycline analogue, anhydrotetracycline (aTc) was added to the assay cultures at the concentrations specified. For circuits using the rhaBAD promoter, the inducer l-rhamnose was added to the assay cultures at the concentrations specified. For circuits using the Pm promoter, the inducer m-toluic acid was added to assay cultures at the concentrations specified. Samples were taken at specified time points for optical density and GFP fluorescence measurements. A FLUOstar Omega platereader (BMG Labtech) was used for all absorbance measurements of optical density (OD) at 600 nm and GFP fluorescence measured using an Attune NXT flow cytometer (ThermoFisher Scientific). Cells were gated using forward and side scatter, and GFP fluorescence (excitation and emission wavelengths: 488 and 525 nm [with 20 nm bandwidth] respectively) was measured. Histograms of fluorescence intensity were plotted, and geometric mean statistics extracted. Coefficients of variation were extracted from the gated population as follows. FCS files were created from the gated cells from flow cytometry and raw reads extracted using the FCS Extract Utility created by Earl F. Glynn at the Stowers Institute for Medical Research. The resulting log-normally distributed data was analysed using Equation (A600 nm) of 0.05 and the plate incubated at 37°C with rapid orbital shaking (700 rpm) in a FLUOstar Omega platereader (BMG Labtech). Optical density and GFP fluorescence (excitation and emission wavelengths: 485 and 530 nm [with 20 nm bandwidth] respectively) were measured every 15 min, with fluorescence normalized by optical density and plotted over time. Error bars shown represent the standard deviation of three independent biological replicates. Statistical significance was determined using a one-way ANOVA, followed by a Tukey's multiple comparison test assuming unequal variance.Equation  (SI 1.4)tet-repressor (tet promoter (Ptet) which controls the transcription of a gene encoding the TetR repressor, which can bind to Ptet reducing tetR transcription. The tetracycline analogue, anhydrotetracycline (aTc) is an inducer of the Ptet-TetR system, preventing TetR binding to the promoter, thereby increasing expression from Ptet. This provides one external input to the system with which to change the output steady state. In our system, the tetR gene is fused to the gene encoding superfolder GFP (sfGFP) allowing easy quantification of the TetR protein concentration. A strong, synthetic RBS with a predicted translation initiation rate (TIR) of 50 000 was designed and inserted upstream of tetR, and a plasmid backbone with a copy number of 15–20 chosen. The performance of the resulting plasmid, pCK200 . As a starting point, a plasmid encoding an autorepressor based on the well-studied epressor ,43–46 wa, pCK200 , was tesE. coli strain MG1655 containing pCK200 was grown in both minimal and rich media and its fluorescence monitored over time. Its dynamic response in minimal media (u1), anhydrotetracycline (aTc) is clearly demonstrated  and the second designed to bind 24 nucleotides including all of the probable Shine Dalgarno sequence upstream of the tetR-sfGFP open reading frame (SD). Both sequences were computationally analysed for binding to the tetR-sfGFP mRNA and for off-target binding to the wild-type E. coli (MG1655) ribonucleome using IntaRNA  was predicted to be more energetically favourable than that of the Start sRNA , implying stronger binding. Binding to off-target mRNAs was substantially less favourable than either of the target sequences  and pCK220 (Start). Next the ability of these sRNA plasmids to inhibit translation of the tetR-sfGFP mRNA was tested. E. coli strain MG1655 containing pCK210  and either pCK209, pCK220 or pAH23  were grown in rich media supplemented with different concentrations of aTc until late exponential phase   that targets the inciples ,84,86. I IntaRNA . Bindingequences  and therase 5 h;  and fluo) Figure  and showd Figure . Encourad Figure . The SD-E. coli rhaBAD promoter (PrhaBAD) . It was surprising that even with such a high concentration of inducer the level of translation inhibition was less than with either of the two constitutive promoter sRNA plasmids. Further inspection of rhaBAD promoter revealed the inclusion of five additional bases after the transcriptional start site (TSS +1), an artefact that could affect sRNA-mRNA interaction. Additionally when spread across two plasmids, the autorepressor and sRNA constructs could be at different copy numbers.We next explored the tunability of the sRNA inhibition system, by replacing the static constitutive promoter with an inducible promoter. This introduces a second external input to the autorepressor feedback loop, allowing the ratio of sRNA to mRNA to be varied precisely. The well-characterised PrhaBAD) ,101 was , JW3876  was usedase 4 h;  and fluoase 4 h; . As experhaBAD promoter  and the SD sRNA-encoding fragment were inserted into pCK210, resulting in pCK221 , and the first input  would allow fine tuning of the circuit output to a greater degree than with the first input alone. We created a simple a priori model of this circuit using nominal parameters  or u2 (l-rhamnose)  or u1 at concentrations above 10 ng/ml . The simulations suggest that extrinsic noise  contributes more to the overall noise in our system, than intrinsic noise (resulting from stochasticity of reactions within the system) . When th) Figure . Coefficl Figure , which il Figure .The performance and analysis of the sRNA-tuned autorepressor demonstrates that engineered sRNAs can play a valuable role in synthetic negative feedback circuits. We next wanted to build and test a negative feedback circuit where the sRNA is in closed-loop feedback with the output of the circuit Figure . The useu3) induces expression of a translation-inhibiting sRNA, which itself targets the mRNA encoding the transcriptional activator and thus reduces production of the activator protein. We investigated this circuit's anticipated behaviour using a simple model with nominal parameter values  reduced the system's output and coefficient of variation. We set out to validate the general behaviour of this circuit experimentally. The two halves of the circuit were split across two plasmids. The first plasmid, pAH12 contains rhaS, the gene encoding the transcriptional activator of the rhaBAD promoter, fused to the gene encoding sfGFP. A plasmid backbone was chosen with a mean copy number of 17 and the strong constitutive proD promoter  and the other targeting the Shine-Dalgarno of the rhaS-sfGFP mRNA (SD). Both sRNAs were analysed using IntaRNA, with the SD sRNA found to bind more favourably than the Start sRNA to the rhaS-sfGFP mRNA  and no substantial off-target binding predicted  and pAH16 (SD). To test the performance of this circuit, JW3876 cells (which lack RhaS) containing pAH12 and either pAH15, pAH16 or pAH23 (negative control plasmid) were grown in rich media supplemented with 0.2 mg/ml l-rhamnose in a microplate reader and optical density (measured at 600 nm) and fluorescence compared at mid-exponential phase (6 h). Both sRNA plasmids were capable of reducing the amount of RhaS-sfGFP produced   Figure , with th) Figure .l-rhamnose was tested. Simulations predicted that in contrast to the sRNA-tuned autorepressor circuit, the response of the closed-loop sRNA feedback circuit should saturate while the output is still high  via flow cytometry. As L-rhamnose concentrations increase from 0 to 0.0008 mg/ml, an almost linear reduction in output level is observed .Next the response of this circuit to increasing concentrations of the inducer h Figure . This red Figure . As concrhaS mRNA, varying the steady state of the output must be realised through changing the strength and rate of rhaS-sfGFP transcription. Replacing the constitutive promoter with an inducible promoter would allow the setpoint of the output to be dynamically controlled through the use of an external input, and the variability in this output controlled via the sRNA feedback loop. To this end the constitutive proD promoter was replaced with the m-toluic acid-responsive Pm promoter and transcriptional activator XylS from Pseudomonas putida (m-toluic acid (inducer u3) and l-rhamnose (inducer u2) and steady state fluorescence measured at late exponential phase  and gradually decreases with increasing concentrations of u3 (m-toluic acid)  or increasing rhaS-sfGFP expression (u3)  Figure . Coeffic) Figure , both oftet-TetR autorepressor through the use of aTc is difficult due to the sigmoidal shape of the input–output response curve. Step-response simulations of the autorepressor with different concentrations of aTc highlight another limitation of this circuit architecture. As increasing concentrations of aTc are supplied, the steady-state output indeed increases, but the response time also increases proportionally , response times are slower than when the sRNA feedback has been established through the supply of l-rhamnose (u2 = 2)  Figure  and D, w) Figure .u3) but insensitive to changes in the second input (u2) . For the sRNA-tuned autorepressor, the dynamic simulations show that within a certain range of inducer concentrations, the introduction of the sRNA to the autorepressor circuit reduces the sensitivity of the circuit to fluctuations in either inducer, whilst maintaining the same mean output . For theput (u2) . Additiom Figure .In this paper we report the first use of translation-inhibiting sRNAs in synthetic negative feedback circuits. The first circuit introduces an sRNA-mRNA summing junction into the feedback loop of an autorepressor. We have shown that the level of sRNA expression can be controlled using inducible promoters and the use of this sRNA dial refines the coarse sigmoidal input–output response of the autorepressor and its associated input, allowing more precise tuning of the circuit output. The performance of the circuit was predicted using a deterministic model and found to accurately capture the mean output of the circuit in response to both inputs. The second circuit uses translational regulation via an sRNA in the central feedback loop of the circuit. Inducible promoters are used to control the transcription of both the output-encoding gene and the sRNA, allowing two external inputs to manipulate the system. One input is primarily used to set the steady state of the circuit output and the other input used to establish feedback through sRNA expression. The use of the sRNA in closed-loop feedback ensures the desired mean output is restricted to a small range and this behaviour is in excellent agreement with simulations based on deterministic model of the circuit. In the course of revising this manuscript, the modelling of a similar sRNA-based circuit has been described, with many of the desirable attributes that have been shown experimentally in this paper simulated .rhaS-sfGFP gene, and the overall CoV levels are higher than the CoV levels of the sRNA-tuned autorepressor. This is likely due to the use of m-toluic acid inducer (u3), perhaps due to inconsistent uptake by all cells, which could be caused by dissociation of the organic acid at the slightly basic pH of the EZ media (approximately pH 7.4) as suggested previously .The dynamic simulations of both circuits performed in this work showed a number of new insights into circuit behaviour. The step-response simulations show that the use of an sRNA to tune the effective feedback strength of an autorepressor allows a decoupling of response time from steady-state output. This contrasts with previously-described feedback circuits, including a recent transcriptional negative autoregulation feedback circuit, where the use of an RNA transcriptional attenuator did not allow decoupling of output and response time . The dynhttps://doi.org/10.1101/336271). The avoidance of a protein-based controller in negative feedback circuits such as these should be beneficial in these applications, through the expected reduction in cellular burden associated with using an sRNA over another resource-sapping protein. Indeed although not the focus of this work, no difference in growth was observed between cells containing either circuit in the presence or absence of sRNA (While the designs outlined in this paper are very simple and use well-characterised, commonly-employed components, their interconnection in a feedback fashion demonstrates the advantages of different architectures, as predicted by mathematical modelling. Although the output of both feedback circuits was a transcription factor fused to a fluorescent reporter protein, this fluorescent reporter protein could be replaced with almost any other protein of interest. Modified versions of these circuits could potentially be used to regulate and optimise the commercial production of protein therapeutics and industrially-relevant enzymes, or in metabolic engineering, to ensure the concentration of an important pathway enzyme is maintained. The use of negative feedback circuits has been proposed as a method to ensure the reliable interconnection of two genetic circuit modules, where the output of one circuit forms the input of another (doi:  of sRNA . This impCK200 MH822873pCK210 MH822874pCK221 MH822875pCK226 MH822876pCK209 MH822877pAH17 MH822878pCK220 MH822879pAH12 MH822880pAH15 MH822881pAH16 MH822882pCK222 MH822883pCK227 MH822884pAH23 MH822885Genbank Accession Numbers of Plasmids:Supplementary DataClick here for additional data file.


Many studies investigated the use of mAb-targeted imaging for a variety of purposes, for instance to monitor disease progression and to predict response to a specific therapeutic agent. Herein, we highlighted the application of mAb-targeted imaging in three different types of pathologies: autoimmune diseases, oncology and cardiovascular diseases. We also described the potential of molecular imaging strategies in theranostics and precision medicine. Due to the nearly infinite repertoire of mAbs, molecular imaging can change the future of modern medicine by revolutionizing diagnostics and response prediction in practically any disease.Molecular imaging modalities hold great potential as less invasive techniques for diagnosis and management of various diseases. Molecular imaging combines imaging agents with targeting moieties to specifically image diseased sites in the body. Monoclonal antibodies (mAbs) have become increasingly popular as novel therapeutics against a variety of diseases due to their specificity, affinity and serum stability. Because of the same properties, mAbs are also exploited in molecular imaging to target imaging agents such as radionuclides to the cell of interest  On December 22, 1895, the first-ever X-ray picture was taken by Wilhelm Röntgen, who used a Crookes tube to image the hand of his wife, soon after he discovered that Crookes tubes generate a new type of radiation, which he called X-rays th century, medical imaging was taken one-step further by the addition of probes that facilitate imaging of a specific target site. This resulted in a new discipline called molecular imaging. This new discipline combines traditional medical imaging techniques with novel targeting molecules to visualize the function of various biological processes in vivo. Molecular imaging opens new avenues in disease management and personalized treatment as it possesses several advantages compared to traditional medical imaging. One major advantage is the ability to measure response to a treatment at relatively early time points. For example, during cancer treatment, it can take months before anatomical imaging modalities such as CT or MRI are able to measure changes in tumor volume. While still mainly in a pre-clinical stage, molecular imaging has the potential to detect the abundance of specific molecular targets (e.g. VEGFR). Thus, in the near future, it will be feasible to assess therapeutic responses by measuring changes in expression of these molecular targets. This response is detected at an early time point before morphological changes are visible in conventional anatomical imaging. Some other advantages include the ability to measure the molecular marker profile of a tumor in a non-invasive manner thereby eliminating the need for biopsies Medical imaging is not only used to diagnose a disease, it is also crucial in disease management Medical imaging involves a wide variety of imaging technologies such as positron emission tomography (PET), magnetic resonance imaging (MRI) and ultrasound (US). Table Imaging probes are the key elements in molecular imaging as they facilitate the emission of a signal specifically from the target site, which is in turn detected by the various imaging modalities. Imaging probes generally consists of two parts: a signal agent (e.g. radionuclide) and a targeting agent (e.g. mAb) that binds to the tissue/cell of interest. While the targeting moiety binds the target, the signal agent produces a signal (e.g. gamma-emitting radionuclides or fluorophores). A linker might be required to bridge these two subunits together. The type of linker can affect the functionality of the targeting moiety and the bio-distribution. Variables in linker design that should be considered include linker length, hydrophobicity, presence of charge and presence of cleavable domains Each imaging modality requires a different type of imaging agent. Generally, there are three main types of imaging agents: contrast agents , optical probes such as fluorophores (FLECT) and radionuclides . Contrasts agents can be targeted to specific sites were they attenuate the imaging signal (e.g. X-ray attenuators are used in CT). Optical agents can include fluorophores for fluorescent imaging or bioluminescent proteins in luminescent imaging. Radionuclides can be targeted to specific tissues and they directly or indirectly emit photons caused by nuclear decay (mechanism depending on the radionuclide used) Specifically for CT, the use of targeted contrast agents is problematic as large amounts of contrast agents are required to see a change in X-ray attenuation. However, several research groups published a molecular imaging approach using CT by for instance using gold nanoparticles Classical MRI imaging uses non-targeted contrast agents such as superparamagnetic iron oxide (SPIO) or paramagnetic gadolinium (Gd) to complement natural tissue contrasts. These contrast agents can be targeted using nanoparticles or monocloncal antibodies and can thereby extend MRI to a functional imaging modality whereas traditionally it is a rather anatomical modality In PAI, contrast agents need to have a high molar extinction coefficient in order to create sufficient contrast by absorbing light. Ideally, contrast agents absorb in the near-infrared window which is more suitable for imaging in deep tissues. Additonally, the absorbance peak should be a sharp peak to ease spectral unmixing 2S quantum dots For optical fluorescence imaging popular imaging agents include near-infrared fluorophores such as IRDye 800CW 99mTc, 68Ga or 18F, which have half-lives between 1-6 hours. Larger probes, on the other hand, have longer serum half-lives, especially monoclonal antibodies as they undergo FcRn-mediated recycling resulting in serum half-life times of several weeks 123I or 111In for SPECT imaging and 64Cu or 124I for PET imaging. The half-life of these radionuclides ranges from half a day to several days For nuclear imaging techniques such as SPECT and PET, many different radionuclides are available. A key parameter that defines which radionuclide is suitable for a specific approach is the half-life of the radionuclide. As radionuclides decay automatically, they have a specific half-life. Ideally, the half-life of a radionuclide is matched to the half-life type of the probe that should be radiolabeled. Smaller compounds such as peptides have a relatively short serum half-life and can therefore best be conjugated with radionuclides such as in vitro affinity maturation enabled researchers to develop antibodies with even higher affinities to their targets making these antibodies desirable in many fields of research Antibodies are of particular interest in biomedical research because of their well-defined structure, relative stability, high specificity and high affinity. The remarkable diversity of naturally occurring B-cell receptors is achieved by a complicated process of V(D)J recombination in the developing B-cell's heavy and light chain genesin vivoThe high affinity and wide repertoire made antibodies of particular interest in targeting strategies such as in the field of molecular imaging. Antibodies are available against a variety of targets such as growth factors, cytokines and cell surface receptors making antibodies useful in molecular imaging in a variety of disease models. An important prerequisite of antibodies is that the target needs to be available extracellularly (e.g. at the outside of cell membranes or as a free molecule in the blood) as targeting of intracellular targets with antibodies is particularly complicated A major challenge in monoclonal antibody-based imaging is the conjugation of the mAb with the imaging agent. While various techniques can be used, only a fraction of these can be used without compromising the functionality and orientation of the mAb. For radionuclide-based imaging, various chelators are often employed as a linker between the mAb and the metallic radionuclide. This is crucial as the short half-life time of the radionuclide does not allow the time required to conjugate the radionuclide directly to the mAb. Therefore, mAb-chelator conjugates are prepared in advance. This makes metallic radionuclides favorable over non-metallic radionuclides.While mAbs are relatively stable as compared to other proteins, chemical modification might adversely affect the affinity of the mAb. A major benefit of the use of mAbs in terms of conjugation strategies is the well-defined structure and the presence of functional amino acids for bioconjugation purposes such as thiol groups that are available on the cysteine residues upon reduction of the antibody. We will briefly describe several conjugation strategies that are frequently employed to conjugate the mAb to a suitable chelator.Examples of chelators include NOTA , DTPA(diethylenetriamine pentaacetate), DOTA  and THP (tris(hydroxypyridinone)). In a 2017 study, Moreau et al. compared four different bifunctional chelators in their ability to bind Fab antibody fragments under relatively mild conditions. Their results demonstrated that the chelator MANOTA (Methyl Amino triazacycloNOnane Triacetic Acid) was the most efficient. Their conjugation strategy was based on an isothiocyanate group and involved linkage to the free amine on the lysine residues Another common approach is the use of carboxyl and amine chemistry. For example, p-NH2-Bn-BFC is used for conjugation to antibodies, in which BFC stands for the bifunctional chelator of choice (e.g. DOTA). This chemical reaction involves the use of EDC (1-ethyl-3-(3-dimethylaminopropyl) as a cross linker which facilitates the covalent linkage of the BFC to the carboxylate residues on the antibody (for instance on Glu and Asp residues)Click chemistry, a group of reactions that are performed fast and easily by the use of a modular approach in vivo do accumulate automatically in certain tissues such as the liver and kidneys. While passive targeting of tumors uses the EPR effect or active targeting to other tissues are designed to minimize the non-specific accumulation in e.g. the liver, residual non-specific accumulation is still unavoidable. The longer the half-life, the more material accumulates non-specifically in other tissues, giving rise to increased background signals that could nullify the target signal. Specifically in PET imaging there is a demand for the use of antibody fragments that are cleared from the circulation more quickly. This is due to the high sensitivity of PET imaging, combined with the high affinity of antibodies; the long circulation time increases the background signal significantly. Furthermore, longer lasting radionuclides are required in PET imaging with full length mAbs which in turn increases radiation exposure in patients.Full-length monoclonal antibodies are popular in research and in medical applications. Antibody production and purification technologies have eased the entire production process and yields increased dramatically. Antibodies are physically stable and experience a prolonged serum half-life due to their interaction with the neonatal Fc receptor, FcRn, resulting in an average half-life of several weeks 2 fragments, or by papain to generate fragment antigen-binding (Fab). Another option is to genetically engineer antibodies to generate a variety of products such as scFv or affibody in vivo kinetics and clearance mechanism .Therefore, several antibody-derived products are developed for several different applications. Each antibody-derived product has a different size, bio-distribution and serum half-life. Full-length antibodies can be digested enzymatically either by pepsin, to generate F(ab')Examples of applications of antibody-derived products in molecular imaging include the use of a scFv against the ion channel hERG1 for cancer optical imaging The smallest antibody derivative is the affibody, which consists of merely 58 amino acids residues that form three helix bundles. Affibodies combine high affinity with rapid uptake and quick clearance which make them useful for PET imaging by creating a high contrast. For instance, a recent study in 2019 reported the use of an affibody against HER2 in PET imaging While most studies that utilize antibody's fragments emphasize the superiority over IgGs by their faster clearance while maintaining similar binding affinities, the benefits of high binding affinities are also being questioned. Fujimori and colleagues, back in 1990, suggested that in tumor imaging the targeting moiety with the highest affinity might not be the best choice We believe that it is clear that in nuclear imaging, smaller antibody fragments such as affibodies are probably going to replace the full-length IgG molecules. In other imaging modalities, it still needs to be seen whether full-length IgGs or derivatives are more suitable. Some emerging optical imaging technologies such as FLECT might be well suited to IgGs while for PET imaging affibodies could well be the new standard. Overall, achieving high contrast, homogenous, fast and reproducible imaging data with high sensitivity and resolution is a complex interplay between targeting proteins and imaging agents. While most labs focus on achieving the highest possible affinity, the binding site barrier theory suggests that proteins with a lower affinity might be better suited for homogenous tumor imaging.Autoimmune diseases affect around 3-5% of the general population. There are many types of autoimmune diseases such as Crohn's disease, multiple sclerosis and Graves' disease. Generally, autoimmune diseases are caused by an overactive immune system and a lack of immunological tolerance Disease biomarkers can be detected in the urine or blood but the most conclusive way of diagnosis relies on biopsies. Biopsies are analyzed by either light microscopy, immunofluorescence microscopy or electron microscopy 99mTc-infliximab while non-inflamed joints did not show any uptake. More importantly, after treatment with unlabeled infliximab the uptake of 99mTc-infliximab decreased, indicating that the treatment reduced inflammation and TNF-α expression resulting in less available targets for 99mTc-infliximab 99mTc-adalimumab specifically labeled inflamed joints while 99mTc-adalimumab uptake in non-inflamed joints was absent. To test the dependency on TNF-α expression, the researchers did a competition experiment by co-administrating excess unlabeled adalimumab. This decreased the signal in inflamed joints by 25%, indicating the dependency on the presence of TNF- α in the affected joints. Treatment with corticosteroids, which are known to reduce the inflammation, resulted in lower 99mTc-adalimumab uptake, proving the suitability of this approach in the assessment of therapy response. The 99mTc-adalimumab uptake was not evident in all inflamed joints, most likely because not all inflamed joints are characterized by TNF-α expression, this is important for pre-selection of patients for anti-TNF-α therapy. A mini review compared studies using both 99mTc-infliximab and 99mTc-adalimumab and concluded that both antibodies are suitable for therapy decision-making purposes in RA 99mTc-adalimumab showed more therapeutic benefit when treated with unlabeled anti-TNF mAb Therapeutic antibodies against the important pro-inflammatory cytokine TNF-α were approved already in the 1990s for use in rheumatoid arthritis (RA). A study from 2005 demonstrated successful radiolabeling of a chimeric anti TNF-α antibody, infliximab, by reducing the disulfide bonds and conjugating the reduced antibody with methylene diphosphonate which complexes with radioactive technetium, ⁹⁹ᵐTc. Using scintigraphy, the authors could accurately image inflamed joints using the 111In. The labeled antibody was injected intravenously in pigs which were subsequently analyzed by scintigraphy. The results showed specific accumulation of the labeled antibody in the inflamed knee and this was significantly higher than a labeled isotype control antibody 99mTc-labeled 1.2B6 Fab was compared with 111In‐labeled 1.2B6 F(ab′)2 in human patients. The 111In-labeled F(ab')2 fragment of 1.2B6 already turned out useful in RA diagnostics as shown previously99mTc because 111In is an expensive radionuclide and it exposes the patient to relatively high radiation doses. A Fab fragment of 1.2B6 was chosen to test the suitability of 99mTc due to its fast blood clearance, which limits the signal from non-target tissues. The 99mTc-labeled 1.2B6 Fab turned out as effective as the 111In‐labeled 1.2B6 F(ab′)2 and both methods were superior to conventional bone scanning techniquesAnother study explored the possibility of using E-selectin as a target. E-selectin is an adhesion molecule that is expressed on vascular endothelium. It is overexpressed by pro-inflammatory signals such as IL-1 during inflammatory disorders. Keelan et al. published in 1994 an animal study in which an anti-E-selectin antibody (clone 1.2B6) was radiolabeled with +, CD4+ and CD20+ cells. The murine mAb OKT-3 against CD3 was employed several times in human studies to test the effectiveness in disease management. Marcus et al. showed that 99mTc-anti-CD3 mAb can be used to assess therapeutic effectiveness in RA patients. All painful joints demonstrated 99mTc-anti-CD3 mAb uptake, however, some unexpected side effects occurred (most likely cytokine release syndrome related) limiting its future applicability 99mTc was shown to be successful by Malviya et al. The researchers demonstrated uptake of the radionuclide by B-cells in a variety of autoimmune diseases, proving the universal nature of this approach. Over time, the role of B-cells in mediating chronic inflammation in autoimmune disorders became more apparent. The role of B-cells in these processes range from the production of auto-antibodies to T-cell activation and pro-inflammatory cytokine production As leukocytes are a central part of the immune system and as they are indispensably involved in the inflammation, targeting leukocytes could accurately image autoimmune disorders. Several strategies were devised to accomplish this by targeting radionuclides to either CD364Cu to image the disease using microPET imaging. As the homing receptor α4β7 integrin is a crucial receptor of leukocytes homing to the gut, the first study utilized an antibody against β7 integrin. This rat mAb, FIB504.64, against human and mouse β7 integrin was conjugated to the bifunctional chelator DOTA. After inducing experimental colitis in C57BL/6 mice using dextran sodium sulfate (DSS), FIB504.64-DOTA was labeled with 64Cu and injected intravenously. At 48 hours post injection, the mice were imaged with a microPET imaging device. The researchers were able to detect a radioactive signal in the inflamed gut while the level of radioactivity was less profound in healthy mice 4β7 integrin named DATK32 and the F(ab')2 and Fab fragment of the FIB504.64 mAb. The rationale was that the DATK32 should provide more specific targeting while the F(ab')2 and Fab fragments of FIB504.64 should be cleared from the blood more quickly thereby eliminating unspecific uptake. The FIB504.64- F(ab')2 fragment demonstrated the highest differential between mice with colitis and healthy mice and was therefore the best candidate for potential future imaging studies of colitis. The DATK32 mAb showed lower intestinal uptake and targets probably a too specific subpopulation of leukocytes for in vivo imaging purposes In 2010 and 2016, Dearling et al. published two studies in collaboration with our group to image colitis, an autoimmune disorder involving the gastrointestinal tract. These studies employed + T-cells in the gut with a radiolabeled anti-CD4 antibody (111In-DOTA-anti-CD4 mAb). Despite the fact that they demonstrated that the CD4+ cells only represent a small fraction of the total lymphocyte population, they were able to image the CD4+ cells in the gut specifically in mice with colitis using SPECT/CT+ cells can represent both helper T cells and regulatory T cells. By only targeting CD4, we believe that one cannot conclude whether the cells that have taken up the radionuclide are pro- or anti-inflammatory.Although the initiation of DSS colitis is mainly driven by neutrophils and macrophages + cells possess an inflammatory phenotype or not.While Kanwar et al. injected 350 µg of radiolabeled-antibody; in 2018 Freise et al. reported similar results with doses as low as 2 µg. Flow cytometry results revealed that the percentages of CD4+ cells in the mesenteric lymph nodes (MLN) were lower in DSS colitis mice than in healthy controls. However, as the MLN in mice with colitis were significantly increased, the total cell number was much higher than in healthy controls. Therefore, the uptake of radiolabeled antibody in the MLN was higher than in healthy controls as was shown in the imaging data In contrast to antibodies targeting cytokines or leukocytes, which are bound in the bloodstream, targeting solid tumors depends greatly on the diffusion of the antibodies from the vasculature into the tumor. The enhanced permeability and retention (EPR) effect has been thoroughly investigated, being a unique property of tumors that enhance diffusion of macromolecules through the leaky vasculature in the tumor area. Due to the rapid growth of tumors, the surrounding blood vessels have a defective architecture and in some cases produce various permeability factors to ensure sufficient influx of oxygen and nutrients Besides using mAbs for therapy, their long circulation time makes mAbs excellent targeting molecules for molecular imaging modalities . Several strategies regarding molecular imaging of tumors will be briefly discussed in the rest of this chapter.111In-DTPA-trastuzumab 188Re (I)-trastuzumab 111In-DTPA-trastuzumab-IRDye800 64Cu-DOTA-trastuzumab-IRDye800 111In 89Zr 64Cu Human epidermal growth factor 2 (HER2) was identified in the 1980s as an oncogene in breast cancer 111In‐cetuximab  to 125I‐cetuximab . Inidum-111 turned out more successful in terms of tumor uptake in all time points, probably due to the internalizing properties of cetixumab Two other growth factor related proteins that have been intensely investigated in oncology are EGFR and VEGF. For both targets, FDA-approved monoclonal antibodies are available for cancer treatment and nowadays these antibodies are also tested in their predictive abilities when used in molecular imaging. For instance, Nagengast et al. demonstrated how radiolabeled bevacizumab can target VEGF in the tumor area of an ovarian tumor xenograft A study from 2018 by Tsai et al. 111In-R1507 111In-R1507 is a predictive marker for response by bone sarcoma patients to anti-IGF-1R therapy. Their results demonstrated that mice with tumors positive for IGF-1R that did not respond to anti-IGF-1R therapy showed 111In-R1507 uptake similar to mice harboring IGF-1R negative tumors and only mice positive for IGF-1R that responded to anti-IGF-1R therapy had a significant uptake of 111In-R1507  is a leading cause of mortality and hospitalization globally. The main cause of CVD is atherosclerosis leading to for instance myocardial infarction, stroke and heart failure. Atherosclerosis is a chronic disease characterized by the accumulation of lipids and fibrous elements in the main arteries leading to plaque formation and lesions. While advanced lesions can grow gradually to eventually block the blood flow, most clinical complications are the results of acute occlusions as a result of plaque rupture. The disease is divided in multiple stages, starting with lipoprotein entrapment in the sub endothelial matrix, resulting in lipoprotein aggregation, which ultimately forms foam-cells (a form of macrophages) and fibrous lesions 125I can detect lipid-rich, oxidation-rich plaques. This is important, as the presence of highly oxidized LDL in plaques is associated with plaque vulnerability 125I-MDA2 can detect changes in the content of oxidized LDL in plaques and therefore predict plaque stability To assess the risk of an individual for the development of CVD, accurate assessment of plaque stability is crucial. Molecular imaging enables the non-invasive assessment of atherosclerotic plaques' stability and can thereby determine the severity of the disease. A study back in 2004 described how oxidation-specific antibodies labeled with Ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles are a novel class of contrast agents in MRI. A study by Wen et al.As the immune system plays a causal role in plaque rupture, other imaging approaches focus on determining the expression levels of endothelial cell adhesion molecules (ECAMs) such as ICAM-1 VCAM-1 or selectins at plaque vulnerable regions to predict the influx of leukocytes. For example, a study in 2010 demonstrated the detection of lesion-prone vascular sites in the early pathogenesis of atherosclerosis. The researchers could detect vascular abnormalities before the development of fatty streaks when monocytes just start to accumulate at the affected site. This study utilized contrast-enhanced ultrasound, which employs microbubbles that accumulate at sites rich in ECAMs. The microbubble accumulation in ECAM-rich sites was achieved by the conjugation of the microbubbles with monoclonal antibodies against P-selectin and VCAM-1Because atherosclerosis is a chronic, latent disease, it is beneficial to diagnose patients at an early stage to prevent severe symptoms that occur during acute injury at a later stage. However, once an acute event eventually takes place, a rapid diagnosis is required to prevent as much damage as possible. Therefore, Davidson et al. 18F- Fluciclatide which targets αVβ3 integrin . Fibrin is of particular interest for detecting thrombosis as it is highly concentrated in the thrombus and it plays an important role in thrombus formation. Fibrin can be targeted by peptides as this was demonstrated in several studies that employed PET, SPECT and several optical modalities In the field of cardiovascular diseases, targets of interest are P-selectin, αVβ3 integrin and fibrin. There is a strong relation between elevated P-selectin levels and the development of thrombosis 18F-GP1, a novel PET probe that targets glycoprotein IIb/IIIa receptors A major milestone was achieved with the development of To conclude, the recent development in molecular imaging holds great potential in the field of cardiovascular diseases. Individuals develop atherosclerosis over a period of decades without the presence of any clinical symptoms This review covered the application of monoclonal antibodies in a variety of molecular imaging modalities. Three major applications were highlighted  to demonstrate how universally applicable molecular imaging is. This universality relies on the high affinity, high specificity and wide repertoire that monoclonal antibodies offer. While antibodies are popular in bio-medical research, they can still be improved using genetic engineering to generate a variety of fragments or fusion protein with different properties. Each application demands its own tailor-made probe and solely relying on native, full-length monoclonal antibodies limits the full potential that antibody engineering has to offer. The combination of therapy and diagnostics, theranostics, prompted much attention in recent years due to its high potential in personalized treatments and disease management. The one-size-fits all approach slowly becomes out of fashion as biologics offer new tools to tailor each treatment to each patient and to each state of the disease. The latter is especially relevant to molecular imaging as the state of the disease changes constantly and with it the expression of certain markers/targets. A routinized theranostic modality that continuously monitors the state of the disease (e.g. in terms of receptor expression) and adjusts the therapy accordingly, holds great potential for the future of precision medicine.Despite the overwhelming amount of research in small animals, clinical translation of molecular imaging modalities is limited, especially in more complex multimodal imaging. While nuclear imaging has been the leading technology in medical imaging for many years, new alternatives are explored, as nuclear probes possess some drawbacks. Clinical facilities require sufficient budget and should be in close proximity to a cyclotron in order to perform routine imaging of patients. The short half-life of radionuclides limits the timeframe and this already present complexity is getting more complicated when conjugating radionuclides to proteins.Radionuclide-protein conjugates add an extra layer of complexity, as they are not only limited by half-life but also require specific conditions to remain stable . Furthermore, the intolerance of proteins to chemical modifications requires novel technologies to readily attach proteins to radionuclides without compromising functionality. Some examples of these were described in this review and provide innovative solutions to such limitations (e.g. adding reactive groups during protein translation by using genetic code expansions). However, the implementation of such innovative ideas from small animal imaging to clinical studies takes time. With the development of fluorescence emission computed tomography (FLECT) or fluorescence molecular tomography (FMT), the field of optical imaging got a boost. These, fluorescence-based optical imaging modalities utilize fluorophores (mainly in the near-infrared region) which are less difficult to manufacture than radionuclides and remain stable for a longer period.Another limitation of the translation of molecular imaging to the clinic is the higher complexity in image analysis as compared to classical medical imaging. To exploit the full potential of  molecular imaging, a multidisciplinary team of physicians/scientists is required to understand the biological process underlying the medical image. However, new developments in machine learning and big data can overcome these hurdles. We believe that the development of new imaging modalities and novel conjugation approaches combined with machine learning programs that use big data to learn to detect abnormalities in patients will drive the transition to clinical applications.In an ideal scenario, the future of molecular imaging involves repertoires of molecular probes that are tested not only in patients but also in healthy individuals to allow for early disease detection by routine screening. These molecular probes should employ multimodal imaging to take benefits of each available imaging modality. Computer programs that learned to detect even the smallest abnormalities analyze the enormous amounts of generated 3D images and reveal physicians with unprecedented accuracy what the state of the disease is.


Frailty status is a well-known predictor of adverse health outcomes and functional performance. An assessment tool based on a wearable sensor was developed to quickly assess frailty using an upper extremity flexion and extension test. However, the current tool has relied on conventional frailty assessment to classify the frailty status of the participant. The aim of this study is to operationalize the frailty index based on wearable sensor to classify frailty status of older adults. 104 older adults were recruited for the study (age=78.6 ±9.7 years old). Participants were asked to perform a quick 20-second upper flexion and extension task while wearing a gyroscope on the wrist. A sensor-based frailty index (FI) was derived using parameters extracted from the sensor. Participants were also assessed using the Fried Phenotype Criteria (FC) and were classified into three groups: robust, pre-frail, and frail. Mean-shift clustering algorithm was used to operationalize the FI by identifying the cut-off point for each group. Grip strength and physical activity level were used as functional outcome measures. Regression analysis (r) was used to compare the correlation of the FC and FI with the identified metrics. Bivariate analysis show that grip strength was highly associated with the sensor-based frailty classification (r=-0.547) and FC (r =-0.503). The sensor-based classification was significantly associated with walking activity (r=-0.355). The results showed that the sensor-based frailty assessment tool could be used to quickly classify frailty status in older adults and eliminated the need for subjective and time-consuming evaluation.


Activated protein C (APC) is a vitamin-K dependent plasma serine protease, which functions as a natural anticoagulant to downregulate thrombin generation in the clotting cascade. APC also modulates cellular homeostasis by exhibiting potent cytoprotective and anti-inflammatory signaling activities. The beneficial cytoprotective effects of APC have been extensively studied and confirmed in a number of preclinical disease and injury models including sepsis, type-1 diabetes and various ischemia/reperfusion diseases. It is now well-known that APC modulates downstream cell signaling networks and transcriptome profiles when it binds to the endothelial protein C receptor (EPCR) to activate protease-activated receptor 1 (PAR1) on various cell types. However, despite much progress, details of the downstream signaling mechanism of APC and its crosstalk with other signaling networks are far from being fully understood. In this review, we focus on the cardioprotective properties of APC in ischemic heart disease and heart failure with a special emphasis on recent discoveries related to the modulatory effect of APC on AMP-activated protein kinase (AMPK), PI3K/AKT, and mTORC1 signaling pathways. The cytoprotective properties of APC might provide a novel strategy for future therapies in cardiac diseases.  Protein C (PC) is a vitamin K-dependent serine protease zymogen that is primarily synthesized by the liver and circulates in plasma with a concentration of 4–5 μg/mL . The genUpon activation by the thrombin-TM complex, APC can dissociate from its membrane-bound receptor cofactor, EPCR, and bind to its circulating vitamin K-dependent plasma cofactor, protein S, on a negatively charged membrane surface, to initiate its anticoagulant function by proteolytically inactivating factors Va and VIIIa, the essential procoagulant cofactors for thrombin generation in both intrinsic and extrinsic pathways of the blot clotting cascade ,13,14. IIn addition to functioning as an essential anticoagulant protease in the clotting cascade, APC also exhibits potent anti-inflammatory and cytoprotective signaling activities when it remains associated with EPCR on the membrane surface . It has The EPCR- and PAR1-dependent anti-apoptotic activity of APC is partially mediated through the down-regulation of expression of the typical intrinsic apoptosis pathway genes including p53 and Bax and up-regulation of the expression of the oppositely functioning gene Bcl-2 . Additio2+ channel is disturbed because the cell compensatory pH regulating system leads to an increase in Ca2+/Na+ exchange ..2+/CaMKKAMPK is a stress and energy sensitive kinase that can be activated by ATP depletion under ischemic stress ,71. AMPKInterestingly, both wild-type APC and the signaling-selective APC-2Cys also increased the autophagic flux in the heart following I/R . This waAPC has been shown to induce the activation of AKT1 in the myocardium, thereby improving the endothelial function of the coronary artery in a global ischemic reperfusion rat model . AKT is Mounting evidence in the literature assigns a key cardioprotective role for AMPK in various cardiovascular disease models . It has In addition to its cardioprotective properties, APC exhibits potent cytoprotective and anti-inflammatory effects in a number of other acute and chronic diseases including sepsis , ischemiIschemic heart disease and heart failure are the leading causes of morbidity and mortality worldwide. There is an urgent need for developing new therapeutic strategies for heart diseases. Results of studies with APC in I/R and HF models are encouraging and warrant further investigation. Because of its potent anticoagulant activity, APC-therapy has been found to also be associated with an increased risk of bleeding. The findings that the signaling-selective APC, lacking anticoagulant function, has a similar cardioprotective function in the I/R injury and HF models may open a new avenue for further investigating the life-saving effects of APC in heart disease without increasing the risk of bleeding.


Infectious bursal disease (IBD) is a highly infectious disease in chicken, and vaccination is the best way to prevent outbreak of infectious bursal disease virus (IBDV). In this study, we isolated a variant IBDV strain from a chicken farm with vaccinated chickens. The full genome of this IBDV strain was determined and analyzed. Infectious bursal disease (IBD) is a highly infectious disease in chicken, and vaccination is the best way to prevent outbreak of infectious bursal disease virus (IBDV). In this study, we isolated a variant IBDV strain from a chicken farm with vaccinated chickens. The full genome of this IBDV strain was determined and analyzed. Infectious bursal disease (IBD) is a highly infectious disease that poses a large economic loss to the chicken industry. Infectious bursal disease virus (IBDV) infects mainly the bursa of Fabricius and the lymphocytes, resulting in immunosuppression and subsequent secondary bacterial infection .Avibirnavirus of the family Birnavirideae  was isolated from a 25-day-old diseased chicken from a farm in Shandong, China. To characterize the virus, the full genome of the isolate was determined. Briefly, total viral RNA was extracted from the allantois fluid of a chicken embryo infected by strain S18 and subjected to one-step reverse transcription-PCR (RT-PCR) (GQ452269) isolated from China . Segment B encodes a VP1 polymerase protein that is relatively conserved. The VP1 amino acid sequence of S18 showed 99% identity with that of the reported IBDV strains in China, e.g., the HLJ-7 and Gx strains  with the HLJ-7 strain (GenBank accession number  strains . The S18In conclusion, we report here the full genome sequence of a virulent IBDV strain isolated from a chicken farm, and the data indicate that a new vaccine matching the IBDV strains circulating in China is warranted to prevent IBD in chickens.MK472711 and MK472712.The complete genome sequence reported here was deposited in NCBI GenBank under the accession numbers