DNA double-strand breaks (DSBs) jeopardize genome integrity and can—when repaired unfaithfully—give rise to structural rearrangements associated with cancer. Exogenous agents such as ionizing radiation or chemotherapy can invoke DSBs, but a vast amount of breakage arises during vital endogenous DNA transactions, such as replication and transcription. Additionally, chromatin looping involved in 3D genome organization and gene regulation is increasingly recognized as a possible contributor to DSB events. In this review, we first discuss insights into the mechanisms of endogenous DSB formation, showcasing the trade-off between essential DNA transactions and the intrinsic challenges that these processes impose on genomic integrity. In the second part, we highlight emerging methods for genome-wide profiling of DSBs, and discuss future directions of research that will help advance our understanding of genome-wide DSB formation and repair. Out of the thousands of DNA insults that hit our cells every day , double-In Part I of this review, we describe how DSBs can form during essential DNA transactions—especially during DNA replication, transcription, and 3D genome folding—after which we touch upon the undesired outcomes of DSB repair and their possible consequences. In Part II we attempt to provide a comprehensive overview of the diverse methods that have emerged to profile genome-wide DSB landscapes and we hypothesize how future efforts can help to answer some of the open questions that exist in the field. Part I—Endogenous Causes of DSBsDuring the S phase of the cell cycle, thousands of replication forks work in a highly coordinated manner to precisely replicate the entire genome exactly once. In the absence of perturbations, a replication fork proceeds undisturbed until it approaches a neighboring fork traveling in the opposite direction. At this point, the two forks fuse, the replication machineries disassemble, and the catenated sister chromatids separate ,21,22,23Replication forks are forced to slow or stall in a global manner when resources required for faithful completion of replication run out . EssentiIndividual replication forks can be brought to stall when an obstacle on the DNA prevents helicase or polymerase activity of the replisome A. If staTranscription bubbles present a natural obstacle for the replication fork ,46,47 FB, and coGenome-wide transcription rates are inherently heterogeneous ,58, and A backtracked transcription complex—composed of a dangling 3’ end of the nascent RNA and an inactive RNAPII that might slide backwards—presents another direct impediment to replication fork progression . BacktraIn general, sensing of replication stress leads to activation of the replication checkpoint, temporary cell cycle arrest, and orchestration of the DDR. Diverse DNA repair pathways can then engage to attempt stabilization of the stalled replication fork—aimed at preventing irreversible fork collapse—and promote removal or repair of blocking impediments or lesions. Ultimately, to restart replication the stabilized fork can be reprimed or, if this fails, replication can be rescued by firing nearby dormant replication origins ,80,81,82Stalled replication forks are predominantly stabilized by components of the HR repair pathway, which also protect nascent DNA at the stalled fork and support repair of DSBs induced by replication stress . FurtherAfter removal of the source of replication stress, restart of the replication fork is believed to occur with the help of HR factors and via repriming mediated by local ssDNA, although the precise underlying fork remodeling and reversal mechanisms remain to be elucidated further . When fork stabilization and restart fail the persistently stalled replication fork can collapse, which terminally inactivates the replisome and can result in the formation of a single-ended DSB ,27,78. FDSBs associated with replication stress occur at higher frequency in certain regions of the genome such as common fragile sites (CFSs). CFSs are defined as chromosomal cytobands that appear broken in metaphase spreads following induced replication stress ,101,102.In contrast to CFSs, early replicating fragile sites (ERFSs) represent a different class of fragile genomic regions that replicate early, reside in gene-rich accessible chromatin, and overlap with sites of recurrent translocations and rearrangements observed in cancer . In a laAlthough still poorly understood, the connection between replication timing and both fragility and the outcome of erratic repair might in part relate to inherent differences between early and late replication. Regions with early versus late replication timing exhibit roughly mirroring patterns of DNA accessibility, gene activity, and nuclear positioning—in line with A/B compartmentalization. Simultaneously replicating regions may thus reside in closer spatial proximity ,112. TheAlthough we already briefly touched upon how transcription bubbles contribute to replication stress, accumulating evidence also connects the act of transcription activation itself to elevated levels of DSBs and genomic alterations. In the following section we will therefore focus further on the relationship between transcription and DSB formation. Besides its share in hampering replication fork progression and generating roadblocks in the shape of R-loops and backtracked transcription complexes, transcription is in itself, in a replication-independent manner, considered to be a source of DSBs ,120,121.The genome-wide frequency of DSBs is not only elevated in CFSs, ERFSs, and long, late-replicating genes, but also in accessible chromatin and, in particular, near transcriptionally active genes, with a significant enrichment around the transcription start site (TSS) ,125,126.In contrast to previous work that related DSBs and, more generally, DNA damage to the suppression of transcription ,131, theExamples of transcriptional activation through DSBs—formed as a consequence of DDR pathways—are plentiful . For exaTranscription-induced DSBs near activated genes in physiological contexts, such as stimulated NSPCs, have been attributed to the action of type II topoisomerase isoforms α (TOP2A) and β (TOP2B), and TOP2-induced DSBs have been suggested to be needed for the transcription of some or all genes ,139,140 TOP2 forms transient DSBs that help resolve positive DNA supercoiling, which builds up ahead of transcription and replication forks, halting their progression or leading to strand breaks ,143,144.Despite the vital function of TOP2 in removing supercoils, its activity is a double-edged sword. On the one hand, TOP2-induced DSBs are thought to be rapidly religated, thereby securing genome integrity ,145,146.In the previous two sections we have discussed relevant work that shows how strongly transcription and formation of endogenous DSBs are interconnected—with and without the interplay with DNA replication. Furthermore, we have seen how transcription activation frequently involves the formation of a transient DSB by TOP2. In the next section we will focus on another functional role of TOP2: resolving topological issues that arise during 3D genome folding. Topological or torsional stress brought about by DNA overwinding not only affects replication or transcription, in which traversing of the linear genome must be made possible, but it can also impact processes involved in organizing the higher-order three-dimensional folding of the genome. Furthermore, supercoils have been hypothesized to transit through chromatin structure and as such, influence larger stretches of chromatin . In thisIn addition to its binding to gene promoter regions, TOP2B has been identified at cis-regulatory genomic elements bound by CCCTC-binding factor (CTCF) or by both CTCF and cohesin ,154. TheAccordingly, chromatin loop borders and loop anchor points are increasingly associated with enhanced levels of DSBs and genomic breakpoints underlying structural variants (SV), and as a result, genome instability and cancer ,160,161.The topological stress that results from chromatin loop extrusion and other chromatin-related remodeling activities can, besides directly impacting strand integrity, in turn also affect the transcription machinery. As torsional stress can contribute to transcription regulation ,165, supIn the sections above, we have covered DNA replication, transcription, and 3D chromatin folding as important endogenous contributors to genome-wide DSB landscapes. However, this overview of endogenous causes is far from complete, as we have deliberately omitted several specialized processes that induce programmed DSBs in specific gene classes or during specific moments in development. We refer elsewhere for excellent literature on programmed physiological DSBs , which fAlthough the DDR can in principle repair programmed and accidental DSBs quickly and faithfully, and rescue stalled replication forks to prevent their collapse, DSBs are drivers of most of the structural genomic rearrangements observed in human (cancer) genomes . In the Imbalances in the regulatory circuitries of the nucleus can cause DNA replication, cell cycle checkpoints, and DDR to go haywire and invoke an avalanche of increasing replication stress, DNA damage, and genomic instability ,173. DurIn the following subsections, we first focus on various processes known to be involved in the transformation of a DSB or a stalled replication fork into potentially harmful non-natural junctions. Then, we describe how profiling of the resulting repair signatures—encountered with variable frequencies in cancer genomes—have become a tool to better understand cancer etiology, and predict disease and therapy outcome. Copy number variation (CNV) is widespread in the human genome and underlies natural variation and evolution, but also cancer and developmental and neurological disorders ,178. CNVAlthough HR is considered to be more faithful than C-NHEJ because it utilizes homologous sequences for repair, homology-directed repair is intrinsically mutagenic. In contrast to healthy cells, where HR is strictly controlled  and miniIn C-NHEJ, DSB ends are repaired without the need for homology, and although the resulting junctions are mostly accurate or have small deletions, free DNA may be inserted or translocations can be formed . In contAlthough most of the mechanisms underlying copy number gains involve replication-based mechanisms, segment amplifications can also arise in a non-replicative manner via breakage-fusion-bridge (BFB) cycles, in which the loss of extensively homologous (sub)telomeric regions induces unstable dicentric chromosomes. It is believed that BFB cycles play a major role in cancer, and the same process has been invoked to explain segmental duplications frequently observed in breast and ovarian cancer ,175. For many years, patterns of single nucleotide variants (SNVs) have helped uncover distinct mutational signatures in cancer genomes, which has led to improved understanding of the underlying causative processes in certain cancer types. Increasingly, a similar approach is applied to multi-nucleotide structural rearrangements or copy number changes in cancer genomes, based on the identification of junctions between genomic sequences that are not naturally together in the reference genome . AlthougAlthough genome-wide profiling of structural rearrangements enables investigation of repair signatures and errors—based on identification of non-linear junctions—these approaches typically reveal past events that have occurred at some point in the history of the cell. In the next section of this review, we change gears back towards the lesions that underlie most of the rearrangements described here, and provide an overview of the various methods that are available for the profiling of genome-wide DSB landscapes.Part II—Studying Genome-Wide Fragility LandscapesWith DSBs structurally underlying most types of structural genomic alterations, insight into DNA fragility landscapes and their associated processes—such as transcription and replication, but also recruitment of repair proteins—is essential to broaden our understanding of the genome, particularly in the context of cancer. Over the past few years, various methods for genome-wide DSB detection and identification have been developed, with the aim of obtaining insight into genome fragility and its molecular basis. These methods can be broadly classified into indirect and direct DSB detection methods , dependiThe first genome-wide DSB landscape was mapped in yeast, using chromatin immunoprecipitation (ChIP) on a tiled microarray (ChIP-chip) based on antibodies against the phosphorylated form of histone variant H2AX, γH2AX —a ubiquiAlthough highly insightful, exploiting proteins as a proxy for DSBs has the disadvantage of being indirect, and assay outcome may be affected by unspecific binding  and spreAmong the earliest in vivo capture approaches for genome-wide identification of DSBs is integration-defective lentiviral vector (IDLV) capture. IDLV employs in vivo NHEJ-assisted incorporation of lentiviral vectors into DSB sites, which are then amplified by linear amplification-mediated PCR (LAM-PCR) and sequenced ,196. IDLIn order to overcome some of these limitations, genome-wide unbiased identification of DSBs enabled by sequencing (GUIDE-seq) was developed . SimilarRather than identifying DSBs genome-wide via local insertion of an ectopic sequence, several groups have developed techniques that exploit the inherent threat of DSBs being converted into genomic translocations. Translocation-capture sequencing (TC-Seq) ,207 and Although more direct and with higher resolution compared to ChIP-seq, the applicability of IDLV, GUIDE-seq, and HTGTS-based methods is limited by the need for transfection or transduction to enable integration or translocation. This can become especially disadvantageous when aiming to map DSB landscapes in primary cells or tissues, with poor or unknown transfection efficiency and possibly toxic effects of transfection or transduction. Furthermore, as these methods critically depend on active DSB repair, their applicability may be limited when working with cells with impaired DSB repair pathways, such as cancer cells. In IDLV, GUIDE-seq, and HTGTS-based methods, all identified DSB sites have undergone active repair, either locally, with ODN or IDLV sequences being integrated into the broken site, or over larger distances, with translocations onto the DSB bait in HTGTS. This suggests that the identified DSB sites only represent a subgroup of all the DSBs present at a given moment—those that are actively but not faithfully repaired. DSBs that are mostly perfectly and rapidly repaired, for example those induced by TOP2 at promoters, may be largely missed. Furthermore, translocation-based DSB identification methods such as HTGTS can only uncover DSBs in genomic regions that are prone to translocate, and can thus underestimate the actual DSB frequency and the propensity of certain DSBs to give rise to genomic alterations different from translocations. In line with this, the dependency of DSB detection methods on NHEJ repair implies that these methods can miss DSBs repaired through a different pathway .The first method to directly capture and identify DSBs in fixed cells in situ was Breaks Labeling, Enrichment on Streptavidin, and Sequencing (BLESS) . BLESS cTwo years prior to BLESS, a method for direct labeling of DNA ends in yeast was published. In contrast to BLESS, damaged DNA immunoprecipitation (dDIP) —among thBiotin labeling of the 3’ ends of DSBs in isolated high-molecular-weight (HMW) gDNA is also performed in DSB-Seq ,213, butAiming to overcome some of the limitations of BLESS, DSB-seq, and Break-seq, DSBCapture  and END-To improve the sensitivity and specificity of BLESS, Breaks Labeling In Situ and Sequencing (BLISS) was recently developed . In BLISSince the structure of DSB ends is believed to affect DSB repair pathway choice , DSB ideFrom the previous two sections it becomes clear that a variety of methods is now available for generating genome-wide DSB maps, with DSB ends identified at (near-)nucleotide resolution . HoweverWhen resources permit and when not restricted by cell number or the nature of a clinical specimen, the combination of a genome-wide assay with nucleotide resolution, such as BLISS, with a repair-based assay such as HTGTS, could help to place the results in a broader context. Together, these assays may provide a complementary picture in which both the fragility at a certain moment and then those DSBs able to engage in translocations or insertions over a period of time can be assessed. However, when working with clinical specimens where cell numbers are generally limited and transfectability may be suboptimal and variable between successive samples, it may be favorable to work with cell fixation and in vitro tagging of DSBs.Another important aspect worth considering is how the generated data will be analyzed to grasp the factors that shape the genome-wide landscape of DSB frequency and distribution in a particular cell type. Fragility scores can for example be correlated to maps of replication timing, transcriptional activity, or chromatin accessibility, preferentially generated in parallel on the same cell type. Furthermore, the frequency of DSB events can be analyzed in light of genome-wide maps of R-loops generated with DNA-RNA immunoprecipitation combined with sequencing (DRIP-seq) , bindingIn this review, we have highlighted several endogenous processes that can lead to the formation of DSBs—either planned or as a result of local disturbances. Furthermore, although some endogenous DSBs play vital physiological roles—for example, in the context of planned genomic recombination, transcription, or replication fork rescue—any type of DSB represents a threat to the stability of the genome when faithful repair fails. The variety of methods to profile DSBs discussed in Part II, as well as their diverse applications, illustrates the complexity and diversity of the field of DSB identification, especially when one considers that genome-wide nucleotide-resolution methods only represent one of several angles to approach DSB biology. In the years to come, to deepen our understanding of variation in DSB susceptibility and cellular implications, integrative approaches will be needed to help decipher how genome-wide landscapes of endogenous DSBs, mapped through one of the methods outlined above , are shaIn addition to certain genomic regions being more prone than others to form DSBs under specific conditions, the possibility of compartmentalized DSB repair and the sequestering of some DSBs to dedicated nuclear regions in mammalian cells is now emerging ,233,234.DSB repair compartmentalization might allow for spatiotemporal uncoupling of different DSB repair pathways in a cell cycle phase-dependent manner ,241, potAlthough endogenous DSBs can be induced by cell stimulation with hormones or transcription factors, or by applying mild replication stress, the effects on fragility are generally not homogenous throughout the cell population, which makes it challenging to draw general conclusions about their effects on transcription or repair. While not valid as endogenous DSBs, the frequently used cell system DIvA  and its To our knowledge there is, at present, no method that allows for 100% efficiency of DSB induction at defined sites in the human genome. Although RNA-guided endonucleases are an arguable exception, it has been suggested—as mentioned above—that the cellular response to CRISPR/Cas9-induced DSBs may not be indicative of regular repair of endogenous DSBs . In lineFurthermore, we envision methods that combine identification of endogenous DSBs with, for example, concurrent assessment of local features such as protein identity or 3D genomic neighborhoods. Another venue of future technology development will be the implementation of combinatorial assays to simultaneously map DSBs and key epigenomic features in the same sample and even single cells, over time. In line with this, single-cell approaches to DSB identification will not only be key to deciphering cell-to-cell variation in fragility landscapes, repair outcomes, and genome folding, but also to helping advance our knowledge of the heterogeneous nature of DSB landscapes in cancer cells and tumor populations underlying tumor evolution. Despite the variety of assays available to map genome-wide DSB landscapes, several issues have not been solved so far. As pointed out previously , due to In line with the emerging relevance of repair signatures in cancer genome studies, exploring the levels of ongoing genome fragility by mapping DSBs directly in clinical specimens might provide new insights into the mechanisms that shape cancer genomes and their evolution. Ongoing fragility underlies most of the future CNAs and SVs, and especially in the context of a cancer cell, DSB landscapes may hold clues on highly fragile regions that underlie rearrangements frequently associated with a tendency to metastasize. In line with that, comparing DSB profiles with the landscape of rearrangements at a later timepoint during tumor evolution, as recently attempted , will be


A peptide tool, called Bcl-2/IP3 receptor disruptor-2 (BIRD-2), was developed to disrupt Bcl-2/IP3R complexes, triggering pro-apoptotic Ca2+ signals and killing Bcl-2-dependent cancer cells. In DLBCL cells, BIRD-2 sensitivity depended on the expression level of IP3R2 channels and constitutive IP3 signaling downstream of the B-cell receptor. However, other cellular pathways probably also contribute to BIRD-2-provoked cell death. Here, we examined whether BIRD-2-induced apoptosis depended on extracellular Ca2+ and more particularly on store-operated Ca2+ entry (SOCE), a Ca2+-influx pathway activated upon ER-store depletion. Excitingly, DPB162-AE, a SOCE inhibitor, suppressed BIRD-2-induced cell death in DLBCL cells. However, DPB162-AE not only inhibits SOCE but also depletes the ER Ca2+ store. Treatment of the cells with YM-58483 and GSK-7975A, two selective SOCE inhibitors, did not protect against BIRD-2-induced apoptosis. Similar data were obtained by knocking down STIM1 using small interfering RNA. Yet, extracellular Ca2+ contributed to BIRD-2 sensitivity in DLBCL, since the extracellular Ca2+ buffer ethylene glycol tetraacetic acid (EGTA) blunted BIRD-2-triggered apoptosis. The protective effects observed with DPB162-AE are likely due to ER Ca2+-store depletion, since a similar protective effect could be obtained using the sarco/endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin. Thus, both the ER Ca2+-store content and extracellular Ca2+, but not SOCE, are critical factors underlying BIRD-2-provoked cell death.The anti-apoptotic protein Bcl-2 is upregulated in several cancers, including diffuse large B-cell lymphoma (DLBCL) and chronic lymphocytic leukemia (CLL). In a subset of these cancer cells, Bcl-2 blocks Ca The anti-apoptotic protein Bcl-2 is upregulated in a large number of cancer cells, including B-cell lymphomas like chronic lymphocytic leukemia (CLL) and diffuse large B-cell lymphoma (DLBCL)3. Bcl-2 prevents apoptotic cell death by neutralizing pro-apoptotic family members, including the executioner proteins Bak and Bax and the BH3-only protein Bim, at the mitochondria5. BH3-mimetic compounds, like venetoclax, disrupt the binding between Bcl-2 and pro-apoptotic BH3-only proteins, thereby triggering apoptotic cell death in cancer cells that depend on Bcl-2's function at the mitochondria for their survival7. Furthermore, the Bcl-2 protein is also located at the endoplasmic reticulum (ER), the main intracellular Ca2+ store9. There, Bcl-2 binds with its Bcl-2 homology 4 (BH4) domain to the central, modulatory domain of the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R)10. In this way, Bcl-2 blocks excessive, pro-apoptotic, IP3R-mediated Ca2+ release from the ER, thereby preventing mitochondrial Ca2+ overload and subsequent apoptotic cell death10. Based on the binding site of Bcl-2 on the IP3R, a peptide tool was developed in an attempt to target pro-survival Bcl-2 proteins at the ER in cancer cells11. This cell-permeable peptide, called Bcl-2/IP3R disruptor-2 (BIRD-2), is capable of stripping Bcl-2 from the IP3R, without affecting Bcl-2/Bim complexes. BIRD-2 was shown to kill Bcl-2-dependent cancer cells, like DLBCL and CLL cells, by eliciting spontaneous, pro-apoptotic Ca2+ signals13. On the other hand, the survival of normal peripheral mononuclear blood cells was not affected by the peptide tool. Furthermore, follicular lymphoma and small-cell lung cancer cells could be killed by BIRD-2 as well and the peptide even decreased the in vivo tumor growth of human myeloma cells in xenografted mouse models15. Interestingly, in DLBCL cells BIRD-2 sensitivity correlated to the expression level of isoform 2 of the IP3R, which is the isoform with the highest sensitivity towards its ligand IP312. DLBCL cells with high IP3R2 levels, like SU-DHL-4 cells, were very sensitive to BIRD-2, whereas cells with low IP3R2 expression levels, such as OCI-LY-1, appeared to be rather resistant to the peptide. On the other hand, OCI-LY-1 cells are very sensitive to BH3-mimetic drugs, like venetoclax16. Recent work from our group showed that there exists an opposite correlation between the susceptibility of DLBCL cells to BIRD-2 and venetoclax16. Additionally, constitutive IP3 signaling also underlies BIRD-2 sensitivity in B-cell cancers17. DLBCL and primary CLL cells could be protected from BIRD-2-triggered apoptosis by blocking constitutive phospholipase C and IP3 signaling.Cell death and survival is regulated by the Bcl-2-protein family, which consists of pro-apoptotic and anti-apoptotic family members2+ oscillations in B-cell malignancies13, which eventually result in Ca2+ overload via IP3R-mediated Ca2+ fluxes12. In many cells, Ca2+ oscillations are maintained through the concerted action of Ca2+ release from the ER and Ca2+ influx from the extracellular milieu. Therefore, we assessed whether extracellular Ca2+ and Ca2+ entry mechanisms such as store-operated Ca2+ entry (SOCE) contributed to BIRD-2 cytotoxicity. SOCE is an important Ca2+-influx pathway that is activated upon ER-store depletion18. It is mediated through STIM and Orai proteins23. STIM proteins are present in the ER membrane where they serve as luminal Ca2+ sensors, while Orai proteins are located in the plasma membrane and function as Ca2+-influx channels22. Upon depletion of the ER Ca2+ store, STIM1 proteins form oligomers that translocate to parts of the ER membrane that are in close contact with the plasma membrane. There, STIM1 activates Orai1 channels to trigger Ca2+ influx24. The BIRD-2 peptide provokes IP3-induced Ca2+ release in cancer cells that depend for their survival on the function of Bcl-2 at the ER. Hence, we hypothesized that BIRD-2 treatment results in SOCE activation, subsequently enabling Ca2+ influx that is contributing to intracellular Ca2+ overload and thus cell death. Here, we focused on SU-DHL-4 cells to test our hypothesis, since this DLBCL cell model displays the highest sensitivity towards BIRD-216. Our data indicate that despite the fact that extracellular Ca2+ is important for BIRD-2-induced cell death in these cells, SOCE was dispensable as pharmacological SOCE inhibitors YM-58483 and GSK-7975A and Stim1 knockdown failed to protect against BIRD-2-induced apoptosis. Interestingly, DPB162-AE, a SOCE inhibitor that also depletes the ER Ca2+ stores, did protect against BIRD-2-induced cell death. However, this is likely through its action at the ER, since ER Ca2+-store depletion brought about by the sarco/ER Ca2+-ATPase (SERCA) inhibitor thapsigargin (TG) too suppressed BIRD-2 toxicity.However, it is not clear whether other cellular factors contribute to BIRD-2-induced cell death in cancer cells. In particular, we found that BIRD-2 provoked spontaneous Ca3R complexes, resulting in excessive Ca2+ signaling16. Since BIRD-2 triggers Ca2+ release from the ER, we wondered whether SOCE occurs upon BIRD-2 application and thus contributes to the cell death-inducing properties of the peptide tool. We explored this hypothesis by focusing on the BIRD-2-sensitive DLBCL cell model, SU-DHL-416, in which we studied the impact of the SOCE inhibitor DPB162-AE on the BIRD-2-induced Ca2+ rise and cell death response. In this cell model, DPB162-AE acts as a potent SOCE inhibitor , which was significantly reduced in cells pre-treated with DPB162-AE  and maximal (free [Ca2+] 3.16 µM) [Ca2+] in COS microsomes overexpressing SERCA2b  against STIM1 Fig. . Mock-trion Fig. , as doneion Fig. . On the IM1 Fig. , indicatlls Fig. , indicatlls Fig.  and timells Fig. . Moreovells Fig. . About 4lls Fig. . Upon tr2+ by chelating Ca2+ from the extracellular environment with ethylene glycol tetraacetic acid (EGTA). Therefore, SU-DHL-4 cells were treated with 3 mM EGTA 30 min prior to the addition of 10 µM BIRD-2. After 2 h of BIRD-2 treatment, apoptotic cell death was measured via Annexin V-FITC and 7-AAD staining of the cells , which cause ER Ca2+-store depletion, should mimic the effects of DPB162-AE and thus suppress BIRD-2-induced cell death. To determine the ability of these compounds to trigger ER Ca2+ depletion, the cytosolic Ca2+ responses were measured in Fura-2 AM-loaded SU-DHL-4 cells upon addition of 1 and 10 µM TG and 10 µM CPA , which occur upon SERCA inhibition/ER Ca2+-store depletion, another ER-stress inducer that did not act through ER Ca2+ dysregulation was used. Therefore, we applied tunicamycin, which induces ER stress and activates the UPR by blocking N-linked glycosylation. Treatment for 4 h with 5 µg/ml tunicamycin triggered ER stress, which was observed as an increase in p-eIF2α, whereas total eIF2α levels remained unaffected  channels, more particularly TRPC channels, have been proposed to be activated by ER store depletion31. Third, ectopic Bcl-2 overexpression has been implicated in downregulation of SOCE in both HeLa and LNCaP prostate cancer epithelial cells, which could be attributed to Bcl-2's ability to reduce ER Ca2+ loading33. Recent work confirmed that overexpression of wild-type Bcl-2 resulted in decreased SOCE, though accompanied by an increase in steady-state ER Ca2+ levels, protecting against TG-induced cell death34. This Bcl-2 property could be abrogated by the α5-helical Bcl-2 mutant 144WGR146/144AAA146, which partially depleted ER Ca2+ stores by itself and boosted SOCE after TG treatment, resulting in aggravated TG-induced cell death. Yet, it remains unclear whether such SOCE modulation can occur by endogenously elevated Bcl-2 proteins, such as in the B-cell cancer cell model used here. Importantly, in this study we showed that Ca2+ present in the extracellular environment contributes to BIRD-2 toxicity, as chelating extracellular Ca2+ with EGTA conferred protection against BIRD-2-provoked cell death in SU-DHL-4 cells. Moreover, the cytosolic Ca2+ oscillations provoked by BIRD-2 treatment were abruptly suppressed by the addition of the extracellular Ca2+ chelator. Hence, although SOCE is not activated by the peptide tool, our results indicate that BIRD-2 does not solely depend on the intracellular Ca2+ levels for its pro-apoptotic effects. This study revealed a hitherto unappreciated role for extracellular Ca2+ in BIRD-2-induced cell death. Yet, the exact molecular mechanisms by which extracellular Ca2+ enters BIRD-2-sensitive cells and contributes to BIRD-2-induced cell death remain elusive.There are several plausible reasons why SOCE may not be activated upon BIRD-2 application, despite the fact that BIRD-2 provokes Ca2+, the ER Ca2+ stores also determine apoptotic cell death mediated by BIRD-2 in DLBCL cancer cells, as we previously showed that IP3R inhibition suppresses BIRD-2 sensitivity12. Indeed, treatment of SU-DHL-4 cells with the SERCA inhibitors TG and CPA protected against BIRD-2-induced cell death. As such, we hypothesize that the protective effects of DPB162-AE against BIRD-2-induced cell death are due to the ability of DPB162-AE to lower ER Ca2+ levels and not due to its SOCE-inhibitory properties. Importantly, the observed protection is a direct consequence of ER Ca2+-store depletion, and is not caused by ER stress/UPR activation, since tunicamycin, an ER-stress inducer that does not directly target the Ca2+-signaling machinery, could not reduce BIRD-2-induced apoptosis. Hence, our work implicates that the proper filling of the ER Ca2+ stores is a critical factor underlying the susceptibility of DLBCL cancer cells towards BIRD-2 exposure. This is in line with previous work showing that the ER Ca2+-filling state is a critical determinant in the apoptotic sensitivity of cells towards cell death inducers36.Besides extracellular Ca3R2, the isoform with the highest sensitivity to its ligand IP3, are very sensitive to BIRD-2, whereas cells with low IP3R2 expression levels are less susceptible to BIRD-2-induced cell death12. Second, we have recently shown that constitutive IP3 signaling is an additional determinant of the sensitivity of B-cell cancers, including DLBCL and CLL, to BIRD-217. Constitutive IP3 signaling has a pro-survival role in cancer cells, but it can be switched into pro-death signaling by disturbing Bcl-2/IP3R-complex formation with BIRD-2. Finally, here we show that Ca2+-mediated cell death induced by BIRD-2 depends on the Ca2+ levels of the ER as well as on the presence of Ca2+ in the extracellular environment, but not on Ca2+ influx via STIM/Orai signaling. Thus, our data indicate that both extracellular Ca2+ and the ER Ca2+-store content contribute to cell death induced by BIRD-2.We had previously shown that BIRD-2-induced apoptotic cell death in DLBCL cancer cells depends on multiple cellular determinants. First, DLBCL cells with high expression levels of IPl-glutamine  and penicillin and streptomycin . Cells were cultured at 37 °C and 5% CO2. All human cell lines used in this study have been authenticated using autosomal short tandem repeat profiling performed by the University of Arizona Genetics Core and fully matched the DNA fingerprint present in reference databases.DLBCL SU-DHL-4 cells were kindly provided by Dr. A. Letai  and cultured in suspension in RPMI-1640 medium . Human cervical carcinoma HeLa cells were cultured in Dulbecco's modified Eagle's medium medium . All media were supplemented with 10% heat-inactivated fetal bovine serum, 2) , Fura-2 AM , tunicamycin , and YM-58483 . GSK-7975A was a gift from GlaxoSmithKline . DPB162-AE was kindly provided by Dr. K. Mikoshiba . BIRD-2 peptide (sequence: RKKRRQRRRGGNVYTEIKCNSLLPLAAIVRV) was purchased from LifeTein  with a purity of >85%.The following antibodies were used for immunoblotting: anti-GAPDH antibody , anti-STIM1 antibody , anti-total eukaryotic initiation factor 2α (eIF2α) antibody , anti-phospho-eIF2α (p-eIF2α) antibody , anti-vinculin antibody . Reagents were as follows: EGTA , TG , CPA , calcium chloride  program P-005. Briefly, 5 × 102+ levels were monitored with Fura-2 AM as previously described37. Briefly, Fura-2 AM measurements were performed by seeding the SU-DHL-4 cells in 96-well plates (Greiner) with poly-l-lysine coating. The cells were loaded for 30 min with 1.25 µM Fura-2 AM at room temperature in modified Krebs solution , 11.5 mM glucose and 1.5 mM CaCl2), followed by a de-esterification step of 30 min in the absence of Fura-2 AM. During the de-esterification cells were treated with vehicle, 3 µM YM-58483, 3 µM GSK-7975A, or 10 µM DPB162-AE. Fluorescence was monitored on a FlexStation 3 microplate reader  by alternately exciting the Ca2+ indicator at 340 and 380 nm and collecting emitted fluorescence at 510 nm. EGTA , TG , CaCl2 , or BIRD-2  were added as indicated. All traces are shown as the ratio of emitted fluorescence of Fura-2 (F340/F380).Cytosolic Ca2+ measurements were performed as previously described38. In brief, SU-DHL-4 cells were loaded with Fura-2 AM as described above. Single-cell imaging was performed using a Zeiss Axio Observer Z1 Inverted Microscope equipped with a 20× air objective and a high-speed digital camera . Data are shown as the ratio of emitted fluorescence of Fura-2 (F340/F380).Single-cell cytosolic Ca2+ measurements were performed as previously described39. ER Ca2+ levels were measured with the genetically encoded Ca2+ indicator G-CEPIA1er, which was kindly provided by Dr. M. Iino 26. The G-CEPIA1er construct was introduced into HeLa cells utilizing X-tremeGENE HP DNA transfection reagent  according to the manufacturer’s protocol. A Zeiss Axio Observer Z1 Inverted Microscope equipped with a 20× air objective and a high-speed digital camera  were used for these measurements. Changes in fluorescence were monitored in the GFP channel (480/520 nm excitation/emission). To chelate extracellular Ca2+, EGTA was added to a final concentration of 3 mM. One minute later, 1 µM TG or different SOCE inhibitors were added. All traces were normalized (F/F0) where F0 is the starting fluorescence of each trace.Single-cell ER Ca2+-dependent ATPase activity experiments were performed on microsomes of COS cells overexpressing SERCA2b using an endpoint colorimetric assay responding to the released inorganic phosphate, as previously described40. The SOCE inhibitors were pre-incubated with the microsomes for 30 min at 4 °C, followed by 5 min at 37 °C. The Ca2+-dependent ATPase activity was measured at maximal (free [Ca2+] 3.16 µM) and submaximal (free [Ca2+] 0.316 µM) [Ca2+].Ca5 cells/ml) were treated for 2 h with 10 µM BIRD-2 with or without a pre-treatment of 30 min with 3 µM YM-58483, 3 µM GSK-7975A, 10 µM DPB162-AE, 3 mM EGTA, 1 µM TG, 10 µM CPA, or 5 µg/ml tunicamycin. Subsequently, cells were pelleted by centrifugation and incubated with Annexin V-FITC  and 7-AAD . Cell suspensions were analyzed with an Attune Acoustic Focusing Flow Cytometer (Applied Biosystems). Cell death by apoptosis was scored by quantifying the population of Annexin V-FITC-positive cells using the FlowJo version 10 software. Data are plotted as the ∆ apoptotic fraction (=apoptotic fraction in treated cells − apoptotic fraction in untreated cells).SU-DHL-4 cells  and 1 tablet complete EDTA-free protease inhibitor ) for 30 min on a head-over-head rotor. Cell lysates were centrifuged for 5 min at 4000 × g and analyzed by western blotting as previously described41.SU-DHL-4 cells were washed with phosphate-buffered saline and incubated at 4 °C with lysis buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1.5 mM MgClt test or a one-way analysis of variance (ANOVA) with a post hoc Tukey’s test, as appropriate. Differences were considered significant at p < 0.05.Results are expressed as the mean ± SEM. The number of independent experiments is always indicated. Significance was determined using a two-tailed paired Student’s


In vivo effects of drugs are complicated by several factors and little is known of the direct effects of statins on pulmonary arteries. This study investigated the direct effects of simvastatin on pulmonary arteries isolated from rats with or without monocrotaline-induced PAH. Simvastatin suppressed contractions evoked by the thromboxane A2 receptor agonist U46619 (30 nM), the α1–adrenergic agonist phenylephrine (5 μM) and KCl (50 mM) by ~50% in healthy and diseased arteries, but did not reduce contraction evoked by sarco/endoplasmic reticulum ATPase blockers. It relaxed hypertensive arteries in the absence of stimulation. Removing the endothelium or inhibiting eNOS did not prevent the inhibition by simvastatin. Inhibiting RhoA/rho kinase (ROCK) with Y27632 (10 μM) suppressed contractions to U46619 and phenylephrine by ~80% and prevented their inhibition by simvastatin. Y27632 reduced KCl-induced contraction by ~30%, but did not prevent simvastatin inhibition. Simvastatin suppressed Ca2+ entry into smooth muscle cells, as detected by Mn2+ quench of fura-2 fluorescence. The calcium antagonist, nifedipine (1 μM), almost abolished K+-induced contraction with less effect against U46619 and phenylephrine. We conclude that simvastatin relaxes pulmonary arteries by acting on smooth muscle to interfere with signalling through G-protein coupled receptors and voltage-dependent Ca2+ entry. Its actions likely include inhibition of ROCK-dependent Ca2+ sensitisation and voltage-gated Ca2+ channels. These are likely to contribute to the beneficial effects of simvastatin in animal models of PAH.Simvastatin reduces pulmonary arterial pressure and right ventricular hypertrophy in animal models of pulmonary arterial hypertension (PAH) and is thought to restore endothelial dysfunction.  To minimise animal use, multiple vessel segments from the 3 largest lung lobes were used for different experiments performed simultaneously. Vessels were left for >20 min to recover from dissection and stability checked by challenging three times with 50 mM KCl. Simvastatin (5 μM) was added to some chambers for 1 hr before experiments started and remained throughout the experiment. Vessel constriction was induced with 50 mM KCl, the α(109 nM) . ROCK acle cells .−8–10−5 M) was applied to evoke endothelium-dependent relaxation and sodium nitroprusside  to deliver NO directly to smooth muscle: drugs were applied to vessels constricted with 30 nM U46619 once tension reached a plateau. In some experiments, endothelial function was blocked by passing distilled water through the vessel lumen or applying N(G)-nitro-L-arginine methyl ester  to inhibit eNOS activity. Loss of function was confirmed by the absence of vasodilation to 10 μM ACh. Experiments employed intact arteries unless otherwise indicated.Acetylcholine , cOmplete protease inhibitor cocktail  and PhosSTOP phosphatase inhibitor cocktail . Homogenates were centrifuged at 16,000g for 5 min at 5°C and the supernatant collected for analysis. ROCK activity was determined using an immunoassay kit that measured phosphorylation of the Rho kinase-specific substrate, MYPT-1 (Merck), according to the manufacturer’s instructions. Lysate activity was measured by comparison to a ROCK2 standard provided in the kit and expressed relative to total protein, measured using the Bio-Rad 2+-sensitive fluorescent indicator fura-2 AM . Fluorescence was excited in individual cells by illuminating alternatively at 340 nm and 380 nm  using a monochromator-based fluorescence system , integrated with a Nikon Eclipse inverted microscope. The fluorescence emitted at 510 nm was recorded using a photomultiplier tube (Cairn Research) and pClamp software . Cells were incubated for 1 hour with vehicle or 5 μM simvastatin (during and after fura-2 loading) before experiments began. To evoke depolarisation and Ca2+ channel activation without Ca2+ influx, cells were incubated with PSS modified to contain 50 mM K+ and no added Ca2+. Osmolarity was maintained by equimolar substitution of Na+. These conditions evoke smooth muscle depolarisation to open Ca2+ channels while minimising Ca2+-dependent fluorescence. Cell fluorescence was recorded for 1 min before adding 1 mM MnCl2, allowing Mn2+ to enter the cell and quench fura-2 fluorescence [2+ quenching was measured from the fluorescence excited at 380 nm.Pulmonary artery smooth muscle cells were isolated as previously described  then increscence . The ratmax) and drug concentration producing half of the Emax (EC50) were estimated from individual concentration–response relationships. Mean concentration-response relationships are plotted with nonlinear regression fits of the Hill equation  superimposed. Values are expressed as mean ± standard error of mean (S.E.M.) for the number of animals (n) used in each experiment. Data were tested for normality using the Shapiro-Wilk and Kolmogorov-Smirnov tests and comparisons made with parametric or non-parametric tests as appropriate. Significance was corrected for multiple comparisons using the test recommended by Prism for each data set. Tests used are indicated next to the comparisons made. P<0.05 was considered statistically significant.The data and statistical analyses comply with the recommendations on experimental design and analysis in pharmacology . ContracU46619 was obtained from Enzo Life Sciences . All other drugs and reagents were from Sigma-Aldrich .50 values of 31 ± 5 nM and 61 ± 20 nM. The maximum contraction to phenylephrine dropped from 2.2 ± 0.1 mN (control) to 1.0 ± 0.4 mN  after simvastatin treatment, with EC50 values of 112 ± 8 nM and 187 ± 59 nM, respectively. Simvastatin (5 μM) had no effect on baseline tension when applied to control vessels, but in vessels from MCT rats it produced a concentration-dependent loss of tone (PAs from control and MCT-treated rats contracted upon application of U46619 (30 μM), in the absence or presence of 5 μM simvastatin A and 2B. of tone .Removing the endothelium did not prevent simvastatin from inhibiting KCl, U46619 or phenylephrine contractions, which were all roughly halved . L-NAME As shown in 2+ influx. Nifedipine was less effective in control vessels stimulated by PE or U46619, where it caused around 50% inhibition, although its effects were enhanced in arteries from MCT rats , almost abolished the contraction to KCl , confirmMCT rats A. This dothelium . In separescence , implyinvastatin .Inhibition of SERCA by 3 μM thapsigargin  or 30 μM50 = 6.9 ± 0.1 and Emax = 71 ± 11% (n = 6), values that were not significantly changed by simvastatin . In contrast, the maximum relaxation achieved by 10 μM ACh in MCT vessels was only 7 ± 10% (n = 9), but it reached 34 ± 4%  after simvastatin treatment.Acetylcholine (ACh) evoked concentration-dependent relaxation of U46619-constricted vessels from control animals A but had50 = 7.8 ± 0.2 and Emax = 87 ± 4% (n = 6) at ~1 μM, and following simvastatin treatment the values were unchanged . In MCT arteries, the SNP concentration-response curve was shifted to higher concentrations. As the maximum was not always clear, sensitivity to SNP was compared in control and MCT vessels using the SNP concentration that gave 50% reduction of the U46619-induced tone: 15 ± 0.4 nM (n = 6) in control vessels and 104 ± 2 nM (n = 8) in MCT vessels. Thus, there was a 7-fold loss of potency in vessels from MCT-treated rats (Mann-Whitney U Test). Simvastatin significantly inhibited responses to SNP in MCT vessels (two-way RM ANOVA), but only at SNP concentrations ≥100 nM.Relaxation to SNP was also reduced in MCT vessels, but not restored by simvastatin B. SNP re1-adrenoceptor activation (phenylephrine), TP receptor activation (U46619) and eNOS inhibition (L-NAME). Simvastatin had the same effects on healthy and diseased vessels, except that it additionally reduced basal tension in diseased arteries. The inhibition of vasoconstrictor responses did not require an intact endothelium or functional eNOS, but resulted from direct interaction with smooth muscle cells. The ability of Y27632 to both mimic and prevent the effects of simvastatin on phenylephrine and U46619-induced contractions indicates the involvement of ROCK inhibition. This is supported by the loss of ROCK activity in simvastatin-treated arteries. The relative resistance of KCl-induced contraction to Y27632, despite inhibition by simvastatin, implies an additional action of simvastatin, independent of ROCK activity. The additional action could be inhibition of L-type Ca2+ channels, as suggested by simvastatin inhibition of Mn2+ quenching of fura-2 fluorescence. In contrast, simvastatin failed to inhibit contraction resulting from the blockade of SERCA (thapsigargin and CPA), which raises myocyte Ca2+ concentration via a mechanism that bypasses Ca2+ channels or receptor activation [Exposure of rat pulmonary arteries to simvastatin caused pronounced inhibition of vasoconstrictor responses to smooth muscle depolarisation (KCl), α1-adrenergic receptors are G-protein coupled receptors (GPCRs). Both couple to Gq/11 [2+-sensitisation of the contractile proteins, which maintains contraction during agonist stimulation [2+-evoked contraction caused by inhibiting SERCA. Statins can disrupt GPCR signalling by interfering with the prenylation of Gγ subunits [TP and αto Gq/11 , which pto Gq/11 ,35 and dto Gq/11 ,37. Thusto Gq/11 . By intemulation . Simvastsubunits  and this2+-sensitisation is less important for contraction mediated by smooth muscle depolarisation. This was confirmed by the smaller effect of Y27632 on responses to KCl compared with receptor activation, in agreement with previous studies [2+ influx through voltage-gated, L-type Ca2+ channels. Consequently, the L-type Ca2+-channel blocker, nifedipine, almost abolished responses to KCl, while reducing phenylephrine and U46619 contraction by only 50% in control arteries. Simvastatin slowed the rate at which Mn2+ quenched fura-2 fluorescence in K+-depolarised pulmonary artery smooth muscle cells. As Mn2+ can only enter the cell to quench fluorescence when Ca2+ channels are open [2+ influx. Thus, simvastatin most likely inhibited L-type Ca2+ channels as reported previously for basilar artery smooth muscle cells [Ca studies ,11. Addiare open , this inle cells , cardiacle cells  and pancle cells .2+ entry through L-type channels have been shown to mediate contraction induced by L-NAME [2+ channels and RhoA/ROCK-mediated Ca2+ sensitisation. Thus, nifedipine and Y27632 relaxed unstimulated arteries from MCT-treated animals, while having no effect on healthy vessels. The raised basal tone in hypertensive arteries was also suppressed by simvastatin, consistent with it inhibiting ROCK and Ca2+ channels.Simvastatin also inhibited contractile responses to L-NAME, which occur only in vessels with an intact endothelium –44. L-NAy L-NAME ,45, so iy L-NAME ,44; thisy L-NAME  and inhiy L-NAME , implyinWe found no evidence that simvastatin suppressed contraction by stimulating AMPK and constitutive eNOS activity, as proposed to explain similar actions on mesenteric arteries . While sin vivo studies , simvastatin exposure appeared to improve the dysfunctional endothelium that developed in PAH [Consistent with the findings of d in PAH –20, althd in PAH . Longer 2+ sensitisation and inhibition of Ca2+ entry through L-type Ca2+ channels.The main effect of acute simvastatin exposure is to promote vasodilation through a direct interaction with smooth muscle. Simvastatin prevents vasoconstrictors from contracting smooth muscle and relaxes hypertensive arteries even in the absence of a constrictor stimulus. We propose that these effects are mediated by loss of ROCK-dependent CaS1 Fig(PDF)Click here for additional data file.


Treponema pallidum subsp. pallidum dissemination. First, intramuscular injection of the flagellin encoded by the plasmid DNA into New Zealand rabbits elicited both humoral and cellular immune responses. Total IgG production increased in response to flagellin. In addition, serum IFN-γ secretion and CD8+ cells were substantially greater in the rabbits immunized with the plasmid encoding flagellin FlaB3 than those in the rabbits immunized with recombinant flagellin. The flagellin encoded by the plasmid DNA induced significant upregulation of serum IL-6 and IL-8 compared to that of the control rabbits. Subsequently, intradermal challenge of the vaccinated New Zealand rabbits with 1 × 107T. pallidum resulted in a significant reduction of the bacterial organ burden in the blood, liver, spleen, and testicles in the flagellin plasmid DNA-vaccinated rabbits. Furthermore, the histopathological analysis demonstrated that the rabbits immunized with the plasmid DNA-encoded flagellin (FlaB3) showed better immune protection. These findings provide evidence that plasmid DNA-encoded flagellin (FlaB3) may be useful as a potential immunization route for future development of a vaccine to inhibit T. pallidum dissemination in related animals.Plasmid DNA encoding flagellin FlaB3 was used as a vaccination candidate for the evaluation of immunogenicity and protection against  Treponema pallidum subsp. pallidum  and is most commonly reported in low- and middle-income countries2. Human infection with T. pallidum usually occurs via mucous membranes or injured skin4. The reported per annum estimated frequency of infectious syphilis is 36 million cases and more than 11 million new infections5. However, the number of cases reported is believed by some to be a significant underestimate. Thus, this disease represents an important global public health burden6.As a sexually transmitted infection, syphilis typically occurs as a result of infection with T. pallidum have included an inactivated vaccine, live attenuated vaccines, recombinant protein vaccines and DNA vaccines9. γ-Irradiated T. pallidum, which is an attenuated syphilis spirochete, has been used as an attenuated vaccine to induce a protective antibody response against T. pallidum in vivo, although the relative efficacy of this vaccine has not been well defined in animal trials8. The T. pallidum laminin-binding adhesin Tp0751 has been reported to induce high titers of specific antibodies and results in delayed lesion development, inhibition of T. pallidum dissemination and increased cellular infiltration at lesion sites10. In another example, sera from immunized rabbits were reported to have anti-endoflagellar antibody titers that were fivefold greater than the serum titers from infected immune rabbits and thus provided some level of passive protection12. Typically, the use of recombinant antigens as vaccination candidates induces powerful humoral antibodies but may not induce some form of cell-mediated immune response13. However, sera from T. pallidum-infected patients display a superiority of IL-2, IFN-γ, and TNF-α (Th1 or Th1-like cytokines) production and low IL-4 and IL-5 (Th2 or Th2-like cytokines) levels14. Due to the presence of protective humoral antibodies, T. pallidum is able to invade the skin or genital mucosa, penetrate the tissue barrier, and gain rapid entry to the bloodstream3. If not completely eliminated from an infected individual, T. pallidum can cause diverse clinical manifestations, including skin rashes, meningitis, ocular disease, and cardiovascular and neurological complications15. More seriously, T. pallidum can permeate both the placental and blood–brain barriers and increases the risk of acquiring and transmitting human immunodeficiency virus (HIV)17. Therefore, effective syphilis vaccine development needs to target treponemes within the vasculature to inhibit T. pallidum seeding into a wide variety of tissues and organs9.To date, developed vaccines against 20. Our previous study showed that T. pallidum flagellins upregulated IL-6 and IL-8 via the TLR5 and MAPK/NF-ƙB signaling pathways in THP-1 cells21. In another study, T. pallidum flagellin  stimulation augmented the production of MMPs  in HaCaT cells via the MyD88-dependent TLR5 signaling pathway22. Furthermore, the filament core protein FlaB3 is homologous to the N-terminal and C-terminal regions of the flagellins of Salmonella enterica and Escherichia coli24. With these encouraging observations, flagellin has been targeted as an immunogenic agent for the development of a vaccine to inhibit T. pallidum dissemination.Bacterial flagellin has emerged as a potent immunostimulator that is capable of activating the MyD88-dependent signaling pathway and NF-κB-mediated production of proinflammatory cytokines and nitric oxide (NO) in vivo, which contribute to immediate clearance of pathogens from the host25. Nucleic acid vaccines against hepatitis B virus, influenza virus, and Mycobacterium tuberculosis are in the preclinical stage of evaluation or have entered clinical trials28. Hence, this study was designed to evaluate the relative immunogenicity and inhibition of T. pallidum dissemination in New Zealand rabbits by intramuscular injection of a plasmid DNA vaccine carrying the T. pallidum flagellin flaB3 gene.DNA vaccines are capable of inducing long-term antibody responses, which are the principal agents of immune protection against most viruses and bacteriapcDNA3/FlaB3 was transfected into HeLa cells. After 48 h, the total proteins from these transfected cells reacted with anti-flagellin sera by western blotting and gave a specific immunoreactive band of 34 kDa Fig. . The resT. pallidum infection33. T cells of both the CD4 and CD8 lineages are needed for protective anti-T. pallidum immunity32. Hence, we assessed the serum levels of IFN-γ and CD8+ cells in the pcDNA3/FlaB3-immunized rabbits  per ml in 0.9% saline at each of eight sites on their shaved backs. Previous syphilis studies have found that monitoring chancre development provides a metric of vaccine efficacy9. Hence, skin lesions were observed and measured in the infected sites every 3 days. On day 14, lesions (rash and ulcerative) were present on all animals, but the control animals presented 100% lesion induration on days 6–8, whereas the animals immunized with pcDNA3/FlaB3 or FlaB3 matched the level of induration observed in the control animals until days 11–12  was used to assess the T. pallidum burdens in the primary lesion sites, blood, spleen, liver, and testicles on day 21 postinoculation. The results showed a slight trend toward increased T. pallidum concentrations at the primary lesion sites in the flagellin plasmid DNA-vaccinated rabbits compared to those of the recombinant protein-vaccinated rabbits . Our results suggest that flagellin plasmid DNA immunization in rabbits is an effective vaccine strategy against T. pallidum dissemination and is significantly better than vaccination with the recombinant protein.To validate whether flagellin plasmid DNA immunization conferred protection against its Fig. . SomewhaT. pallidum. Cellular infiltration of the skin  was significantly higher among the rabbits immunized with pcDNA3/FlaB3 than among the rabbits immunized with either recombinant flagellin or vaccinated with vector DNA alone , flagellin can be recognized by the cell surface through TLR5 and the cytosolic NOD-like receptor protein 4 (NLRC4) inflammasome receptor NAIP5, which contributes to activation of inflammatory responses and secretion of proinflammatory cytokines and chemokines during bacterial infections48. The T. pallidum flagellin induced MMP-9 and MMP-13 expression in keratinocytes, which are involved in activation and regulation through the NF-κB and MAPK signaling pathways22. These results suggest that the T. pallidum flagellin may be crucial for triggering inflammation, which may contribute to skin inflammatory responses and keratinocyte-mediated ECM degradation during T. pallidum infection. Hence, the ability of FlaB3 to stimulate nonspecific host resistance may be relevant for anti-T. pallidum protection. In particular, inflammatory cytokines induced by the plasmid encoding the FlaB3 gene may significantly contribute to protection due to their well-known ability to activate host phagocytes for efficient control of bacterial invasivity36. Histological analysis of the primary lesion sites in the control and immunized animals revealed a significant increase in cellular infiltration in the DNA-vaccinated rabbits at day 21 postinfection. Strikingly, abundant lymphocytes, macrophages and plasma cells were discovered within the primary lesion sites. Significant inflammatory responses were observed in the rabbits vaccinated with vector DNA alone but never in the testicular tissues of the rabbits immunized with pcDNA3/FlaB3 and recombinant flagellin. In line with the trend observed by darkfield microscopy, the primary lesions from the immunized animals had few treponemes present per field, and these organisms were nonmotile. The results of our study also provide evidence that the FlaB3-specific antibodies produced by these immune cells facilitate T. pallidum retention at the primary lesion sites by inhibiting treponemal dissemination. In particular, this protective effect was more pronounced in the New Zealand rabbits immunized with pcDNA3/FlaB3 than in the rabbits immunized with recombinant flagellin. However, these histological analyses were performed at a single time point and therefore did not represent the dynamic properties of cellular infiltration.Phagocytosis of T. pallidum pathogenesis is vascular dissemination, and therefore effective vaccination needs to target treponemes within the vasculature to inhibit T. pallidum seeding into secondary sites9. Analysis of the treponemal burden in the blood, liver, spleen, and testicles using qPCR revealed that pcDNA3/FlaB3 immunization inhibited T. pallidum invasion into distant tissue sites. In particular, the most significant decrease in the T. pallidum DNA concentration was observed in the blood, spleen, or testicles between the immunized and control animals. Previous investigations have confirmed that Tp0751 is capable of mediating spirochete attachment to endothelial cells when heterologously expressed by a noninfectious Borrelia burgdorferi strain49. Moreover, Lithgow et al. reported that animals immunized with Tp0751 displayed attenuated lesion development, inhibition of T. pallidum dissemination and increased cellular infiltration at lesion sites10. Our work reported here used a recombinant flagellin to stimulate augmented production of MMPs which are involved in the degradation of various components of the extracellular matrix (ECM) to facilitate cell migration and invasion22. Therefore, the qPCR results clearly demonstrate that pcDNA3/FlaB3 is a promising vaccine candidate, because immunization with this protein significantly reduces T. pallidum dissemination within the host. However, these results must be interpreted with caution because the qPCR is capable of detecting DNA from both live and dead organisms.The key component in T. pallidum-elicited infections may convert from an initial acute infection to a form of long-term latent infection by this microbe50. If the bacterial loads present in the liver, spleen, blood, or testicles in this study are not be removed completely following initial infection, the limited survival of T. pallidum in the organs will be a negative factor. Thus, further investigation is needed to evaluate the relative antimicrobial activity among immunized rabbits with different genetic backgrounds.Notably, the statistical data in this study are not generally representative due to the small sample size and the outbred nature of the rabbits used as the animal model. However, the findings from this study can be used to guide experiments in the future as a parameter to determine an adequate sample size. Some T. pallidum was constructed to express the recombinant flagellin protein in E. coli BL21 (DE3); the protein was expressed and purified as described in a previous report12. The mammalian expression vector pcDNA3/FlaB3 was constructed from pET28a/FlaB3 using appropriate restriction sites flanking the flaB3 gene. The T. pallidum subsp. pallidum, Nichols strain was propagated by intratesticular inoculation of adult New Zealand White rabbits as previously described52. All animal experiments were approved by the Animal Welfare Committee of the University of South China and conducted in accordance with the institutional regulations.The pET28a/FlaB3 plasmid encoding the flagellin gene of TM 2000 Reagent  according to the manufacturer’s instructions. Transfected HeLa cells were harvested and detected using immunoblotting analysis with antibodies against FlaB3. The primary anti-FlaB3 antibodies were provided by the Institute of Pathogen Biology at the University of South China. The secondary antibody was horseradish peroxidase-conjugated secondary goat anti-rabbit IgG . Finally, the ECL Prime Western Blotting Detection Reagent  was used as recommended to visualize the protein bands with a G:BOX Chemi XX9  digital imager.HeLa 229 cells  were transfected with either pcDNA3 or pcDNA3/FlaB3 using the LipofectamineWe used a cohort of eight male SPF New Zealand White rabbits  with negative VDRL and FTA-Ab serology. Two experimental groups of rabbits with three animals per group were injected intramuscularly with either purified recombinant flagellin (150 μg) emulsified in complete Freund’s adjuvant  or pcDNA3/FlaB3 (250 μg) in PBS. Two animals randomly selected from the cohort of eight rabbits were injected intramuscularly with pcDNA3 (150 μg) in PBS. The experimental group rabbit were immunized three times at 2-week intervals. As a control, the data derived from the rabbits injected with PBS gave the same result as the pcDNA3 control group and therefore were not shown in this report. Serum samples were collected by ear vein bleeding 14 days after the commencement of immunization.7T. pallidum subsp. pallidum (Nichols strain) per ml in 0.9% saline in each of eight spots53. The challenge sites were monitored daily for erythema, induration, and ulcerative lesions and measured every 3 days to assess the lesion diameters. Aspirates were taken from the lesions 21 days post-challenge and examined by darkfield (DF) microscopy  for the presence of treponemes.At 21 days after the final immunization and 10 days after the control animals were bled, the rabbits’ backs were shaved, cleansed with 70% ethanol and injected intradermally with 0.1 ml of 1 × 10A small volume (1 ml) of blood was collected from the rabbit ear veins, followed by centrifugation for serum separation. The serum samples were tested for specific antibodies using the indirect ELISA method. Purified recombinant flagellin (pET28a/FlaB3) was used as the coating antigen. Horseradish peroxidase-conjugated secondary goat anti-rabbit IgG (1:1000) was used as the secondary antibody. ELISA was performed to examine the A value at the 450 nm wavelength . Each experiment was repeated three times.The induction of IFN-γ/CD8+ cells was evaluated using a rabbit interferon-γ (IFN-γ) ELISA kit  and a rabbit cluster of differentiation 8 (CD8) ELISA kit  according to the manufacturers’ instructions. The quantity of IFN-γ was reported as pg/ml and CD8+ cells was reported as ng/ml according to the standard curves.The IL-6 and IL-8 concentrations in sera from the immunized rabbits were determined using a specific enzyme-linked immunosorbent assay (ELISA) kit from Cloud Clone Inc. (USA) according to the manufacturer’s instructions. The quantity of each secreted cytokine was reported as pg/ml according to the standard curve.T. pallidum-challenged rabbits using a method adapted from the Qiagen QIAamp DNA Mini Kit . Lysis Buffer  was added to ~25 mg of tissue (~10 mg for spleen) and homogenized using the Qiagen TissueLyser LT operating for 40 s at 30 Hz. Proteinase K (5 mg) was added, and the digestion was performed overnight at 56 °C. Then, the samples were treated with RNase A (400 µg), followed by incubation at 70 °C for 10 min. Three tissue samples from each rabbit organ were analyzed for reproducibility. The tissue extracts were eluted in 100 µl of Gibco PCR Grade Distilled Water (Thermo Fisher Scientific) and stored at −80 °C. DNA was quantified using a Beckman Coulter DU 730 Life Science UV/Vis Spectrophotometer .Genomic DNA (gDNA) was extracted from tissues collected from T. pallidum-challenged rabbit tissues using a SYBR green I assay. The T. pallidum gDNA was quantified using primers targeting a 285-bp region of the endoflagellar sheath protein (flaA) gene (GenBank accession number M63142). The sense primer (5′-AACGCAAACGCAATGATAAA-3′) annealed to bases 475 to 494 and the antisense primer (5′-CCAGGAGTCGAACAGGAGATAC-3′) annealed to bases 738 to 759 of flaA. The rabbit gDNA was quantified with primers targeting a 267-bp region of exon 1 of the collagenase-1 precursor (MMP-1) gene (GenBank accession number M17820). The sense primer (5′-TTGCTTCTTCACACCAGAATGCTGT-3′) annealed to bases 300 to 324 and the antisense primer (5′-GCGTGATCAGGCACTATGTAGCAAT-3′) annealed to bases 542 to 56610. All primers were ordered from Integrated DNA Technologies . The quantitative real-time PCR reactions were performed in 20-μl reaction volumes according to the manufacturer’s instructions . The amount of gDNA isolated was variable between tissue types but was normalized within each tissue type based on the lowest gDNA concentration obtained from the spectrophotometric measurements. A standard curve was created for flaA using a 10-fold serial dilution from 107 to 101 copies of linearized plasmid DNA with an efficiency of 99.2% and an R2 value of 0.990. A standard curve was created for MMP-1 using a twofold serial dilution of rabbit gDNA from 100 to 0.0488 ng μl−1 with an efficiency of 99.1% and an R2 value of 0.995. The assays were run on the LightCycle 96 apparatus . The PCR conditions for flaA were as follows: 95 °C for 8 min, followed by 40 cycles of 95 °C for 15 s, 56 °C for 20 s, and 72 °C for 30 s, an additional extension step at 84 °C for 10 s, and a final denaturation step of 15 s at 95 °C; additionally, a melting curve was performed from 60 to 95 °C over 20 min. The PCR conditions for MMP-1 were as follows: 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, 55 °C for 20 s, and 72 °C for 40 s, and a final denaturation step of 15 s at 95 °C; a melting curve analysis was performed from 60 to 95 °C over 20 min. Each assay was run with the following four controls: (1) no template control; (2) no amplification control (no Taq polymerase); (3) no primer control; and (4) positive control with a known concentration or copy number of rabbit gDNA or linearized flaA plasmid DNA, respectively.Quantitative real-time PCR was performed on gDNA extracted from the Biopsy punch samples (2 mm) were taken from a single lesion and testicle from each rabbit at day 21 postinfection and sent to the University of South China. The samples were fixed in formalin, mounted, bisected, and vertically sectioned with a 4-mm thickness. The samples were processed with hematoxylin and eosin staining for microscopic analysis to determine the abundance of immune cells, including lymphocytes, macrophages, and plasma cells.t-tests and nonlinear regression with an extra sum of squares F-test to investigate any apparent differences between the test and control groups. Differences were considered significant at the P < 0.05 level. The GraphPad Prism 5.0 software  was used for statistical analysis of the data.The data were analyzed using Student’s Fig S1Fig S2Fig S3


Meiosis is an essential cell-division process for ensuring genetic diversity across generations. Meiotic recombination ensures the accuracy of genetic interchange between homolous chromosomes and segregation of parental alleles. Programmed DNA double-strand breaks (DSBs), catalyzed by the evolutionarily conserved topoisomerase VIA -like enzyme Spo11 and several other factors, is a distinctive feature of meiotic recombination initiation. The meiotic DSB formation and its regulatory mechanisms are similar among species, but certain aspects are distinct. In this review, we introduced the cumulative knowledge of the plant proteins crucial for meiotic DSB formation and technical advances in DSB detection. We also summarized the genome-wide DSB hotspot profiles for different model organisms. Moreover, we highlighted the classical views and recent advances in our knowledge of the regulatory mechanisms that ensure the fidelity of DSB formation, such as multifaceted kinase-mediated phosphorylation and the consequent high-dimensional changes in chromosome structure. We provided an overview of recent findings concerning DSB formation, distribution and regulation, all of which will help us to determine whether meiotic DSB formation is evolutionarily conserved or varies between plants and other organisms. In flowering plants, reproductive cells develop in the ‘sporophytic generation’ and then differentiate into the gamete-forming ‘gametophytic generation’ ,2. DurinSaccharomyces cerevisiae (S. cerevisiae), nine DSB accessory proteins have been characterized, namely Ski8, Rec114, Mei4, Mer2, Rec102, Rec104, Mre11, Rad50 and Xrs2 [The programmed induction of DSBs along chromosomes is catalyzed by Spo11, which is a homolog of the A subunit of an archaeal topoisomerase (TopoVI), and this is a general mode for initiating meiotic recombination in fungi, invertebrates, mammals, and plants ,42. Howeand Xrs2 ,44,45,46and Xrs2 ,48,49. Tand Xrs2 ,52.Schizosaccharomyces pombe (S. pombe), seven DSB accessory proteins have been identified in addition to the Spo11 ortholog Rec12 [S. cerevisiae, such as Rec14 (Ski8), Rec24 (Mei4), Rec7 (Rec114) and Rec15 (Mer2), while the other three, Rec6, Rec10 and Mde2, have no clear homologs in S. cerevisiae or other organism [S. cerevisiae and S. pombe. Rec12, Rec14 and Rec6 form the DSB catalytic core (commonly known as DSBC) in S. pombe [S. cerevisiae [S. pombe, Red1 is dispensable in S. cerevisiae [S. cerevisiae, is only required for DSB repair and not for DSB induction in S. pombe [In og Rec12 . Some oforganism ,56,57,58S. pombe . Rec7, Rrevisiae ,56,59,60S. pombe .S. cerevisiae, and this subcomplex co-localizes with chromosome axes via the interaction between IHO1 and HORMAD1, which are key components of the cohesin complex [In mice, except for the conserved protein SPO11, another five proteins are indispensable for DSB formation, including REC114, MEI4, IHO1, MEI1 and TOPOVIBL ,63. The  complex ,65,66. M complex ,68,69. T complex .Arabidopsis thaliana (Arabidopsis) has three Spo11 homologs, namely AtSpo11-1, AtSpo11-2 and AtSpo11-3. Two of them, AtSpo11-1 and AtSpo11-2, are essential for DSB formation [Arabidopsis, explaining the non-redundant nature between AtSpo11-1 and AtSpo11-2 [The model plant ormation ,72,73,74ormation ,76. TopotSpo11-2 ,77. In atSpo11-2 ,78,79. BtSpo11-2 ,80,81. HtSpo11-2 .comet/OsBVF1. Although the participation of OsSPO11-1, OsSPO11-4 and OsMTOPVIB in DSB formation is conserved, as is their homologs in other species [Arabidopsis, which are SDS and PCH2, respectively, are not required for DSB formation [comet was first identified to be involved in the spindle assembly checkpoint in human cells [comet, also named OsBVF1 in an independent study, was unambiguously demonstrated to be required for DSB formation [In rice, six DSB-forming proteins have been identified so far, namely OsSPO11-1, OsSPO11-4, OsMTOPVIB, OsSDS, OsCRC1 and P31 species ,84,85,86ormation ,88. Thesan cells . Howeverormation ,91. TakeArabidopsis  and more than 50 cyclins [Arabidopsis [In  cyclins ,130,131,bidopsis ,131,132,bidopsis ,131. Howbidopsis . SDS, a bidopsis ,132,133.bidopsis ,135. TheS. cerevisiae, Tel1 and the related protein kinase Mec1 act as key factors that synergistically fine-tune the number of DSBs [S. cerevisiae have revealed a cis-regulatory machinery, termed DSB interference, that reduces the frequency of coincident DSBs at the region adjacent to the preexisting DSB [trans-inhibition, defines the ability of a DSB to occur on a single chromosome, and this suppresses DSB designation on its homolog and sister chromatid at the same locus [Once DSBs are made, there are several mechanisms to maintain the proper number of DSBs within a chromosome region ,140,141. of DSBs ,144. Lac of DSBs . In cont of DSBs ,146. Mec of DSBs ,148. In ting DSB ,142,143,ting DSB ,149. On me locus ,150. MorArabidopsis, Tel1 and Mec1 homologs exist, termed ATM and ATR, respectively. However, ATM and ATR play synergetic roles in maintenance of genomic stability in meiotic cells by processing Spo11-dependent DSBs rather than influencing DSB formation [In ormation ,154,155.S. cerevisiae, for example, Spp1, a PHD finger domain protein and Set1 COMPASS complex member, binds to H3K4me2/3 adjacent to DSB-prone sites on loops and interacts transiently with axis-bound Mer2, establishing a spatial linker between DSB loci and the DSB-forming machinery, thus promoting DSB formation on chromosome axes (S. cerevisiae Spp1 [Spatiotemporal control of DSB formation takes place within a specialized chromosomal structure featuring replicated sister chromosomes organized into linear looped arrays that emanate from a central proteinaceous axis ,157,158.ome axes A [158,16iae Spp1 , which ciae Spp1 ,166. Arabidopsis [comet in rice [In plants, although the formal ‘tethered-loop/axis complex’ system remains to be established, there are many indications that this may also be the case. Recently, using super-resolution microscopy, Spo11-1 and DSBs sites were found to be associated with chromosome axes in maize . Additiobidopsis , CRC1 an in rice ,91 and D in rice . HoweverIn recent years, much progress has been made toward understanding meiotic DSB initiation and distribution in plants. These advances allowed researchers to translate acquired knowledge from model species to various crops, which facilitated breeding programs . However


The pressure-volume loop, isolated heart, and Ca2+ transients triggered by field stimulation or caffeine were used to analyze the hemodynamic mechanism of rolipram. The results demonstrated that rolipram  significantly increased the in vivo rat heart contractility by enhancing stroke work, cardiac output, stroke volume, end-systolic volume, end-diastolic volume, end-systolic pressure, heart rate, ejection fraction, peak rate of rise of left pressure (+dp/dtmax), the slopes of end-systolic pressure-volume relationship (slope of ESPVR) named as left ventricular end-systolic elastance, and reduced the slopes of end-diastolic pressure-volume relationship (slope of EDPVR). Meanwhile, the systolic blood pressure, diastolic blood pressure, and pulse pressure were significantly enhanced by rolipram. Also, rolipram deviated normal ventricular-arterial coupling without changing the arterial elastance. Furthermore, rolipram  also exerted positive inotropic effect in isolated rat hearts by increasing the left ventricular development pressure, and +dp/dtmax in non-paced and paced modes. Rolipram (10 μM) increased the SERCA2a activity, Ca2+ content, and Ca2+ leak rate without changing diastolic Ca2+ level. Rolipram had significant positive inotropic effect with less effect on peripheral vascular elastance and its underlying mechanism was mediated by increasing SERCA2a activity. PDE4 inhibition by rolipram resulted in a positive inotropic effect and might serve as a target for developing agents for the treatment of heart failure in clinical settings.This study was designed to investigate the hemodynamic effect of rolipram, a phosphodiesterase type 4 (PDE4) inhibitor, in normal rat hearts both  Over 60 cyclic nucleotide phosphodiesterase (PDE) isozymes have been grouped into 11 classes based on their hydrolysis substrates and biochemical features . Of the 2+ channel  in Shanghai (China) with the catalog number of HY-16900. All other chemicals were purchased from Sigma-Aldrich (USA). Rolipram was dissolved in DMSO at room temperature and used on the same day.max (peak rate of rise of left ventricular pressure), Slope of linear regression’ ESPVR (end-systolic pressure-volume relationship) which is also named as left ventricular end-systolic elastance, Slope of linear regression’ EDPVR (end-diastolic pressure-volume relationship). Furthermore, the systolic blood pressure, diastolic blood pressure, pulse pressure, arterial elastance which is the ratio of end-systolic pressure to stroke volume, and ventricular-arterial coupling were analyzed.The investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health . Male Sprague Dawley rats (300–350 g)  were used in this study. Experiments were performed as previously described . Brieflyex vivo experiments were the same as that of in vivo experiment described above and the methods used were described previously . Rolipram was dissolved in DMSO and then transferred to perfusion solutions to reach the final concentrations of 0.1, 1, and 10 μM, respectively. The control perfusion solutions contained the same concentrations of DMSO as that in rolipram groups. During the experiments, these different concentrations of rolipram from low to high concentration order were added to the perfusion solution and infused via retrograde perfusion of the coronary artery. The hemodynamic parameters of control or rolipram  were recorded when isolated heart contraction reached constant level with rolipram perfusions longer than 10 min for each concentration. Their intraventricular pressures under non-isovolumic condition of contraction were measured by a pressure and volume microtip catheter  inserted into the left ventricle via the left atrium. The hearts were beating spontaneously (non-paced) or stimulated at a rate of 4 Hz (paced). The paced isolated hearts were stimulated between left and right atrium at 5 V. Left ventricular developed pressure was calculated as the difference between the peak systolic pressure and end-diastolic pressure. Also, the heart rate only in non-paced setting and + dp/dtmax were calculated.The selection and anesthesia of rats for eviously . Briefly2 1.8, MgCl2 1.7, NaHCO3 4.4, NaH2PO4 1.5, HEPES 20, Glucose 11 gassed with 95% O2 plus 5% CO2 (pH 7.4 with NaOH). The heart was then perfused with the same buffer with the addition of 2.0 mg/ml collagenase type II and 0.1 mg/ml Protease Type for 50 min. Following removal of both atria and the right ventricle, the left ventricular myocytes were gently separated with forceps in the buffer without collagenase. Rod-shaped non-contracting cells with clear striations were used.For isolation of adult rat left ventricular myocytes, the rat ventricular myocytes were obtained by enzymatic dissociation as previously described . The exc2+ transient recording triggered by field stimulation, adult rat left ventricular myocytes were loaded with the membrane-permeable acetoxymethyl ester form of the fluorescent Ca2+ indicator Fluo-4 AM (5 μM) for 30 min at 37°C. Fluo-4 was excited at 488 nm  and emitted fluorescence measured with a 515-nm-long pass filter. The region of interest was restricted to a single cell with the aid of an adjustable window. The Ca2+ transient amplitudes were calculated as a difference of the peak and diastolic fluorescent values. Background fluorescence levels were used to correct raw fluorescence data. Ca2+ spark fluorescent intensity of Fluo-4 loaded in myocytes was recorded at imaging frequency of 100 Hz during electrical pacing  with two platinum electrodes. The extracellular solution contained (in mM): NaCl 117, KCl 5.7, NaHCO3 4.4, MgCl2 1.7, HEPES 20, Glucose 20, Taurine 20, CaCl2 1.8, pH 7.4 with NaOH. After the constant imaging of control was recorded, rolipram (10 μM) in the extracellular solution was perfused for longer than 5 min and then its constant Ca2+ transient effect was recorded. Images were recorded with an ANDOR ZYLA-5.5-CL3 CCD camera  connected to an inverted microscope  that was synchronized by a real-time analog-digital processor unit and Meta fluor acquisition software .For Ca2+ transient involves two components, which are a fast component dominated by sarcoplasmic reticulum (SR) Ca2+-ATPase type 2a (SERCA2a) and slow component dominated mainly by Na+/Ca2+ exchanger (NCX) and sarcolemmal Ca2+-ATPase (PMCA). Isolation of two components was analyzed by the theory and method of plasma concentration-time curve with two exponential fitting following intravenous administration of a drug in human.The decaying phase of Ca2+ transient by field stimulation is mediated mainly by SERCA2a, NCX, and PMCA. The NCX and PMCA control the decaying phase of caffeine-evoked Ca2+ transient in normal Tyrode’s solution (NT) in which caffeine prevents the effect of SERCA2a on SR Ca2+ concentration gradient formation by widely opening RyR2. Also, PMCA function can be isolated from decaying phase of the caffeine-evoked Ca2+ transient in the condition of Na+, Ca2+-free solution which deactivates NCX activity. The rate constants of decline of caffeine-evoked Ca2+ transient in normal Tyrode’s solution and Na+, Ca2+-free solution were used to analyze the functions of mixed NCX plus PMCA and PMCA alone. The detailed methods were previously described  was perfused to myocytes in normal Tyrode’s solution to obtain the decaying rate constant of combined activity of NCX and PMCA. To calculate the decaying rate constant of PMCA alone, Ca2+ leak rate, and Ca2+ content, Na+,Ca2+-free solution was perfused before caffeine (20 mM) perfusion. The normal Tyrode’s solution contained (in mM): NaCl 134, KCl 4, MgCl2 1, HEPES 10, Glucose 11, CaCl2 1, adjusted to pH 7·4 with NaOH. The Na+, Ca2+-free solution contained (in mM): LiCl 130, HEPES 10, Glucose 11, MgCl2 1, KCl 4, adjusted to pH 7.4 with KOH. For control experiment, normal Tyrode’s solution was used to record functional isolations of NCX and PMCA. Rolipram (10 μM) was added to recording chamber for at least 10 minutes to maximize its effects on NCX and PMCA. The effects of control or rolipram on combined function of NCX plus PMCA or PMCA only were performed in different group cells and t-test was used to analyze significant differences.The decaying phase of Caescribed . Brieflyt-test and one-way ANOVA were used as appropriate and data analysis was performed using SPSS 11.0. Differences with p < 0.05 were deemed statistically significant.All values are expressed as mean ± SEM. max, and decreased the end-systolic volumes, end-diastolic volume compared with control  without significantly changing end-diastolic pressure. Also, the slope of ESPVR (Ees) was increased and the slope of EDPVR was reduced as shown in To examine the hemodynamic effects of rolipram on the rat heart and peripheral vessels, the pressure-volume loop (P-V loop) technique was used to measure the left ventricular pressure-volume and aortic pressure simultaneously. Rolipram  significantly enhanced the stroke work, cardiac output, stroke volume, end-systolic pressure, heart rate, ejection fraction, and +dp/dtmax in non-paced mode in a concentration-dependent manner. In paced mode, rolipram  also increased in a concentration-dependent manner the left ventricular developed pressure and +dp/dtmax with a fixed heart rate of 240 beats per min.To further investigate the direct effect of rolipram on the heart, the isolated non-paced and paced rat hearts were used to analyze the inotropic and chronotropic effects of rolipram. The pacing was used to rule out the frequency-induced inotropic effect and 4-Hz pacing was chosen to match the non-paced heart rate as shown in 2+ transient from rat left ventricular myocytes without changing the diastolic baseline fluorescence intensity  as shown in Rolipram (10 μM) significantly enhanced the amplitude of Ca2+ transient, caffeine-induced Ca2+ transient experiment was performed. In this condition, SERCA2a activity was abolished due to the opening of RyR2 by caffeine and the decaying phase mainly was mediated by NCX and PMCA. The result in NCX + PMCA) from 0.190 ± 0.006 in control to 0.190 ± 0.008 . The representative curves of caffeine-induced Ca2+ transients in normal Tyrode’s solution before and after rolipram (10 μM) injection are shown in To further analyze the decreased rate constant of NCX and PMCA activities in field stimulation-induced Ca+ and Ca2+-free extracellular solution was used to block the activity of NCX. In this condition, the Ca2+ transient decaying phase is mainly mediated by PMCA. Rolipram (10 μM) had no effect on PMCA activity based on insignificant rate constant (rPMCA) change from 0.025 ± 0.002 in control to 0.025 ± 0.002  as shown in 2+ content from 559.0 ± 40.2 in control to 662.0 ± 44.2  and enhanced the diastolic Ca2+ leak rate from 6.9% ± 0.3 in control to 9.1% ± 0.7 . The representative Ca2+ transient curves induced by caffeine in Na+ and Ca2+-free extracellular solution are presented in To further isolate the activity of PMCA by rolipram from combined activity of NCX and PMCA, the Na0 ± 44.2   are expressed in rat heart . Althougbstrates . In thisbstrates .in vivo positive inotropic effect of rolipram is under the condition of basal β-adrenergic tension by neurohumoral regulation. To elucidate whether this positive inotropic effect is independent of basal β-adrenergic stimulation, the isolated rat heart perfusion experiment was used. The results demonstrated that rolipram significantly enhanced rat left ventricular developed pressure, heart rate, and +dp/dtmaxin vitro in a concentration-dependent manner. This effect occurred in both non-paced and paced models, and was independent of heart rate as reported  and NCX . However, RyR2 activity which mediates the Ca2+ transient rise phase was not analyzed due to the limitation in this study with slow sampling of 10 ms. Even though, the peak shape of field stimulation induced-Ca2+ transient demonstrated that there was a sharp peak in control and a blunt one in rolipram-treated groups (data was not shown). The peak shape is determined by decay “tail” RyR2 Ca2+ release, Ca2+ leak, and start of SERCA2a activity. The underlying mechanism still needs further investigation. Many studies have reported that PDE4 inhibition increased the PLB phosphorylation which enhances the activity of SERCA2a and RyR2 phosphorylation level (2+ content of cytoplasm (To investigate the mechanism underlying the positive inotropic effect, the Ca channel . Third, on level . Moreoveytoplasm . In our in vivo and in vitro and its underlying mechanism was mediated by enhancing the SERCA2a activity, however, there are still a few limitations in this study. First, PDE4 constitutes a large fraction of the total PDE activity in rodent heart than in human heart (Although this study addressed the inotropic effects of rolipram both in an heart , suggestin vivo and in vitro without insignificant peripheral arterial elastance, which resulted in the mismatched ventricular-arterial coupling in normal rats. The inotropic effect was mediated by Ca2+ handling in which rolipram enhanced SERCA2a activity and Ca2+ leak and reduced NCX and PMCA activities. However, rolipram had no direct effects on rate constants of NCX and PMCA. These results indicated that this PDE4 inhibitor may serve as a potential positive inotropic agent for the treatment of heart failure.In conclusion, rolipram, a PDE4 inhibitor, exerted positive inotropic effect both This manuscript contains previously unpublished data. The name of the repository and accession number are not available.LC, HH, and MX contributed to study design. WL, RW, and KC carried out literature research. HH, MX, LG, WZ, XZ, and YW performed experiments. HH and MX contributed to data analysis and photographs analysis. LC contributed to manuscript preparation, and MB contributed to manuscript revision.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.