Despite progress, acute myeloid leukemia (AML) remains one of the deadliest cancer diseases. The identification of novel molecular targets may allow developing innovative and alternative treatment options for AML. Using public data from genome-edited cancer cells, we identified factors that are specifically essential for AML cell growth. We validated the critical role of the transcription factor IRF8 and demonstrated that it modulates the function of the cells by regulating important signaling molecules. These results support that IRF8 may be a suitable molecular target for the treatment of AML.Personalized treatment of acute myeloid leukemia (AML) that target individual aberrations strongly improved the survival of AML patients. However, AML is still one of the most lethal cancer diseases of the 21st century, demonstrating the need to find novel drug targets and to explore alternative treatment strategies. Upon investigation of public perturbation data, we identified the transcription factor IRF8 as a novel AML-specific susceptibility gene in humans. IRF8 is upregulated in a subset of AML cells and its deletion leads to impaired proliferation in those cells. Consistently, high IRF8 expression is associated with poorer patients’ prognoses. Combining gene expression changes upon IRF8 deletion and the genome-wide localization of IRF8 in the AML cell line MV4-11, we demonstrate that IRF8 directly regulates key signaling molecules, such as the kinases SRC and FAK, the transcription factors RUNX1 and IRF5, and the cell cycle regulator Cyclin D1. IRF8 loss impairs AML-driving signaling pathways, including the WNT, Chemokine, and VEGF signaling pathways. Additionally, many members of the focal adhesion pathway showed reduced expression, providing a putative link between high IRF8 expression and poor prognosis. Thus, this study suggests that IRF8 could serve as a biomarker and potential molecular target in a subset of human AMLs. Acute myeloid leukemia (AML) is a genetically heterogeneous myeloid disease caused by the hyperproliferation of undifferentiated myeloid progenitor cells . AML is The conventional AML treatment via chemotherapy inhibits DNA synthesis, cell division, and mitosis, which affects both cancerous and normal cells, leading to side effects, including bone marrow suppression. Analyses for mutations in genes and their combination can offer a prognostic tool and help to create personalized treatment plans . MoleculTranscription factors are often mis-regulated in cancer, but they just recently emerged as potential drug targets in AML. In contrast to enzymes, which can be inhibited by targeting the active center, transcription factors were considered unsuitable as potential drug targets in the past. However, technical progress led to the discovery of novel drugs that can impair the function of transcription factors, allowing to utilize alternative treatment options ,7. The iIRF8 belongs to the transcription factor family of interferon regulatory factors (IRFs) that play a variety of roles in the hematopoietic system and are involved in the type I interferon response . It reguTo validate a critical role of IRF8 in AML, we studied its influence on cellular properties, its genomic targets, and its role in gene regulation in the AML cell line MV4-11. Our work suggests that IRF8 may be a promising molecular target in AML.6 cells in triplicates in 500 µL medium on 24-well plates and counting of cells in three-day intervals. Cells were stained with trypan blue and counted using a Neubauer counting chamber.HEK-293-T cells were cultured in DMEM/F-12, GlutaMAX™ Supplement  supplemented with 10% fetal bovine serum  and 1% Penicillin/Streptomycin (Sigma Aldrich). MV4-11, MOLM13, HL-60, THP1, and NB4 cells were cultured in RPMI 1640 Medium, GlutaMAX™ Supplement  supplemented with 15% fetal bovine serum  and 1% Penicillin/Streptomycin (Sigma Aldrich). Cell growth was assessed by seeding 0.15 × 10Whole cell extract was prepared with RIPA-Buffer  were added directly before use). The protein concentration was measured with the “DC™ Protein Assay” (Bio Rad) at 750 nm and estimated by a standard BSA curve.Fractionation assays were performed to determine the intracellular localization of IRF8 using the “Subcellular Protein Fractionation Kit for Cultured Cells” by Thermo Fisher (78840) according to the manufacturer’s instructions. Six-well plates were used corresponding to a packed cell volume of 20 µL. The same volumes of all fractions of interest were loaded on SDS-PAGE gels.IRF8. Standard FuGENE transfection was used. Supernatant containing virus was collected 48h after transfection.To generate stable knock-out cell lines, MV4-11 were subjected to lentiviral infection. Virus was produced in HEK-293-T by transfection with the packaging plasmids pMD2.g, psPAX2 and lentiCRISPRv2 (Addgene #52961) , containAML cell lines were lentivirally infected and selected with 1µg/mL of puromycin 36h after infection. MV4-11 knockout clones were generated by limited dilution, analyzed for IRF8 expression on protein and mRNA level, and clones were genotyped by PCR of the targeted genomic locus and Sanger sequencing. MV4-11 IRF8 ko clone 1 and 2 were generated by gRNA1 (GCGTAACCTCGTCTTCCAAG) and clone 3 by gRNA2 (GCTTGCCAGCGTGTTTCCAA).The following antibodies were used: IRF8 , β-actin , H2B , SP1 , TubuliCells were harvested and washed twice in 1 × PBS and 1% FBS (washing buffer). Cells were fixed by resuspension in 1 mL washing buffer and slow addition of 4 mL 100% ice-cold ethanol whilst vortexing.The cells were rehydrated by washing twice in 1 mL washing buffer before staining. Then, they were resuspended in 1 mL staining buffer  and incubated 30 min at RT in the dark. Samples were measured with a LSR II flow cytometer. Data were analyzed by FlowJo v10.6.1.4 to 1 × 105 cells were resuspended in 150 µL PBS and centrifuged onto the slides at 500 g for 5 min. The slides were left to dry at 4 °C overnight. Cells were stained in 50% May-Grünwald solution for 7 min and washed in ddH2O until no more color was coming of the slides. Then, they were stained in 10% Giemsa for 25 min, washed again and left to dry at 4 °C. The water used was adjusted to a pH of 6.8 beforehand. Slides were stored long-term at 4 °C and assessed microscopely.To assess the morphology of cells, they were subjected to a Pappenheim’s staining. MV4-11 cells were spun onto a glass slide by cytospin. 8 × 10RNA was isolated using the RNeasy Mini kit  and transcribed into cDNA with the Tetro cDNA Synthesis Kit (Bioline BIO-65043). qPCR was performed with the ImmoMix reagent  and gene expression was normalized to GAPDH expression. RT-qPCR Primers are presented in 5 cells per mL were seeded on three 15 cm plates with 25 mL medium each and cultured for 48 h. To cross-link DNA and proteins, 1%-formaldehyde was added and incubated at room temperature for 10 min. To stop the reaction 125 mM glycine was added for 5 min. Cells were transferred to a 50 mL tube and centrifuged at 2000 rpm and 4 °C for 10 min. After the medium was aspirated cells were washed twice with 10 mL cooled 1 × PBS, thereby bringing together cells of the same cell line in a 50 mL falcon. In-between washing steps, cells were centrifuged at 2000 rpm and 4 °C for 10 min. The pellet was resuspended in 1 mL buffer B , 0.25% Triton X-100) per 15 cm plate, transferred to a 15 mL falcon and centrifuged at 2000 rpm and 4 °C for 5min. The supernatant was aspirated, and the pellet was resuspended in 1 mL buffer C , 200 mM NaCl) per 15cm plate. After 10 min incubation on ice, the suspension was centrifuged at 2000 rpm and 4 °C for 5 min, the supernatant aspirated and the pellet was resuspended in 200–250 µL buffer D , 1% SDS, 1 × Protease Inhibitor Cocktail (PIC) by Roche), depending on the size of the pellet. After an incubation period of 10min on ice, the chromatin was sheered 2 × 6 min in a pre-cooled bioruptor, transferred to a 1.5 mL-tube and centrifuged at 13,000 rpm and 4 °C for 10 min. The concentration of the soluble chromatin fraction was measured at a Nano-Drop. The chromatin immunoprecipitation was performed with the “one day ChIP kit”  and performed according to the manual. Per IP, 60 µL chromatin in the same concentration were used, adjusted to the lowest concentrated chromatin. Eluted DNA was either analyzed by ChIP-qPCR or analyzed by next generation sequencing. ChIP-qPCR primers are presented in For chromatin preparation 5 × 10For ChIP-Seq, chromatin immunoprecipitated DNA was used for indexed sequencing library preparation using the Microplex library preparation kit v2 . Libraries were purified on AMPure magnetic beads (Beckman). For RNA-Seq, total RNA was extracted from MV4-11 cell infected with control vector, or from IRF8 KO clones by using the RNeasy Mini system  including an on-column DNaseI digestion. RNA integrity was assessed on an Experion StdSens RNA Chip . RNA-Seq libraries were prepared using the TruSeq Stranded mRNA Library Prep kit . RNA-Seq and ChIP-Seq libraries were quantified on a Bioanalyzer . Next-generation sequencing was performed on Illumina NextSeq 550.ChIP-Seq data were aligned to the human genome using Bowtie . Peak caFollowing public ChIP-Seq datasets were used: IRF8 (THP1): GSM3514948 ; RNA PolRNA-Seq samples were aligned to the human transcriptome GENCODE v32 using RNA-Star (2.7.2b) . Reads phttp://gepia.cancer-pku.cn/index.htmlGEPIA: https://depmap.org/DepMAP: https://www.gsea-msigdb.org/GSEA: https://www.bioconductor.org/Bioconductor/R: https://usegalaxy.org/Galaxy: https://xena.ucsc.edu/Xena Browser: https://genome.ucsc.edu/UCSC Browser: http://great.stanford.edu/GREAT: https://portals.broadinstitute.org/ccleCCLE: https://orcs.thebiogrid.org/CRISPR Screen Database: Below the links to used databases and tools:t-tests. Other P-values were determined by the used software/tool, such as GSEA [Statistical comparisons of two groups was carried out using unpaired Student’s  as GSEA , HOMER [ as GSEA  and DeSe as GSEA .Previous work on AML have identified several important factors for the development of AML, such as the kinase FLT3, the transcription factor RUNX1 and the GTPase NRAS . These fn = 345)  B. For mon = 345) .Next, we investigated whether the selected genes show an aberrant expression in AML cell lines. Given that no normal tissue exists for AML, we compared the average expression levels of those genes in AML samples versus chronic myeloid leukemia (CML) cells , obtained from the GEPIA  platform . A fractTo gain insights into the role of IRF8 in AML, we had a closer look at publicly available data. We observed that IRF8 is expressed in most tissue types at a high level, but with varying intensities. IRF8 is particularly strongly expressed in lung, intestinal, and blood tissues A, suggesTo address the role of IRF8 in AML cells, we next investigated how IRF8 deletion influences the proliferation of AML cell lines in comparison to other cancer types using the above-mentioned CRISPR screen data . In mostTogether, these results suggest that IRF8 upregulation in a subset of AML cells influences the properties of these cells, which in turn leads to a dependency on IRF8 for their proliferation.To investigate the role of IRF8 in IRF8-dependent AML cancer cells in more detail, we selected six AML cell lines  and analyzed the expression of IRF8 in those cells. HL60 and KG1α cells have not been investigated yet in a CRISPR screen , thus itTo approach the role of IRF8 in MV4-11 cells, we first investigated its cellular localization. We found IRF8 is predominantly present in the nucleus and at the chromatin C, suggesTo gain insights into the gene regulatory role of IRF8, we created IRF8 knockout cells using the CRISPR/Cas9 system, whereby IRF8 was completely deleted. The successful targeting of the IRF8 locus and the absence of IRF8 protein has been confirmed by Western blotting E. AdditiUpon examination of the MV4-11 cells, we observed a strongly impaired proliferation of the IRF8 knockout cells compared to the control cells H. We detTogether these results suggest that the function of the cells is massively compromised upon IRF8 deletion, both at the transcriptional and cellular level. Given the transcriptional function of IRF8, we hypothesized that IRF8 possibly influences the cellular properties by modulating the gene regulatory network.To elaborate the mechanism of how IRF8 modulates the function of AML cells, we next addressed the genomic targets of IRF8 in MV4-11 cells and their role in gene regulation. Via ChIP-seq experiments, we identified almost 70,000 locations with bound IRF8 A. Those Investigation of the IRF8 binding pattern within the MV4-11 genome D revealeMotif analysis demonstrated that the IRF8 peaks are most strongly enriched for a motif that is a composite of the ETS and IRF transcription factor binding motifs F, consisTo identify the biological functions of the genes targeted by IRF8, we made use of GREAT (Genomic Regions Enrichment of Annotations Tool) , which lTogether these investigations demonstrate that IRF8 binds to tens of thousands of loci in the genome of AML cells, which are associated with critical biological pathways, implicating that IRF8 is an important transcriptional regulator in MV4-11 cells.p < 0.01, fold change >2)  A,B. A roange >2) C, suggesTo understand how IRF8 deletion influences the properties of the cell, we used our RNA-Seq data and performed GSEA , with a Both SRC and FAK are regulators of focal adhesion , which dCollectively, our results suggest that IRF8 is critical for enhancing the gene transcription of essential signaling molecules and transcriptional regulators in AML cells, and that the absence of IRF8 leads to an impaired expression of pro-tumorigenic factors involved in cell signaling, focal adhesion and cell cycle H.In this study, we aimed to identify novel AML-specific susceptibility genes. In our approach, we used three distinct criteria to select potential candidates: CRISPR perturbation score from AML cell lines, overexpression in AML patients’ samples, and association with poor prognosis in patients. This approach is similar but distinct from another recently published study . In ZhouTo substantiate the findings of our in silico analysis by in vivo experiments, we characterize the role of one of the candidates in more detail. We selected IRF8, given that the related protein IRF5 can chemically be inhibited , suggestAs consequences, many members of key signaling pathways showed diminished expression, potentially explaining the impaired proliferation upon IRF8 deletion H. In parAlthough our work supports a pro-oncogenic role of IRF8 in AML, it has several limitations. The initial analysis uses public datasets that are either based on cell lines, or on a limited number of patients. Thus, it would be important to further validate the role of IRF8 in human AML with primary cells or patient derived xenograft models. Moreover, the TCGA dataset, where we identified a poorer prognosis for high IRF8 expressing AMLs C, is ratSince our cell-based experiments were mainly performed in the MV4-11 cell line, which shows a very high IRF8 expression, the conclusions about the molecular role of IRF8 in AML is restricted. Although IRF8 has an overall similar chromatin binding pattern in THP1 and MV4-11 cells C, it mayInterestingly, previous studies proposed that IRF8 acts as a tumor suppressor in some leukemia types, while our analyses strongly support an oncogenic function of IRF8 in human AML. Therefore, the question arises how these seemingly contradictory roles of IRF8 in distinct leukemia types can be explained. Most studies that describe a tumor-suppressor role of IRF8 in leukemia refer to CML ,80,81,82Contradictory data can be found for the role of IRF8 in AML. In an early study, low IRF8 expression was detected both in AML and CML patients , but morRecent work suggests that also transcription factors , includiIn summary, our investigation of publicly available data revealed novel factors to be specifically essential in AML. We validated the critical role of IRF8 in a human AML cell line and demonstrated that IRF8 is involved in the regulation of multiple genes that are important for pro-proliferative settings in AML cells. Thus, IRF8 could possibly serve as a molecular marker and might be a suitable drug target for the treatment of AML.


The availability of smart and intelligent sensors has changed the monitoring, control and maintenance of a conventional and advanced cyber-physical system used in public or private sectors of a society. For example, internet of things (IoT)-based health, agricultural and weather management systems. With the emergence of such sensors, along with the new ways to communicate or coordinate with them, we need to analyze and optimize the system construction processes. In this paper, to address the issue of scalability for bigger and complex systems based on sensors, we redefine an incremental construction process with an emphasis on behavior preservation and study the effectiveness of the use of software component models from the component-based development domain. In this paper, to deal with the issue of scalability, we investigate component-based development approaches with respect to our defined process and propose a taxonomy of component models with respect to component/system behavior. Moreover, based on the outcome of our analysis, we recommend the EX-MAN component model as the most suitable approach. We investigate incremental construction in the context of the three main categories of current component models, namely models where components are: (i) objects, (ii) architectural units and (iii) encapsulated components. Furthermore, to evaluate our defined process and selection of EX-MAN, we designed three examples of systems using our proposed process in EX-MAN component model. Technological advancements have made many dreamed autonomous and intelligent systems possible these days. The role of smart sensors  for the In general, there are two kinds of software based systems: systems with changeable requirements and systems with relatively fixed requirements. The system specifications driven by external factors  are best built by following agile methods . HoweverThe concept of an incremental and iterative process to construct systems is very old ,9. In prIncremental system construction aims to build systems iteratively by adding increments in a stepwise manner, starting from a small initial system. Such an approach holds the promise of managing scale and complexity, and should therefore be useful for building large systems. In the context of software development, many approaches based on refinement have been proposed for incremental system construction. In general, these approaches are top-down and not bottom-up. The view taken in this paper is that incremental construction is intrinsically bottom-up and would therefore be easier to achieve by means of component-based development (CBD), which is also intrinsically bottom-up.In this paper, based on the behavior containment and preservation, we define our notion of incremental construction by which we focus only on growing the system in increments, i.e., incremental construction. In our view, an iterative software construction approach is incremental if the functionality of the system under construction is incremented in each successive iteration. This means that in the construction process functionalities are only added, and not altered or deleted. Such an approach is applicable to construct systems with fixed requirements or specifications.This paper contributes a redefinition of the notion of incremental construction with an emphasis on the behavior containment. Next, in the context of incremental construction, we contribute a study of current component models to investigate how incremental construction can be achieved in the three main categories of current component models, namely models where components are: (i) objects, (ii) architectural units, and (iii) encapsulated components. This study has not been presented in the literature before. By studying and comparing the strengths and weaknesses of component models from the three categories, we propose a taxonomy of component models with respect to component/system behavior and select the EX-MAN component model (EX-MAN) ,12 for iThe rest of the paper is organized into six sections. Developing a program/software in steps by following an incremental and iterative process is an old and fundamental approach ,13,14. PIn a published article in year 2003, Larman and Basili highlight the concept of incremental and iterative development (IID) process to construct systems in the historical perspective . These aIn CBD, in a system, interactions between the components is an important element that can help in finding the ways of composing components together . For CPSReuse of existing work in software engineering is highly demanded to reduce the development cost and to gain high quality software . In CBD An object b has pubIn CBD, a component model defines a unit of programs, called component and composition mechanisms, to create bigger composite components from smaller components. The behavior and structure of this bigger unit is defined by the composing mechanism. A bigger system can be created by the composition mechanism if the mechanism is algebraic ; in otheIn containment, to define a component, at least two components are put together for the composite. In extension, a composite is defined by extending the behavior from two existing components. A connection mechanism defines the interactions between two components; a connection can be either to pass messages directly or indirectly. In coordination, to create a composite of two components, a third coordinator program unit defines the coordination of control/data between the composed components.Components in many component models are objects developed in an object-oriented programming language. For example, using Java, an EJB component can be created by using object aggregation/composition or inheritance mechanisms for objects. A COM component model supports a containment composition mechanism . In the We conclude that the composition mechanisms from containment and extension categories, by and large, are used to define larger basic program units from the smaller program units. In contrast, composition mechanisms from connection (except trait–class composition) and coordination categories define interactions among the composed program units in the resultant composite unit; these mechanisms are useful to construct systems .In CBD, a system is a pair  process a to cons process b, relateC) and data (D). An execution of a system’s service may invoke computations from more than one component. Hence, for a service’s execution request, a system’s functionality is the result of executing its computations (according to its control flow) on its data. As with the system, the functionality of a component can be defined as a set of (provided) services exhibited by the component, e.g., component C in The concept of behavior containment is based on the functionality of components and system. Adding further details, the system pair  and D (an increment with one provided service A) of the current system are exhibited by the new system and (ii) the incremented system offers new services  and of the increment . The containment relationship between the two behaviors is expressed as ‘In Out of many demanded features required for the construction of CPS, in this paper, we focus on achieving the feature of scalability by using our defined incremental construction approach. In this era of technology, an IoT-based CPS is constructed from a big set of requirements in steps. In a system construction method, with a support of reuseability in the construction step, smaller units are composed or constructed to make bigger units. The outcome of each such step should be a defined type for further reuse without losing the existing features of the composed units. In other terms, the composition is algebraic; this means the system construction can be achieved recursively. The incremental way of constructing systems would help us to construct systems of systems. IoT-based systems and cloud-based systems are two such examples of bigger systems constructed by composing existing conventional physical systems in order to work together. Keeping in view the complexity of these bigger systems, after each incremental step the outcome can be tested by using certain test cases. As no functionality is removed, test cases of previous partial systems can also be executed on the later partial and final systems; hence, the one direct benefit of our incremental construction process is the ability to automate the automatic testing of the intermediate as well as the final system. This feature of automatic testing is out of scope of this paper.In truly CBD-based approaches, system construction is a bottom-up process, i.e., starting system construction from pre-built system-independent components ,36. For In this section, we investigate the current software component models with respect to the composition mechanisms defined in these models. The purpose of this investigation is to identify the most suitable component model for the construction of large scale systems incrementally. As mentioned in the previous section, current component models are of three kinds. Hence, we investigate these three kinds of models, and then based on a comparative analysis, identify EX-MAN as the suitable candidate for incremental construction.In this section, we go through the composition mechanisms for objects from  to explosys class) is the execution result of the object’s main method, as shown in run method.Objects/classes are composed by containment , by extension (inheritance) and by connection (object delegation) mechanisms. For classes, containment and extension composition mechanisms construct a single class. In contrast, in OOD and CBD paradigms, a system is a composition of two or more objects connected by object delegation (message passing mechanism). The functionality of a single object  must be called within the functionality of the system .Trait is a unit of reuse  which prWe now show an increment based on code for a repetition construct, as shown in calObj . In the next step, using aspect weaving, sObj is added to the system by object delegation, as shown in calObj for a method of sObj. In the next step, we increment the system by inheritance (sObj has a new method from cObj) and add a local method call (again by aspect weaving) in calObj, as shown in For the expression calculator example, we start with one component S and construct the system by programming. In the system class S, first a call to method m1 of sObj is made to get the squared value of a number. Next, a method call to m2 of cObj is made to get the cubed value of a number. Lastly, a method call to method m3 of calObj is made to get the addition of the squared and the cubed values. The system class S is coordinating with the three components.Another way of construction is to create a new coordination class In CBD, many component models are categorized as ADLs . ComposiComponent models support different architectural styles in different ways; for example, filter components in  have datIn CBD, for pipe-and-filter architectural style , a filteK). In order to keep the model simple, we assume that an in-port is related to one out-port. The functionality of a component can also be represented as a set of out-ports; where an out-port is simply a function of in-ports, as shown in K. In the basic pipe-and-filter component model (basic-pnf), a component is an AU with many in-/out-ports a. A compA) is incremented by connecting C) through a pipe to create system AC). Function f5  between two non-similar (one in-port and one out-port) ports by matching the associated data types. The port of a filter component can be connected to one pipe. Composing two or more filters with pipes produces a composite filter d. All uncalc to add two integer values, (ii) sCom to square an integer value and (iii) cCom to cube an integer value. The system is constructed in two steps, as shown in In order to illustrate incremental construction in basic-pnf, we use Modelica  to constMany component models  support the client–server composition style. In this style, two components communicate by passing messages through their ports. The execution of the caller service is paused by making a call and resumed on receiving the response from the called component. In our basic client–server component model (basic-cs), a component is an AU with many required ports (r-ports) and provided ports (p-ports), as shown in K). For a component, an interaction (sending a request message and receiving a response message) on an r-port is initiated from within the computation code of a service in the component. In basic-cs, an r-port of a component may be related to one or many p-ports and a p-port may be related to zero or more r-ports, as shown in K.In basic-cs, a component with at least one p-port and zero or more r-ports is an eligible component. A port represents a message communication channel between a component and its environment. A signature (of a procedure/service) is associated with each port that represents the type of message allowed to be communicated through the port. A component’s functionality is a set of services and a service is a relation between a sub-set of r-ports and one p-port  is incremented C) by port connection and the composite AC is the incremented system In Calc (to add two numbers), sCom (to square a number) and cCom (to cube a number) in ArchJava )To have a system with fixed functionality by using a component model from the ‘Not-Fixed’ category, the construction process may begin with a component without any dependencies . Then an increment is added to the system to create the incremented system such that the system has fixed functionality. In the worst case, the system may be constructed in a single step (big-bang integration) if there is no encapsulated component for constructing a system. In contrast, component models from the other  category can be used to construct the system in the desired way. Pipe-and-filter style systems are data driven systems. A component model with publish–subscribe composition style is more appropriate for building graphical user interface (GUI) software . In bothThere are many methodologies to address the issue of constructing large software systems in steps. In a step, a functionality may be added, amended or deleted. The unique advantage of the proposed approach is the addition of newer functionality in each step. For the construction of system in which the change is minimum, the proposed approach can be applied. After adding each step the system may be tested automatically. With the defined approach, the selected EX-MAN component model is used for the fixed pre-defined connectors. Such a component model makes the use of incremental construction process easier.With a special emphasis on the behavior containment, this paper contributes a redefinition of the notion of incremental construction for the construction of large and complex systems. Next, we contribute a study of applying the defined approach on current component models to investigate how easily large systems can be constructed in CBD. By studying and comparing the strengths and weaknesses of current component models, we propose a taxonomy of component models with respect to component/system behavior and select the EX-MAN component model (EX-MAN) for its comparative suitability for incremental construction. For the evaluation of our scalable approach, in this paper, we discuss three different systems in EX-MAN.In conclusion to our study of achieving incremental in the current component models, we find EX-MAN the most appropriate model for the construction of systems incrementally. However, the majority of the component models in CBD are ADLs; for future work, we would like to work on an ADL with the property to achieve incremental construction with ease. The automatic testing benefit of our defined approach (discussed in


Allium vegetables such as garlic (Allium sativum L.) are rich in organosulfur compounds that prevent human chronic diseases, including cancer. Of these, diallyl trisulfide (DATS) exhibits anticancer effects against a variety of tumors, including malignant melanoma. Although previous studies have shown that DATS increases intracellular calcium (Ca2+) in different cancer cell types, the role of Ca2+ in the anticancer effect is obscure. In the present study, we investigated the Ca2+ pathways involved in the anti-melanoma effect. We used melittin, the bee venom that can activate a store-operated Ca2+ entry (SOCE) and apoptosis, as a reference. DATS increased apoptosis in human melanoma cell lines in a Ca2+-dependent manner. It also induced mitochondrial Ca2+ (Ca2+mit) overload through intracellular and extracellular Ca2+ fluxes independently of SOCE. Strikingly, acidification augmented Ca2+mit overload, and Ca2+ channel blockers reduced the effect more significantly under acidic pH conditions. On the contrary, acidification mitigated SOCE and Ca2+mit overload caused by melittin. Finally, Ca2+ channel blockers entirely inhibited the anti-melanoma effect of DATS. Our findings suggest that DATS explicitly evokes Ca2+mit overload via a non-SOCE, thereby displaying the anti-melanoma effect. Allium vegetables such as garlic (Allium sativum L.) are rich in allyl sulfides that have been shown to prevent human chronic diseases, including cancer cyt) results in the activation of Ca2+/calpain, an intracellular Ca2+-dependent cysteine protease, thereby resulting in the processing of the mitochondrial localized pro-apoptotic molecule, apoptosis-inducing factor, and caspase-independent apoptosis. The activation of Ca2+/calpain occurs during the apoptosis of glioblastoma and neuroblastoma induced by DAS, DADS, and DATS [2+ concentration ([Ca2+]mit) leads to increased permeability of the mitochondrial membrane, mitochondrial dysfunction, and release of pro-apoptotic molecules such as cytochrome c and Apaf-1, resulting in caspase-dependent apoptosis. Store-operated Ca2+ entry (SOCE) is the principal route by which external Ca2+ enters in non-excitable cells. Ca2+ depletion in the endoplasmic reticulum (ER) triggers SOCE. The depletion induces the translocation of stromal interaction molecules 1 (STIM1) to ER/plasma membrane (PM) junctional regions, where the molecule activates the PM channel ORAI1, leading to Ca2+ entry from the extracellular milieu. Recently, Gualdani and colleagues reported that SOCE mediates cisplatin-induced Ca2+mit overload, ROS production, and apoptosis cyt. We used the Ca2+-ATPase inhibitor, thapsigargin (Tg), as a positive control, because it depletes the ER Ca2+ stores, thereby stimulating SOCE. Tg substantially increased [Ca2+]cyt, while DATS at the concentration of up to 200 μM minimally increased it  increased both [Ca2+]cyt and [Ca2+]mit in a dose-dependent manner  containing CaCl2 or EGTA and treated with DATS. At pH 7.4, the Ca2+ removal almost entirely abolished the increase in [Ca2+]mit, while at pH 6.8, it reduced the increase only a modestly . 2+]mit increase in the cells placed in HBSS (pH 7.4) and Ca2+-containing buffer. The [Ca2+]mit rise seen in HBSS (pH 6.8) and Ca2+-free buffer (pH 7.4) after melittin treatment were also comparable /SOCE antagonists 2-aminoethoxydiphenyl borate (2-APB) entirely blocked this Ca2+ response, verifying the onset of SOCE , a smaller but significant [Ca2+]cyt rise was seen following Ca2+ re-addition and was minimally affected by 2-APB (2+ influx (non-SOCE). Meanwhile, at pH 6.8, no substantial SOCE was observed, while the non-SOCE was increased by the acidification  involved Cat pH 7.4 A. Under t pH 7.4 A. 2-APB tly 20%; A. Next,  200 μM; B.2+ dysregulation in the anti-melanoma effect of DATS with a particular interest in the Ca2+ pathways involved. We found that DATS induced caspase-dependent apoptosis in melanoma cells, although relatively high concentrations of DATS are required  opening and the resulting release of apoptogenic proteins [2+ transport pathways participate in the Ca2+mitt overload and apoptosis caused by DATS and melittin –mitochondrial Ca2+ uniporter (MCU) pathway [2+]mit and tethered to mitochondria via a mitochondria-associated membrane; the physical association allows rapid Ca2+ transport through specified microdomains [3R physically links to VDAC1 on the outer mitochondrial membrane and transports Ca2+ into the mitochondrial matrix [2+mit remodeling in osteosarcoma cells was under the control of Ca2+ uptake through the MCU [2+]mit is also regulated by Ca2+mit efflux through several different pathways, including the mitochondrial Na+/Ca2+ exchanger (NCLX), Ca2+/H+ antiporter, and MPTP mand MPTP ,40,41,42ma cells . Therefo2+ transport from the extracellular space and activated following the depletion of Ca2+ stores from the ER. Recently, SOCE has emerged as critical machinery for Ca2+ influx, contributing to various malignant phenotypes in cancer cells [2 to produce arachidonic acid and lysophospholipids and activates SOCE in vascular smooth muscle cells [2+ dysregulation, as cisplatin does. A notable property of SOCE is its sensitivity to acidification; SOCE is suppressed by decreasing extracellular pH in several cell types [2+mit overload induced by melittin was mainly dependent upon extracellular Ca2+ entry and was suppressed by acidification  involved in Ca2+mit overload caused by DATS is challenging. Our preliminary experiments revealed that melanoma cells expressed Cav1.2 and Cav1.3 isoform of l-type VGCCs. Notably, the previous studies demonstrate the occurrence of the coordinate control of STIM1-ORAI1 and Cav1.2 [v1.2 plays a role in Ca2+mit overload via a specific interaction with SOCE. Further studies are necessary to clarify the molecular entity of VGCCs involved and to test this hypothesis.There remain issues to be resolved in future studies. In particular, the identification of voltage-gated Cad Cav1.2 ,51. Ther2+mit overload through the different pathways, possibly VGCE and SOCE. Our findings suggest the involvement of multiple Ca2+ entry pathways in Ca2+mit remodeling in melanoma cells and switching between them, depending on apoptotic stimuli and pH microenvironments.In conclusion, the present study demonstrated that DATS and melittin exhibit the anti-melanoma effects by evoking pro-apoptotic CaAll chemicals were purchased from Sigma Aldrich  unless otherwise specified. Alliin and allicin were obtained from Cayman Chemical Co. . Soluble recombinant human TRAIL was obtained from Enzo Life Sciences . The pan-caspase inhibitor Z-VAD-FMK was purchased from Merck Millipore . All insoluble reagents were dissolved in dimethylsulfoxide (DMSO) and diluted with high glucose-containing Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) or Hank’s balanced salt solution  prior to use.2 humidified atmosphere unless otherwise specified. The human melanoma cell lines A375 (cell number CRL-1619) and A2058 (cell number IFO 50276) were obtained from the American Type Culture Collection  and the Japanese Collection of Research Bioresources (JCRB) Cell Bank of National Institutes of Biomedical Innovation, Health, and Nutrition , respectively. Cells were harvested by incubation with 0.25% trypsin-ethylenediaminetetraacetic acid  for 5 min at 37 °C.Cells were cultured in 10% FBS/DMEM supplemented with 100 U/mL penicillin and 100 μg streptomycin  in a 95% air/5% CO3 cells/well in 96-well plates  and cultured with agents to be tested for 72 h at 37 °C before the addition of 10 μL of cell counting reagent SF and further incubation for 2 h. The absorbances were measured at 450 nm using an ARVO MX microplate reader .Cell viability was measured by WST-8 assay using Cell Counting Reagent SF  as previously described  with mod5 cells/well) were treated with the agents to be tested for 20 h and stained with FITC-conjugated annexin V and PI using a commercially available kit . The stained cells  were analyzed in FL-1 and FL-2 channel of a FACSCalibur flow cytometer (BD Biosciences) using the CellQuest software (BD Biosciences). Four cellular subpopulations were evaluated: viable cells (annexin V-negative and PI-negative); early apoptotic cells (annexin V-positive and PI-negative); late apoptotic cells (annexin V-positive and PI-positive); and necrotic/membrane-damaged cells (annexin V-negative and PI-positive).Cell death was quantitatively assessed by double-staining with fluorescein isothiocyanate (FITC)-conjugated annexin V and propidium iodide (PI) as previously described . Briefly2+cyt and Ca2+mit levels were measured using Fluo4-AM and rhod2-AM , respectively, as previously described [6/mL) were resuspended in HBSS in 96-well plates. The cells were manually added with the agents to be tested. Then, the cells were measured for fluorescence in a microplate reader  with excitation and emission at 485 and 538 nm (for Fluo 4-AM), respectively, and 542 and 592 nm (for rhod 2-AM), respectively. For the analysis of Ca2+ release and SOCE, Fluo4-AM-loaded cells were suspended in a Ca2+-free medium (HBSS supplemented with 1 mM EGTA) and treated with 2 μM Tg for 10 min and then added to 2 mM CaCl2. For analysis of non-SOCE, cells in the Ca2+-free medium were treated with the medium for 10 min and then added to 2 mM CaCl2.Changes in Caescribed . For impescribed , it was . p < 0.05 was considered statistically significant.Data are presented as mean ± standard deviation (SD) or standard error (SE) and were analyzed by one-way analysis of variance followed by Tukey’s post hoc test using add-in software for Excel 2016 for Windows


These changes persist as HF develops.Compensated hypertrophy is accompanied by an increase in 2+ content increases during compensated hypertrophy then decreases in HF. In healthy cells, increases in SR Ca2+ content and Ca2+ transients can be achieved by the same amount of inhibition of the Na+,K+‐ATPase as measured in the diseased cells.SR Ca2+ leak.SERCA function remains constant during compensated hypertrophy then decreases in HF, when there is also an increase in spark frequency and spark‐mediated CaINa,late and a decrease in Na+,K+‐ATPase current and function alters the balance of Ca2+ flux mediated by the Na+/Ca2+ exchange that limits early contractile impairment.We suggest an increase in 2+ and Na+ regulation from the formation of compensated hypertrophy (CH) until signs of heart failure (HF) are apparent using a trans‐aortic pressure overload (TAC) model. In this model, in vivo fractional shortening (FS) remained constant despite HW:BW ratio increasing by 39% (CH) until HF developed 150 days post‐TAC when FS decreased from 70% to 39%. Using live and fixed fluorescence imaging and electrophysiological techniques, we found an increase in INa,late from –0.34 to –0.59 A F−1 and a decrease in Na+,K+‐ATPase current from 1.09 A F−1 to 0.54 A F−1 during CH. These changes persisted as HF developed . Sarcoplasmic reticulum (SR) Ca2+ content increased during CH then decreased in HF (from 32 to 15 μm l−1) potentially supporting the maintenance of FS in the whole heart and Ca2+ transients in single myocytes during the former stage. We showed using glycoside blockade in healthy myocytes that increases in SR Ca2+ content and Ca2+ transients can be driven by the same amount of inhibition of the Na+,K+‐ATPase as measured in the diseased cells. SERCA function remains constant in CH but decreases  in HF. In HF there was an increase in spark frequency and spark‐mediated Ca2+ leak. We suggest an increase in INa,late and a decrease in Na+,K+‐ATPase current and function alters the balance of Ca2+ flux mediated by the Na+/Ca2+ exchange that limits early contractile impairment.We followed changes in cardiac myocyte Ca in vivo fractional shortening (FS) remains constant until heart failure (HF) develops, when FS decreases from 70% to 39%.During compensated hypertrophy INa,late and a decrease in Na+,K+‐ATPase current. These changes persist as HF develops.Compensated hypertrophy is accompanied by an increase in 2+ content increases during compensated hypertrophy then decreases in HF. In healthy cells, increases in SR Ca2+ content and Ca2+ transients can be achieved by the same amount of inhibition of the Na+,K+‐ATPase as measured in the diseased cells.SR Ca2+ leak.SERCA function remains constant during compensated hypertrophy then decreases in HF, when there is also an increase in spark frequency and spark‐mediated CaINa,late and a decrease in Na+,K+‐ATPase current and function alters the balance of Ca2+ flux mediated by the Na+/Ca2+ exchange that limits early contractile impairment.We suggest an increase in  In this study we follow the changes in cellular Ca2+ and Na+ regulation from the initial stages of cardiac overload and the formation of compensated hypertrophy, until signs of heart failure are apparent in an animal whose cardiac cell physiology closely resembles that of the human. We consider a variety of indices that dictate heart function and intracellular Ca2+ and Na+ regulation to build a picture of the progression of cellular Ca2+ pathology. Our results suggest a continuum of change with one of the earliest modifications being a reduction in Na+,K+‐ATPase current.Most studies that have investigated the changes in cellular Caad libitum. All studies were carried out with the approval of the Animal Welfare and Ethical Committee of Imperial College and the Home Office, UK and are in accordance with the UK Home Office Guidance on the Operation of the Animals (Scientific Procedures) Act 1986, which conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health under assurance number A5634‐01.Male Dunkin–Hartley guinea pigs purchased from Marshall BioResources  were used and housed at 21 ± 1°C on a 12h light–dark cycle and provided with standard guinea pig feed and water et al.  to reduce the oral and nasal secretions and facilitate endotracheal intubation. Lidocaine  spray was applied prior to endotracheal intubation which was with a 14G × 64 mm intravenous catheter. After intubation, anaesthesia was maintained with 1.5∼2% isoflurane in oxygen via the endotracheal tube. Animals were ventilated with a Zoovent Mini ventilator  at 70 ml kg−1 and 70 cycles min−1. End tidal CO2, peripheral O2 saturation and pulse rate were monitored with a Capnovet‐10  and pulse‐oxymeter . Prophylactic antibiotic  5 mg kg−1s.c.) and analgesia  5 mg kg−1s.c. and buprenorphine 0.05 mg kg−1s.c.) were given after stable anaesthesia had been achieved. Local anaesthetic was administered  by subcutaneous injection around the incision site and intraoperative hydration was provided with 4 mg kg−1 h−1 saline 0.9% s.c. carprofen  was given orally for 3 days to maintain post‐operative analgesia.Cardiac hypertrophy was induced using the surgical procedure described by Kingsbury et al.  but perfin vivo.M‐mode echocardiographic measurements were carried out on un‐anaesthetized animals to overcome anaesthetic drug influences on heart function. An S‐12 paediatric echo probe on an Agilent HP 5500  cardiac echo machine was used to assess the degree of ventricular function and left ventricular hypertrophy Experiments were carried out on isolated left ventricular myocytes enzymatically dissociated from sham‐operated and aortic‐banded guinea pig hearts as already described  for 25 min at room temperature (RT). Once loaded, cells were not used for at least another 30 min to allow the intracellular fluo‐4‐AM to be de‐esterified.The CaLeft ventricular myocytes were stored in Dulbecco's modified Eagle's medium  at RT until used. During storage, the cells were protected from light to avoid bleaching of the fluorescent indicator. A drop of cell suspension was placed onto a glass coverslip that formed the floor of a fast exchange, open diamond RC24N superfusion chamber . The chamber was mounted on the stage of an inverted Nikon Eclipse or Diaphot microscope and the cells visualized with a Nikon ×40 fluor oil immersion lens (NA = 1.3). Cells were superfused with normal Tyrode solution (composition below) at 37°C. Cells with rounded edges, obvious cytoplasmic vesicles, automatic activity prior to stimulation, major ultrastructural defects, absence of clear striations or cells that were not incompletely isolated (e.g. cell pairs) were not used.2+ sparks were recorded by previously described methods and analysis of the line scan images was performed using ImageJ  with the SparkMaster plugin  with high resistance microelectrodes to minimize cell dialysis so preserving the intracellular milieu. Sharp microelectrodes had resistances of 20–30 MΩ and were filled with a solution that contained 2 m KCl, 10 mm Hepes, 100 μm EGTA, pH adjusted to 7.20 with KOH. The required signals were recorded using pClamp (v 8 or 10) acquisition and analysis software .Action potential duration and Na+,K+‐ATPase currents following pump block and late sodium currents were measured by using the whole‐cell patch‐clamp technique to improve cytoplasmic ionic control. The resistances of the patch‐pipettes ranged from 4 to 7 MΩ. Pipettes for sharp electrodes and patch experiments were pulled from glass capillaries 1.5 mm OD and 0.86 mm ID with an internal glass filament.Reactivation of Nam: NaCl 140, KCl 6, CaCl2 2, MgCl2 1, glucose 10, Hepes 10; pH adjusted to 7.4 with NaOH. Krebs–Henseleit buffer contained, in mm: NaCl 119, KCl 4.7, MgSO4 0.94, CaCl2 1, KH2PO4 1.2, NaHCO3 25, glucose 11.5; pH adjusted to 7.4 with HCl. Tris‐buffered saline and Tween 20 (TBST) for 1 l contained, 100 ml of TBS 10× , 900 ml of distilled water and 1 ml Tween 20. The external solution for Na+,K+‐ATPase reactivation experiments contained, in mm: NaCl 140, KCl 6 , glucose 10, Hepes 10, MgCl2 1, BaCl2 2, NiCl2 5; pH adjusted to 7.4 with NaOH. The pipette filling solution for Na+,K+‐ATPase reactivation experiments contained, in mm: caesium methane sulfonate 100, NaCl 30, CsCl 10, Hepes 10, EGTA 5, Mg‐ATP 5, MgCl2 0.75; pH adjusted to 7.2 with CsOH. The superfusing  solution for late sodium current experiments contained, in mm: NaCl 137, CsCl 5.4, glucose 10, Hepes 10, MgCl2 2, CaCl2 1.8, nitrendipine 0.002; pH adjusted to 7.4 with CsOH. The pipette filling solution for late sodium current experiments contained, in mm: caesium methane sulfonate 100, NaCl 10, CsCl 30, Hepes 10, EGTA 5, Mg‐ATP 5, MgCl2 0.75; pH adjusted to 7.2 with CsOH.Normal Tyrode (NT) contained, in m2+ transients and quantitative determinations of SR Ca2+ content were made using well described techniques currently in use in the laboratory , a standard single exponential equation was used to fit the decay trace of a steady‐state Ca2+ transient to obtain a time constant (τ) that represented the combined contributions of the Ca2+ regulatory mechanisms. Cells were then exposed to NT containing 10 mm caffeine (at a high flow rate) to induce a release of Ca2+ from the SR. The decay phase of this caffeine‐induced transient represented the contribution of Na+/Ca2+ exchange and slow components. Removal of the caffeine followed by external field stimulation at 0.5 Hz ensured the cell returned to steady‐state. Caffeine was rapidly applied a second time but in Na+‐free, Ca2+‐free solution to elicit another caffeine‐induced Ca2+ transient. In this case, Na+/Ca2+ exchange was blocked by the Na+‐free, Ca2+‐free solution, and therefore the decay τ of the second caffeine‐induced transient represented the contribution of the slow components to Ca2+ efflux. The rate constants of each decay phase were calculated as 1/τ.To assess the relaxation contribution from Na+,K+‐ATPase current was measured in cells voltage‐clamped at –80 mV. First a train of preconditioning pulses (square clamp pulses from –80 to 0 mV for 300 ms at a frequency of 1 Hz) were applied for 1 min to ensure steady‐state [Na+]i had been reached. Stimulation was stopped and the superfusate switched to NT containing 2 mm BaCl2 and 5 mm NiCl2 (Ba2+ was used to block K+ channels and Ni2+ to block Ca2+ channels and inhibit the Na+/Ca2+ exchange). After 1 min, 5 μm dihydro‐ouabain (DHO) was applied to inhibit the high affinity component of the Na+,K+‐ATPase and after a further 1 min, 500 μm strophanthidin was applied to produce complete inhibition of the current so allowing measurement of total pump current and that due to the individual isoforms of the Na+,K+‐ATPase.Na+ current  was recorded at RT using a modified protocol established from previous work  to induce Na+ current. This sequence was repeated following superfusion of NT containing 10 μm ranolazine for 3 min. INa,late was obtained by subtracting the trace in the presence of ranolazine from the original trace. INa,late density was determined from the averaged current measured between 210 and 220 ms after the depolarizing step to –20 mV. This time interval was chosen to avoid a contribution of the transient (early) Na+ current which is reported to be completely inactivated within 200 ms  to glass bottom culture dishes  pre‐coated with laminin . They were washed with phosphate buffered saline (PBS) three times then fixed with 4% paraformaldehyde (PFA) for 10 min and permeabilized with Triton X‐100 (0.05% in PBS for 10 min). Non‐specific antibody binding sites were blocked by incubating the cells in PBS containing 5% bovine serum albumin (BSA) and 3% goat serum for 30 min before application of the primary antibodies. These procedures were carried out at RT.+,K+‐ATPase α1, Invitrogen, cat. no. MA1‐16731, lot no. RK2298051 and rabbit anti‐Na+,K+‐ATPase α2, Merck, Kenilworth, NJ, USA; cat. no. AB9094‐I, lot no. 2855648) were added at dilutions (with blocking solution with PBS) of 1:200 and 1:150, respectively, and the cells incubated overnight at 4°C. The myocytes were then washed 3 times with PBS for 5 min. Alexa Fluor 488 conjugated secondary antibodies  were used accordingly at 1:500 dilution and applied to the cells for 3 h. After washing the cells, glycerol‐based mounting medium containing 4′,6‐diamidino‐2‐phenylindole (DAPI)  was added. Image acquisition was performed with a Zeiss LSM780 confocal microscope . The settings on the confocal microscope were standardized at 1 Airy unit and gain and offsets were kept constant for all image collections and measurements for each antibody. Cell images were recorded approximately mid‐way through the cell depth as judged by DAPI staining. Two regions of interest (ROI) (143 × 93 pixels) were analysed using ImageJ; one was placed over the sarcolemmal area and the other in the centre of each cell. The image threshold was set to allow calculation of the area of fluorescence of the antibody, minimizing the acquisition of background staining. The mean area of fluorescence was calculated from the two ROIs for each cell and the measurements pooled with results from at least 10 cells from two hearts of each group.Primary antibodies  at 1:2000 dilution or glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) primary antibody  at 1:10000 dilution for 1 h at RT. Following a series of washing steps with TBST, the membrane was incubated with secondary antibody (anti‐mouse IgG–horseradish peroxidase (HRP), Abcam, Cambridge, UK, cat. no. ab205719, lot no. GR247077‐7) at 1:2000 dilution for 1 h at RT. The membrane was subsequently washed again and the HRP‐conjugated antibody bound to the target protein was visualized by Clarity Western ECL Substrate  and captured by a G Box Chemi XT 16  (4 min exposure) using GeneSnap software version 7.12.06 .The membrane containing the diluted proteins was blocked with blocking grade buffer (Bio‐Rad cat. no. 1706404) overnight at 4°C to prevent non‐specific binding of the detection antibodies during the subsequent steps. The membrane was incubated with either Na+,K+‐ATPase α2 western blot, the membrane was blocked for 1 h at RT and then incubated with the α2 antibody  overnight at 4°C. Following a series of TBST washing steps, the NKAα2 membrane was incubated with anti‐rabbit IgG‐HRP secondary antibody  and the GAPDH was incubated with anti‐mouse IgG‐HRP, both at 1:2000 dilution for 1 h at RT. The membrane was washed again and developed using Clarity Western ECL with exposure time of 5 min. A similar protocol was used for the phospholemman (PLM) blot. The membrane was blocked for 1 h at RT and incubated in primary antibody anti‐rabbit FXYD1  overnight at 4°C. The membrane was washed using TBST and incubated with secondary antibody (anti‐rabbit IgG‐HRP) at 1:2000 dilution for 1 h at RT. The membrane was washed and developed using Clarity Western ECL with exposure time of 2 min.For the Nan = 4 samples for each group investigated. Each blot was repeated three times and the averaged density value for each sample was used to determine differences between means. Representative samples are illustrated in the final western blot. ImageJ software was used to analyse the blots.Western blotting was carried out using a minimum of P values are designated with asterisks: ***P < 0.001, **P < 0.01, *P < 0.05. Either Student's t test or ANOVA was used to calculate P values with Prism 7 software . Experimental values were normally distributed except some Ca2+ spark data that were transformed logarithmically where necessary before running the statistical test. The number of cells used in each study together with the number of hearts from which they were isolated are detailed in the figure legends.Data are presented as mean (SD) or mean  and Experimenters were blinded to the origin of the cells used in the immunocytochemistry experiments but in all other studies experimenters were not blinded.et al. et al. et al. 2+ and Na+ regulation changes as the hearts progress from established hypertrophy to HF.This animal model of pressure overload due to transverse aortic constriction (TAC) has been carefully studied at the point where cardiac hypertrophy is well developed – between 40 and 60 days post‐operatively  also increases 150 days post‐AC. No changes to LW:BW occur at earlier time points. The lung weight changes correlate well with the increase in LVIDd, which is also only different at 150 days post‐AC. The enlarged LVIDd indicates volume retention in the left ventricle and this is likely to produce pulmonary congestion confirmed by the increased LW:BW at this stage.2+ and Na+ regulation in cardiac myocytes we isolated single cells at two time points – at 60 days post‐AC (60AC), corresponding to a compensated hypertrophy stage, and at 150 days post‐AC (150AC), a time when the hearts are beginning to fail.We conclude from these whole heart measurements that between 30 and 60 days post‐AC there is no decrease in heart function, yet heart weight has increased suggesting that compensated hypertrophy has occurred. At 150 days post‐AC the compensation has halted, whole heart function is poor and signs of heart failure are apparent. To assess changes to Ca2+ transients recorded from myocytes isolated from 60‐day AC hearts were larger than those isolated from sham‐operated control cells . With progression of disease, the Ca2+ transients decreased by 40%. The time‐to‐peak of the Ca2+ transients was not different in cells from sham and AC hearts until 150 days when the cells isolated from AC hearts displayed a pronounced slowing of this parameter (from 58 (6.3) ms, to 68 (6.3) ms, P < 0.01, Student's t test).During the compensated hypertrophy stage (60 days) the Ca2+ transients, indicating the kinetics of cytoplasmic Ca2+ removal, was 33% slower in 150AC compared with Sham; however, the rates were similar between Sham and 60AC groups. The rate constant of 150AC was 34% slower compared with 60AC. The rate constants of SERCA, Na+/Ca2+ exchange and other slower transporters remained unchanged between Sham and 60AC  as did the contribution of each system to relaxation .The decline of the Ca+/Ca2+ exchange and a 51% decrease in the rate constant of SERCA compared with those in Sham . 150AC had a 28% decrease in the SERCA contribution to relaxation and 63% increase in the contribution from Na+/Ca2+ exchange .By 150 days AC showed a significant increase in the rate constant of Naham Fig. C. 150AC2+ content measured 60 days post‐AC was greater in myocytes isolated from AC hearts compared with those isolated from sham‐operated control cells . With progression of disease the SR Ca2+ content decreased by 48% . These changes mirror the alterations to the Ca2+ transients and suggest that the Ca2+ transient amplitude at the compensated hypertrophy stage is maintained at least partly by a more Ca2+‐loaded SR.SR Calls Fig. A. With 2+ sparks  and from 150‐day sham‐operated control hearts (upper panel). Dihydro‐ouabain  was applied to inhibit the high affinity component of the Na+,K+‐ATPase (α2 isoform) and 500 μm strophanthidin was applied to produce complete inhibition of the pump current. Total current declines at 60 days post‐AC  corresponding with the increase in SR Ca2+ content and the compensated hypertrophy stage. The Na+,K+‐ATPase current remains low during the progression to heart failure . We did not find any change in the ratio of isoform contribution to total current at any time during the progression to heart failure .One possible mechanism that would explain the positive inotropy, increased Ca+/K+ pump activity can be described in terms of the rate of decline of Na+,K+‐ATPase current following reactivation after a period of pump inhibition. We inhibited the pump in myocytes isolated from sham and 150‐day AC hearts with K+‐free superfusate . The pump was then reactivated by adding back 6 mm K+ and this produced an outward current that decayed to a steady state. The decay phase represents the decrease in intracellular [Na+] that occurs on pump reactivation and therefore indicates net Na+ extrusion. The amount of Na+ pumped from the cells was therefore calculated as the integral of the current to the point where it had reached a steady state .The Naate Fig. A. The ptes Fig. B.+,K+‐ATPase current and Na+ extrusion during the progression towards heart failure. To assess if the decline in these two indices of function may be explained by a decrease in the amount of pump protein, we undertook immunocytochemistry and western blotting experiments.The foregoing experiments present evidence of a decline in NaA shows typical staining patterns obtained from immunolabeling of α1  and α2  Na+,K+‐ATPase isoforms in cells isolated from 150‐day sham‐operated controls and 150‐day AC hearts. To assess the changes in α1 and α2 isoform staining we created two regions of interest. The staining intensities within these areas were calculated and grouped to yield pooled measurements from at least 10 cells from two hearts of each group. These data are shown in Fig. B. The overall staining was greater in sham cells compared with the HF cells.Figure  α1 Fig. Aa and α α2 Fig. Ab Na+,K+,K+‐ATPase are mirrored by the decline in protein levels after 60 and 150 days as assessed by western blotting. This result is shown in Fig. A. GAPDH was used as the loading control and blot densities were normalized to the level of this protein. The 60‐day and 150‐day AC groups had significant decreases in the mean protein levels of the α1 isoform compared with the age‐matched sham groups. The levels of α2 isoform were unchanged.Both the reduction in function and in immunolabeling of the Na+,K+‐ATPase is regulated by phospholemman (PLM) and so we assessed the levels of this protein  in the same samples . There was a decline in expression between Sham and AC at 60 days; however, there was no difference in PLM protein expression between 150‐day sham and 150‐day AC groups.The Nales Fig. B. There+,K+‐ATPase functional data suggest there is about a 50% reduction in pump current in the compensated stage of heart failure. We investigated if this reduction could act as a positive inotropic mechanism to preserve contractile function early in the progression of disease and be sufficient to explain the enhanced SR content and larger Ca2+ transients measured at that stage.Our NaA), we found that application of 10 μm strophanthidin produced a 47 ± 7% reduction of Na+,K+‐ATPase current. This reduction in current increased the Ca2+ transient amplitude by 12%  and the integral of caffeine‐induced inward Na+/Ca2+ exchange current by 37% . These results suggest that inhibition of the Na+,K+‐ATPase current by a similar amount to that at the compensated hypertrophy stage can account for the increase in SR load and the maintenance of the Ca2+ transient. In the setting of developing HF, this could maintain cardiac function that would otherwise be in decline.Following a series of dose–response experiments Fig. A, we fo2+ uptake that occurs in the progression to failure. When Na+,K+‐ATPase was reduced by 50%, an inhibition of SERCA function also by about 50% using 200 nm cyclopiazonic acid . Increasing Na+ influx using 1 μm veratridine in this condition did not augment Ca2+ transients and was pro‐arrhythmic .We also investigated if this inotropy could be altered by partial inhibition of SERCA in healthy cells so reproducing the gradual failure of SR Ca30% Fig. A and B.mic Fig. A and C.et al. et al. et al. A consistent finding in cardiac hypertrophy and heart failure in many studies is prolongation of the action potential duration (APD) . Although very small in comparison to the main Na+ current that produces the AP upstroke, its persistence ensures it contributes to the plateau phase potential, morphology and duration and to Na+ influx. There is evidence that the density of the current increases and its inactivation kinetics slow in heart failure .A second component of Narts Fig. A and B.INa,late 215 ms from the start of 2 s depolarization steps from a holding potential of –120 mV to – 20 mV . INa,late was increased in the compensated stage (60 days) and further increased during disease progression so, when the failing stage was reached, it was about 240% larger than control.We measured ranolazine‐sensitive 2+ pathology from the initial stages of cardiac overload and the formation of compensated hypertrophy, until signs of heart failure are apparent. We do so in an animal whose cardiac cell physiology closely resembles that of the human. It presents a unique insight into the progressive changes that take place that, when studied in isolation, may give rise to areas of confusion and contradiction. We considered a variety of indices that, in our view, are important markers of whole heart function and intracellular Ca2+ and Na+ regulation, to build a picture of the progression of cellular Ca2+ pathology. Our results suggest a continuum of change with the earliest modifications being a reduction in Na+,K+‐ATPase current and an increase in late Na+ current.In this study we follow the development of cardiac cellular Caet al. et al. et al. et al. et al.  has already been used in a number of studies mainly at the point where cardiac hypertrophy is well developed – between 40 and 60 days post‐operatively , their prolongation, and a decrease in SR Ca2+ content, three hallmarks of the cellular phenotype of HF in animal and human studies  stage, bulk cytoplasmic [Na+] had increased by 4–5 mm . We cannot rule out the contribution of other transport processes  and SR Ca2+ leak increases .The increase in  may not reflect alterations in subsarcolemmal [Na+] regulation. It is not inconceivable that there will be differences in co‐localization of ion transporters in disease states that will create microdomains with different functional characteristics.The work demonstrates that the decline in Nak et al. b for re+] biases the bidirectional function of the Na+/Ca2+ exchange, increasing reverse mode and reducing forward mode operation during the cardiac cycle, particularly when APD is prolonged. During the compensated hypertrophy stage, the Ca2+ transients recorded from myocytes isolated from 60‐day AC hearts were larger than those isolated from sham‐operated control cells and we suggest that this effect is due to increased Ca2+ influx by the Na+/Ca2+ exchange that contributes to the transient and also to the observed increase in SR Ca2+ content. This occurs with no change to the rate constant of exchange‐mediated Ca2+ removal. Whilst the rate constants for Ca2+ removal allow a comparison of the contributions of the various Ca2+ regulatory systems to relaxation, they need to be viewed with some caution. It is presumed that the rate constants should be independent of the peak value of [Ca2+]i, i.e. the transport rates are linear functions of free [Ca2+]i. However, Bers & Berlin   provide an activation signal for calcineurin . Mitochondrial Ca2+ homeostasis is a balance of Ca2+ influx through the mitochondrial uniporter and efflux through the mitochondrial Na+/Ca2+ exchange. An increase in cytoplasmic [Na+] causes a decrease in [Ca2+]m and reduces oxidative phosphorylation so slowing mitochondrial ATP production . We also acknowledge use of the Imperial College Facility for Imaging by Light Microscopy (FILM).


Recent research has found that pet rabbits are frequently inappropriately housed, fed, and not routinely provided with healthcare, often suffering painful medical conditions and shortened lifespans as a result. Using an online survey, this study provides a new understanding of rabbit owners’ perceptions of rabbits, how these affect the resources  that rabbits are provided with, and how these resources impact rabbit welfare. Our results found that variation in owner perception influenced provisions of conspecific partners, level of enrichment, diet, and type of housing provided. Welfare scores  were improved with a diet of mainly grass/hay, a larger variety of enrichment, free-roam housing, and increased time spent with owners. These results suggest that a practical approach to improving the welfare standard provided to rabbits may be to target improving owner perceptions. This information would be beneficial in tailoring public education programs to increase provision of welfare enhancing resources, improving the human–animal relationship, and improving the welfare standards for rabbits.Pet rabbit welfare is a hidden crisis: Inappropriately housed, fed, and not routinely provided healthcare—rabbits can often suffer painful conditions and shortened lifespans. This study provides novel understanding of owners’ perceptions of rabbits’ mental capabilities; how this impacts their husbandry; and subsequent effects on rabbits’ welfare. A survey was designed to investigate owner and rabbit demographics, owner perception of rabbits, resources provided, and rabbit behavior. Distributed online and by the Rabbit Welfare Association and Fund, the survey received 1516 responses. It was found that increased owner perceptions of pain, emotions, and intelligence resulted in increased likelihood of providing a partner, increased enrichment variation, and a more appropriate diet and type of housing. Welfare scores were associated with diet, housing, variety of enrichment, and time spent with owners. These results suggest that a practical approach to improving the welfare standard provided to rabbits may be to target improving owner perceptions of the species’ intelligence, emotionality, and experience of pain. This information would be beneficial in tailoring public education programs to increase provision of welfare enhancing resources, improve the human–animal relationship, and thus improve the welfare standards for this species. Pet rabbits are the third most popular pet in the western world , however2. Both Rooney et al. [Several survey-based studies have been carried out within the last decade to collect data on the current state of rabbit welfare. Rooney et al.  ran a lay et al.  and Mully et al.  state thy et al. ,7. This y et al. ,4,5. Wely et al.  carried Recently, Rioja-Lang et al.  gatheredAn appropriately enriched environment  have a significant effect on welfare, as well as the behavior demonstrated between group housed individuals. For example, in a laboratory setting, provision of enrichment within a farmed rabbit setting in the form of wooden sticks to chew lead to a lower incidence of skin injuries, which could be extrapolated to pet rabbits . Mullan Recently, there has been an increasing interest in the mutual benefits of human–animal relationships, and the human attitudes towards animals that impact these relationships ,11. Alonn = 52) by Edgar and Mullan [Although perceptions of rabbit pain, intelligence, and emotionality have not yet been studied, perceptions of rabbit husbandry requirements have been found to significantly affect the human–animal relationship and pet rabbits’ welfare. A small survey-based study ; and how these various resources affect the rabbits’ welfare. Knowledge of such relationships could be beneficial in tailoring public education programs developed by charities, pet shops, and veterinary professionals to increase provision of welfare enhancing resources such as a companion, appropriate diet, and medical care, improve the human–animal relationship and thus improve the welfare standards vital for this species .This project gained ethical approval by the University of Glasgow Medicine, Veterinary and Life Science College Ethics Committee; project code: 200190110.https://tinyurl.com/y3oabznf.A link to a duplicate of the survey distributed: The questionnaire used was developed using a combined approach. Review and summarization of the scientific literature, alongside expert opinion elicitation, were used in the identification of suitable questions to be used in an anonymous online questionnaire.The questionnaire was created in Microsoft Forms (@2020 Microsoft)  in choice, open ended, and rating style formats, based on 4 sections:Section A: Owner and rabbit demographic information—age, gender, country of residence, and rabbit community member status of owner; age, sex, breed, time owned, spay/neuter status, vaccination status, insurance status, and if the rabbit had attended the vet for a non-routine reason. Rabbit information was limited to three rabbits per respondent due to a limitation of question numbers on Microsoft forms (with a limit of 100 questions). If respondents owned more than 3 rabbits, they were instructed to input information about their three most recently acquired rabbits.Section B: Perceptions of pet rabbits—scoring perception of rabbits’ ability to experience pain, emotions, and intelligence from 0–100 ; open-ended questions to elaborate on perception. As a perception study based on rabbits is novel, this scale was chosen based on intelligence-based perception studies by Nakajima et al.  and MercThis section also included five-point Likert scales on strength of agreement (from strongly disagree to strongly agree) that rabbits could form strong bonds with other rabbits, strong bonds with humans, and that rabbits were happy to live alone.Section C: Resources provided to pet rabbits—enrichment ; diet ; housing ; and partner provision  were presented as closed-choice questions. The number of days in the month rabbits would have access to the outdoors, access to indoors, and time spent with rabbits per day (observing them but not directly handling them) were asked as open questions (restricted to a numbered answer).Section D: Individual behavioral frequency closed-choice questions —‘binkies’, ‘flopping’, ‘zoomies’ (positive welfare state); bar biting, chewing furniture, over-grooming (negative welfare state); and self-grooming . These individual behaviors were described in a written format, and also shown as video examples so participants were familiar with behaviors being assessed as follows in Participants were required to fill in an individual behavior section for each rabbit included in Section A. Behavioral frequency questions were pre-determined to be positive or negative welfare indicators using previously established indicators . These wThe online questionnaire was distributed to current rabbit owners via a hyperlink on social media groups  as well as by email to Rabbit Welfare Association and Fund members. The link was presented with information regarding the importance of research to improve animal welfare, a note of appreciation for completing the survey and sharing it with fellow rabbit owners, and the opportunity to enter a prize draw to encourage responses. Reponses were collected between 10 March 2020 and 10 April 2020. Participants were required to certify that they currently owned a rabbit/s, were over the age of 16, and that they understood that participation was voluntary and could be terminated at any time and that informed consent was agreed.Following data collection, the raw data were exported from Microsoft forms into Excel for Mac Version 16.16.20; where all choice and qualitative responses were coded. Positive and negative welfare scores were created by adding the frequencies of behaviors (previously deemed positive or negative indicators of welfare) to provide a score/18. For example, if a participant reported their rabbit to ‘binky’ daily, to ‘flop’ weekly, and to ‘zoomie’ never, their positive welfare score would be calculated as 6 (daily) + 5 (weekly) + 0 (never) to give a positive welfare score of 11/18.All variations of entry for each country were transformed. Cleaned sheets from Excel were then exported to IBM SPSS Statistics Version 26  and descriptive statistics were carried out.Data were not normally distributed; therefore, non-parametric statistics including independent-sample Kruskal Wallis Tests, Mann–Whitney U Tests were performed as an overview of potentially significant variables to enter into multivariate statistical models. The independent variables were the perception scores for rabbits’ intelligence, emotionality, and pain level, and the dependent variables were housing, diet, partner provision, enrichment score, time spent, and welfare scores. Spearman’s Rho correlations were used to test if perception scores correlated with welfare scores, time spent with rabbits, and enrichment score. The independent variables were the perception scores for rabbits’ intelligence, emotionality, and pain level, and the dependent variables were time spent and welfare scores.p < 0.00625 [Box-Tidwell  procedur 0.00625 . Based oMultinomial logistic regressions were run for perceptions and the remaining resources: Housing, diet, and enrichment score. To test the assumption of a linear relationship between the continuous independent variables and the logit of the dependent variable, the independent variables were graphed against the logit of the dependent variables, and visually inspected for linearity. All answers were provided by closed choice answers, so no significant outliers were found.p < 0.0001, R2 = 0.015). Backwards selection regression was used to accept or reject variables as significant, and significant resources were entered into a generalized linear model with positive and negative welfare scores. To verify the model’s assumptions, a residual analysis was performed based on deviance residuals, fitted values, q-q plots, scale-location plots, and Cook’s distance. Results were deemed significant at p < 0.05. Graphs were plotted using Microsoft Excel.The resources against positive and negative welfare scores were found to have a linear fit using Curve Fit in SPSS , 5.4% (82), and 0.7% (11) were female, male, and non-binary, respectively (five responded that they would prefer not to say). The majority of respondents said that they kept their rabbit with another rabbit , whilst 5.1% (78) kept their rabbit with a different animal, and 35.6% (539) kept their rabbit alone. The majority of respondents considered themselves part of a rabbit community , and 10.7% (162) did not. Responses were received from 42 countries with the greatest number  being from the UK, followed by the USA  and New Zealand .Of 2126 rabbits, 79.8% (1697) were vaccinated, 20.2% (429) were not, 24.4% (519) were insured, and 75.6% (1607) were not. The largest groups of breeds entered were: 27.0% (574) of rabbits crossbred, 19.4% (412) mini lop, 11.0% (234) lionhead, 8.8% (187) standard lop, and 23.2% (493) unknown breeds.The majority of participants in this study kept their rabbit in free roam indoor housing, provided a partner to their rabbit, fed a diet of a majority of hay, and provided four different items of enrichment .The median intelligence, emotions, and pain that participants perceived rabbits to experience were 70, 90, and 100, respectively, out of a total of 100 . The majA Spearman’s Rho correlation found a positive correlation of the ranks of perception scores for intelligence, emotions, and pain with positive welfare score, time spent per day, and enrichment score, and a negative correlation of the ranks of perception scores for intelligence, emotions, and pain with negative welfare score .Logistic Regressions.χ2(4) = 27.402, p < 0.0001. The model explained 0.8% (Nagelkerke R2) of the variance in partner provision and correctly classified 62.5% of cases. Of the three predictor variables, only two were statistically significant: Increasing perception of emotions (Exp(B) = 1.005, p = 0.016) and pain (Exp(B) = 1.004, p = 0.028) were associated with an increased likelihood of providing a partner.A binomial logistic regression was performed to ascertain the effects of perception of rabbit’s ability to experience intelligence, emotions, and pain on the likelihood that participants house their rabbit with a partner. The logistic regression model was statistically significant, p < 0.0001). The model predicted that participants were significantly more likely to provide free-roam indoor housing and free roam outdoor housing compared to an outdoor hutch with a higher pain perception score (Exp(B) = 1.015, p < 0.0001 and Exp(B) = 1.017, p < 0.0001, respectively), but not with intelligence nor emotion score.A multinomial logistic regression was run to predict housing provided to pet rabbits according to perception scores for intelligence, emotions, and pain. The model fit against the intercept significantly (p < 0.0001). The model predicted that participants were significantly more likely to provide a diet of majority vegetables compared to muesli with a higher intelligence perception score (Exp(B) = 1.048, p = 0.019).A multinomial logistic regression was run to predict type of diet provided to pet rabbits according to perception scores for intelligence, emotions, and pain. The model fit against the intercept significantly (p < 0.0001). The model predicted that participants were significantly more likely to provide a greater variety of enrichment with a greater pain perception score. Compared to providing no enrichment the likelihood of providing two different forms of enrichment (Exp(B) = 1.049, p = 0.011), three different forms of enrichment (Exp(B) = 1.058, p = 0.003), four different forms of enrichment (Exp(B) = 1.054, p = 0.005), and five items of enrichment (Exp(B) = 1.062, p = 0.002) all increased with increasing pain perception score.A multinomial logistic regression was run to predict the amount of enrichment provided to pet rabbits according to perception scores for intelligence, emotions, and pain. The model fit against the intercept significantly  variables from the model. With regards to positive welfare score, housing, neutered status, and partner were removed as they were not found to have a significant relationship, whilst partner and diet were removed with regards to negative welfare score.Backwards selection was used to remove non-significant , and found a positive welfare score had a very strong positive association with a majority grass/hay diet , majority vegetables diet , and a majority homogenized pellet diet  compared to a majority muesli diet. A positive association was found with positive welfare score and enrichment score  as well as with time spent with pet rabbits  (The GLM had a good fit ( 0.0001) .χ2 = partner 85.37, p < 0.0001), and found negative welfare scores were strongly positively associated with outdoor hutch housing when compared with outdoor free roam housing . Negative welfare score was also positively related to housing in an indoor hutch, compared with free outdoor roam ; and a weak positive association was found between negative welfare score and time spent . Negative welfare score was significantly negatively associated with a higher enrichment score  The GLM had a good fit  and secondary emotions , similar to those attributed by owners to cats and dogs in a study by Martens et al. . HoweverIt is widely accepted that mammals, as sentient beings, experience basic evolutionarily beneficial emotions such as fear and pleasure ; but furWork by Tamioso et al.  concludeBekoff  suggestsThe participants in this study rated rabbits’ ability to experience pain in comparison with humans very highly, with a mode of 100/100. However, answers did range from 0  to 100 , with a mean of 89/100. The key notion mentioned by 26.56% of 800 participants that elaborated on rabbits’ ability to experience pain was that as prey animals, they hide their pain. This indeed makes pain assessment challenging in its subtly and could lead to a misunderstanding that rabbits do not or are not experiencing pain.There is strong evidence to suggest vertebrates experience pain as of their anatomy and physiology, as well as their behavioral reactions to noxious stimuli ,29. FoleHowever, the gold standard for reporting pain in humans remains as self-report. This leaves non-verbal animals, and human infants’ experience of pain, to debate within the scientific community.It has been shown across many laboratory animal pain studies that small mammal pain is often underestimated and under-dosed, leading to a significant welfare impact ,30,31. IBekoff  discusseBertenshaw and Rowlinson  found daChanging perceptions of the emotional and intellectual capacity of rabbits could therefore be key in changing human behavior towards improving the standard of welfare provided to this species. This is corroborated by Tamioso et al. , suggestIn this study, there was a significant positive relationship between perception scores for the intelligence, emotions, and pain experienced by rabbits and time spent with rabbits (without directly handling them), and also with levels of enrichment.These results suggest that perceptions of rabbits may influence their welfare in both a negative and positive sense, so warrant further work within public engagement to further educate the public about rabbits’ ability to experience these elements of sentience. The amount of time a person spent directly observing their rabbits, and the enrichment they provide their rabbits with, seemingly directly influenced their perceptions: Perhaps through the witnessing of more behaviors that imply intelligence and emotionality.“Binkies” and “zoomies” were seen to be an unequivocal sign of joy in pet rabbits by owners, and it was repeatedly reported to also be a great joy for owners themselves to witness.Vocalizing in pain in rabbits was spoken of extremely emotively by those that described it, making pain an unquestionable experience of rabbits to those owners. They spoke of it being ‘unforgettable’ and ‘heart breaking’.The bond between rabbits that had been successfully group housed was spoken of as indescribably strong, and frequently described as ‘love’.The perceived grief experienced by rabbits was frequently mentioned when attributing emotional pain, emotionality, and emotional intelligence, and had a great impact on owners that witnessed it, highlighting the strength of the intraspecies bond.The ability of rabbits to learn tricks, their perceived ability to understand timing and routine, and their spatial memory had clear impact on owner perception of rabbit intelligence.The results of this study highlight the great importance of owner perception of rabbit mentality to improve welfare. It is therefore suggested that public education be tailored to improve the perception of rabbits’ ability to experience, and to promote empathy towards the species. Notable behaviors were mentioned by participants in this study that greatly impacted them and were spoken of very emotively. Perhaps these behaviors could be used to cause an emotional impact on new owners and alter their perceptions of rabbits. For example:As discussed, the roll of pet care professionals and veterinarians in face-to-face education at the point of purchase and onward throughout the life of rabbits has an impact on health care level and resources provided to these animals. Perhaps this public education should not only be provided through rabbit associated campaigns, but also shown to vets and pet care professionals, altering their own perception of rabbits, and thus perhaps influencing them to push for better standards from owners.It was found that housing, neutered status, and partner did not have a significant impact on positive welfare scores, whilst partner and diet had no impact on negative welfare scores.It is widely accepted by key rabbit organizations , that for adequate social interaction and performance of natural behavior, rabbits should be kept in groups of at least two individuals. However, there is very limited published literature on the single or group housing of rabbits within a pet rabbit setting from which to extrapolate an expected positive welfare impact of group housing.Some studies regarding group housing of rabbits in a commercial setting include Szendro et al.  found thAnimal bonding is defined by Abrantes  as a bioOther negative associations were found in rabbit studies; reporting antagonistic behaviors displayed in 78% of rabbits housed with companions ; 25% of Contrary to the studies outlining the welfare concerns of group housing rabbits, Bozicovich et al.  found thTrocino et al.  reinforcWithin a pet rabbit setting, surveys first by Mullan and Main , found tA highlight of the literature surrounding the group housing of rabbits is that evidence is scant for pet rabbit owners, and those that inform them (such as veterinarians and pet care advisors). Future work regarding the group housing of rabbits in a pet setting, and perhaps the characteristics of pet rabbits that form the most reliably strong bonds could be a step forward in advising owners: In order to reduce rehoming and injury, and to improve the longevity and success of rabbit bonding.In this study, data were collected from 1516 participants and fully completed information was provided for 2126 individual rabbits. Survey links were posted on rabbit social media forums and emailed out to members of the Rabbit Welfare Association and fund. This would have likely skewed the data collected towards both particularly enthusiastic rabbit owners, and rabbit owners with time and access to the internet . Indeed,Whilst the skewed population of participants carried a degree of variation in perception scores and in resources provided to analyze their relationship, the responses cannot be generalized to that of the entire rabbit owning population. It must be acknowledged that rabbit enthusiasts may indeed think more highly of their pet’s mental ability, impacting the response figures received. It should also be noted that the authors are also females from the UK within the veterinary profession, which will have had an impact due to bias on the study design.It has been posed in previous survey-based studies that online surveys would skew data by age group, with only younger groups computer literate enough to access the survey . HoweverAs participants were self-reporting information about their rabbits, there may be a discrepancy between stated resources and actual resource provision. Due to logistical constraints, this study did not perform home visits to assess resources or rabbit behaviors. Rooney et al.  specifieDewitt et al.  states iIn future studies, home visits should be considered in order to validate the data provided in responses on resources provided and frequency of rabbit behavior.Notable anecdotal behaviors that imply intelligence and emotionality in this species were shared in open-ended questions that require further investigation to objectively assess rabbit intelligence and emotionality in an objective and species-specific format.Future studies focused on the public’s perceptions of rabbits should aim to receive responses from a more even distribution of owners in terms of gender, country of origin and of rabbit community status to allow for generalization of results.A study showing the impact of perception-targeted public education on resource provision and welfare of rabbits would be useful to confirm the advice arising from the results of this paper that improving perception scores may improve welfare.Work should be carried out regarding the characteristics of rabbit groups that produce the most successful bonds, with the highest incidence of agonistic and lowest incidence of antagonistic behaviors.Higher perception scores of intelligence, emotions, and pain provided by owners were consistent with higher positive welfare scores. This suggests that a practical approach to improving the welfare standard provided to rabbits by owners may be to target improving these perceptions of the species’ features of sentience. These may be directly targeted through public engagement: Demonstrating behaviors of rabbits that infer such qualities or improved through encouraging owners to spend more time with their rabbits, and to provide them a greater range of enrichment, shown in this study to relate to perception scores.It is thought this perception-centric approach could be essential to hone in on improving empathy and compassion towards this species, deemed widely as an inexpensive and replaceable ‘child’s pet’; to move towards rabbits being seen as a sentient, sociable mammal with a wide range of complex and dynamic behaviors that deserve respect and a high welfare standard.