The epicardium, the outer layer of the heart, has been of interest in cardiac research due to its vital role in the developing and diseased heart. During development, epicardial cells are active and supply cells and paracrine cues to the myocardium. In the injured adult heart, the epicardium is re-activated and recapitulates embryonic behavior that is essential for a proper repair response. Two indispensable processes for epicardial contribution to heart tissue formation are epithelial to mesenchymal transition (EMT), and tissue invasion. One of the key groups of cytokines regulating both EMT and invasion is the transforming growth factor β (TGFβ) family, including TGFβ and Bone Morphogenetic Protein (BMP). Abundant research has been performed to understand the role of TGFβ family signaling in the developing epicardium. However, less is known about signaling in the adult epicardium. This review provides an overview of the current knowledge on the role of TGFβ in epicardial behavior both in the development and in the repair of the heart. We aim to describe the presence of involved ligands and receptors to establish if and when signaling can occur. Finally, we discuss potential targets to improve the epicardial contribution to cardiac repair as a starting point for future investigation. The epicardium, a mesothelial layer covering the heart, has been recognized as a distinct structure for a long time, whilst its origin and function remained uncertain. Until halfway through the 20th century, the general consensus was that epicardial cells originated from the myocardium. Even though Kurkiewicz  discoverIn the adult mammalian heart, the epicardium as a single mesothelial cell layer displays little activation. Interestingly, this ‘quiescence’ is not necessarily indefinite. An important observation was made in 2006 when Lepilina et al. found that injury reactivated the epicardial layer in the zebrafish heart. This process resembled embryonic epicardial activation, and was shown to be essential for regeneration of the adult zebrafish heart . To furtDuring cardiac repair in organ-regenerating animals, e.g., the newt and zebrafish, several vital processes such as cardiomyocyte proliferation , and bloThe TGFβ family is a large group of proteins named after the cytokine that was described first , the polTGFβ family ligands are secreted as a complex where the active growth factor is bound to a prodomain. TGFβ family members can signal in a autocrine or paracrine fashion. Upon secretion, TGFβ ligands can be stored in the extracellular matrix (ECM) by binding to heparin, or to latent TGFβ binding protein (LTBP), thereby preventing the association of the ligand with its receptor and controlling ligand bioavailability (reviewed in ). ActivaAfter the ligands are released from the ligand binding proteins, they bind to a TGFβ family receptor complex (reviewed in ). These Of course, there are exceptions to the general division in a TGFβ and a BMP cluster; in some cases, BMPs can induce SMAD2/3 phosphorylation or TGFβ can induce SMAD1/5/8 phosphorylation ,38. ThisBesides signaling in a SMAD-dependent manner, TGFβ is also able to exert biological effects by activation of SMAD-independent pathways see . One of Overall, the TGFβ family of signaling components provides a broad range of responses that can be regulated on multiple levels. The highly regulated availability of ligands, and the presence of specific combinations of receptors on the cell surface determine if a cell is able to activate the signaling cascade. On top of this, type III receptors and interaction with SMAD-independent pathways can modulate the signal.As mentioned previously, the epicardium plays an essential role in the formation of the heart. Cardiac development starts with the formation of a bilayered linear heart tube consisting of a myocardial layer lined by the endocardium on the luminal side. The heart tube follows a sequence of looping events during embryonic day 9 (E9) in the mouse, which corresponds to week 3–4 of human development, ultimately resulting in the formation of a four chambered heart. From E9.5 onwards, cells from the pro-epicardium (PE), a cell cluster located at the venous inflow tract, migrate towards the heart and proliferate to cover the bare myocardium with a third cardiac layer: the epicardium . By E11.In the adult heart, the epicardium does not display epicardial activation markers WT1 and RALDH1 and 2 ,56, suggAppreciating that the epicardium can be stimulated to improve cardiac regeneration led to a focus on pathways involved in the main features of epicardial behavior: EMT and invasion. As was introduced before, key players in regulating these processes are members of the TGFβ family. Given the distinction between TGFβ and BMP signaling, we describe their possible regulation within the epicardium separately, starting with TGFβ signaling.Tgfb2 mRNA is present as early as E9.5 in the PE and remains detectable in the first epicardial cells that appear on the outside of the myocardium at E10.5. A clear epicardial mRNA expression pattern of Tgfb2 is maintained until E12.5, after which it starts to decline. A similar expression pattern was observed in the epicardium of chick embryos at a comparable developmental stage [Tgfb3 is not observed anywhere in the heart at early developmental stages. However, from E11.5 onwards, when the epicardium is established and starts to participate in the formation of the heart, Tgfb3 mRNA expression increases and is pan-epicardially expressed [Tgfb2 and -3, no obvious expression for Tgfb1 was reported in the ventricular epicardium, but Tgfb1 mRNA was found to be localized to the epicardium of the AV sulcus [The first requirement for TGFβ signaling to occur in the epicardium is the presence of the actual proteins. There are three TGFβ ligands, TGFβ1, -2, and -3, that are quite similar in structure and function. However, each of them has a specific spatiotemporal gene expression pattern during cardiogenesis  see Fig. In the al stage . Tgfb3 ixpressed . TGFβ3 hxpressed . In contV sulcus . RemarkaAlk7 but have high levels of Alk4 and Alk5 [TGFBR2 and ALK5, suggesting that TGFβ signaling in epicardial cells can occur [Alk5 [Smad4 [Alk5 and Smad4 are present in the developing mouse heart.Since TGFβ can signal in both an autocrine and paracrine fashion, the expression observed in the epicardial region does not necessarily result in actual signaling within the epicardium. To that end, the presence of the associated receptors is required to be able to determine if a cell is susceptible for signaling. Unfortunately, literature regarding receptor expression in the epicardium is scarce, which might be related to the limited availability of specific antibodies, the very low expression levels, or simply the fact that the epicardium is often overlooked in cardiac research. Interestingly, in vitro studies did reveal that mouse epicardial cells in culture do not express the type I receptor and Alk5 . Furtheran occur . The facur [Alk5  or Smad45 [Smad4  display TGFβ ligands are present, suggesting an important role in epicardial behavior. However, since these ligands can be stored in a latent form in the extracellular matrix of the heart, protein expression does not automatically correlate with spatiotemporal pathway activation. Therefore, determining where phosphorylated SMAD2/3 or other downstream targets are localized within the epicardium would provide better insight into which cells are involved in actual signaling.Tgfb2 deficient embryos. These embryos exhibited a spectrum of cardiac malformations including ventricular septum defects, early trabeculae formation, dual outlet of the right ventricle and dual inlet of the left ventricle [Tgfb3 deficient mice died after birth due to cleft palate [Tgfb3 mutant mice displayed signs of segmentally thick epicardium and an underdeveloped myocardium, indicating that before birth an epicardial defect is present [Tgfb1 deficient embryos displayed inflammatory disease and impaired hematopoiesis, and died of compromised vasculogenesis. Any observed cardiac malformations in these embryos are likely an indirect effect of defective vasculogenesis [Alk5-/− embryos [Tgfbr2−/− embryos [Tgfb1 deficient embryos, suggesting that these proteins are all involved in the same developmental processes. Since TGFβ is critical in a wide variety of developmental mechanisms, it is not surprising that KO mouse models exhibit a range of defects of which it is difficult to determine cause and consequence. However, three conclusions can be drawn. Firstly, there is a specific role for Tgfb2 in cardiogenesis. Secondly, the distinct phenotypes of the KO models of the three ligands demonstrate that TGFβ ligands are not interchangeable in vivo, probably due to the differences in spatiotemporal expression patterns and affinity for receptor binding. And thirdly, the defects observed in Tgfb2−/− and Tgfb3−/− mice are related to developmental processes where epithelial-mesenchymal interactions occur, e.g., cochlear morphogenesis in the inner ear [Tgfb1−/− are more related to impaired growth and inflammation [To determine if the expression of TGFβ members is functionally relevant for cardiac development, multiple conventional knock-out (KO) animals have been generated which are almost all embryonically lethal. Interestingly, severe cardiac defects were only observed in entricle ,66. All t palate  and abnot palate . Interes present . Tgfb1 dogenesis ,71. Alk5 embryos  and Tgfb embryos  displayenner ear , respiranner ear , while mammation ,75,76.Cre-recombinase under control of an epicardial-restricted fragment of the Gata5 promoter [Gata5-Cre mouse was crossed with a floxed Alk5 mouse line to specifically knock-out Alk5 in epicardial cells as early as in the PE at E9.5 [Alk5 deficient mouse embryos at E12; they displayed a detached epicardial layer, a diminished smooth muscle cell coverage of the coronary vessels, and a thinner myocardial wall due to reduced cardiomyocyte proliferation [Smad4 specifically in cells expressing the epicardial marker WT1 did not induce severe cardiac malformations in the mouse embryo. Interestingly, these embryos displayed a reduced number of EPDCs and cardiac fibroblasts [Since TGFβ is crucial for multiple cellular processes in the embryo, cell specific KO studies are essential to determine the role of TGFβ signaling specifically in the epicardium. A more specific model in this regard is a transgenic mouse line expressing promoter ,77. This at E9.5 . The emb at E9.5 , an obse at E9.5 . In contferation . This phferation . Hence, roblasts , indicatAlk5 [Alk5 KO embryo presents with an aberrant epicardial phenotype. The observed detached epicardial layer could be due to the absence of a sub-epicardial mesenchyme as a result of deficient EMT. Whether the Alk5 total KO embryo displays a detached epicardial layer and absent coronary vessel coverage remains elusive, as it has not been described. In the Tgfb1−/− and Tgfb2−/− embryos, the vascular phenotype and overall symptoms are likely to be too severe to determine an epicardial specific defect. Interestingly, in the Tgfb3−/− embryo an abnormal epicardial layer and immature myocardium were reported [The in vivo studies suggest that both ALK5 and SMAD4 are essential for epicardial behavior, yet through an unresolved mechanism. To further unravel the involvement of TGFβ signaling in the different aspects of epicardial behavior, in vitro models are important to be able to independently investigate epicardial EMT and epicardial invasion. Various in vitro models are used to study epicardial EMT. For instance, chick epicardial cells were studied by transferring hearts to a collagen gel allowing the epicardial cells to grow out as a monolayer . After rAlk5  or the uAlk5 ,79,81 prreported , which iFollowing EMT, epicardial cells acquire the ability to invade underlying tissue. Invasion of epicardial cells can be studied in more detail in ex vivo cultured hearts. For instance, when ex vivo E12.5 mouse embryonic hearts with YFP-labeled WT1+ cells were stimulated with TGFβ, the number of YFP+ cells invading the myocardium increased . In a siThere are studies reporting that TGFβ does not induce epicardial EMT and invasion, and even inhibits FGF-induced epicardial EMT and invasion in a chick heart model . They deTo conclude, although there are some opposing findings in literature, likely due to technical differences, there is abundant evidence that in the epicardium TGFβ induced ALK5 signaling is essential for cardiac development by regulating epicardial EMT and invasion of epicardial cells. When the TGFβ pathway is activated, several cell biological mechanisms are set in motion. These involve orchestrating the process of EMT- including changes in cell adhesion molecules and the cytoskeleton-, and regulating invasion of transformed mesenchymal cells into the underlying tissue. The regulation of cell adhesion molecules in epithelial cells is a well-known effect of TGFβ stimulation . In the Concomitantly to alterations in the cell adhesion molecule profile, the cytoskeleton undergoes dramatic changes during the process of EMT. The regulation of the cytoskeleton is often related to TGFβ-induced Rho signaling ,83,86,87Mylk, Rock1 and Tmsb4x. The combination of these factors has been described as an epicardial motile gene program [Changing the cell-adhesion profile and cytoskeleton ultimately results in the transition of an epithelial to a mesenchymal cell type, after which cells are primed to become motile and invade the underlying tissue. Motility can be regulated by many factors, for example  program . Interes program , overall program .Overall, TGFβ signaling appears to orchestrate a range of factors that ultimately result in enhanced motility of the epicardial cell.So far, we mainly described epicardial TGFβ signaling through the ALK5 receptor, a process that leads to phosphorylation of SMAD2/3. Although ALK5 is predominantly described in this context, it is not the only receptor resulting in SMAD2/3 phosphorylation. As depicted in Alk4 was shown to be expressed in epicardial cells in vitro [Not much is known about the role of activin signaling in cardiac development or regarding the epicardial expression of activin. Most of the KO mouse models related to activin signaling do not have a clear cardiac phenotype (reviewed in ). An excin vitro , implyinin vitro ,79,81 poin vitro . Howeverin vitro ,97,98,99in vitro . More reAs schematically shown in Bmp2 deficient mouse and cardiomyocyte specific Alk3 KO mouse displayed defects in the formation of the AV canal and also in the formation of the AV valves [Alk3 in Wt1+ epicardial cells displayed abnormalities in the tissues of the AV junction as well [In the developing heart, BMP ligands are mainly expressed in the myocardium . They arV valves ,105,106.V valves ,107. Mou as well , suggest as well . Recentl as well . Epicard as well . In WT1K as well   deficient of TGFβR3 or their wildtype littermates ,122. Culvimentin , but wervimentin . Furthercapacity  and dimicapacity ,122. Forcapacity , suggestKO cells ,122. Howinvasion . Lastly,and used . Additionhibited ,125. Binnhibited . Indeed,The exact modulation of epicardial TGFβR3 in TGFβ and BMP signaling seems to be complex, as it is bound by an array of ligands, but can also be reduced by several inhibitors. For instance, both inhibitors of ALK4/5/7 and ALK2/3 were able to block TGFβR3 mediated invasion in response to either TGFβ2 or BMP2 respectively . MoreoveThus far, we established a potential contribution of TGFβ and BMP signaling in the epicardium during heart development. However, as described previously, the epicardium is also involved in the injured heart. Cardiac injury arises when an occlusion of the coronary vasculature results in cardiac ischemia and damage of downstream tissue. The contribution of TGFβ signaling in the repair of the injured adult heart has been described extensively. Upon infarction, the protein expression of TGFβ ligands in the mouse heart increases. TGFβ1 and TGFβ2 expression is fast and decreases after 3–7 days, while TGFβ3 expression is induced after a few days and remains for a longer period . ConcomiTo be able to study adult epicardial EMT and invasion, in vitro cell culture models using patient derived epicardial cells were developed. In these human adult cells, it was established that all three TGFβ ligands are expressed, as well as the type I receptor ALK5 , indicattgfβ1, -2 and -3 14 days post-cryoinjury (dpci). Furthermore, strong mRNA expression of alk5b was present in the injured heart 4 dpci, and appeared to be specifically enhanced in the epicardium. This expression decreased over time, but was maintained up to 14 dpci. Although not described by the authors, mRNA expression of alk4 appeared to be present in the epicardium as well at 4 dpci. The presence of relevant ligands and receptors indicates that epicardial TGFβ signaling can take place in the injured heart. Blocking TGFβ signaling with the ALK4/5/7 inhibitor SB431542 resulted in defective regeneration of the damaged tissue. This cannot be directly attributed to the epicardium because besides epicardial expression, injury induces an overall increase of pSmad3 in the zebrafish heart indicating a widespread activation of TGFβ signaling [Thus far, in vitro studies indicate that TGFβ can regulate epicardial EMT via ALK5. Limited knowledge is available on epicardial TGFβ signaling and repair in vivo. It has been shown that the activated adult epicardial cells undergo EMT and form a sub-epicardial layer . From deignaling . The impignaling . In addiignaling , indicatignaling  and ColXignaling . Interesignaling . Since eignaling ,135, theinhbaa, a subunit of Activin, results in less CM proliferation in the regenerating zebrafish heart [inhbaa in the injured heart appeared to be mainly in the epicardial and endocardial layer [inhbaa KO is regulated via, among others, the epicardium.Since SB431542 inhibits ALK4/5/7 signaling, its effect on the repair of the zebrafish heart can also be attributed to defective activin/ALK4 signaling. This is supported by the finding that a zebrafish KO of sh heart , similaral layer , which cTherefore, although so far no direct link between the epicardium and the diminished regeneration of the zebrafish heart after inhibition of TGFβ signaling could be established, combination of the expression levels of TGFβ members in the epicardium see  with thebmp2 ligand expression starts to increase and reaches a maximal level after 3 days where after expression declines. After 7 days, expression of other BMP ligands, such as bmp4, bmp6 and bmp10 appears. For the epicardium specifically, the presence of relevant signaling proteins was revealed in a spatially restricted RNA sequencing experiment [bmp2b and bmp7 were enhanced in the wound border zone and also in the epicardium covering the wound at 7 days post injury (dpi). In addition, single cell sequencing of the zebrafish epicardium identified bmp4 as a specific epicardial marker of the adult zebrafish heart [bmp4 expression in the zebrafish heart was enhanced after injury [bmpr1aa, ortholog of BMP type I receptor ALK3, was enhanced in the viable myocardium and the epicardium 7 dpi. These expression levels indicate that BMP signaling can occur in the injured heart including the epicardium, which was confirmed by the presence of pSmad1/5/8 in the epicardial layer at 3 dpi [wt1b expressing epicardial cells was observed [wt1b was assessed. Therefore, this only shows that BMP signaling does not interfere with epicardial activation, but effects on epicardial EMT and invasion were not described. Expression profiles of BMP receptors and downstream target proteins show that BMP signaling is activated in the epicardium after injury in the zebrafish heart . Theref vessels . Moreove vessels . AlthougAnother opportunity to activate the epicardium of the injured heart could be via BMP4 signaling. During development, BMP4 was shown to be vital for epicardial maturation . Thus faTo be able to investigate these mechanisms in epicardial EMT, our research group has developed an in vitro cell culture system that allows for a direct comparison of fetal and adult human primary epicardial cells. Interestingly, while adult cells need a trigger to undergo EMT, fetal epicardial cells are more active and need incubation with an ALK4/5/7 kinase inhibitor to prevent spontaneous EMT . This obIn this review we aimed to provide a structured overview of the role of multiple ligands and receptors of the TGFβ family in epicardial behavior. In our opinion, the strength of this review is the structured and detailed overview on the potential power of the TGFβ pathway. This also presents a limitation of this manuscript; there are several other signaling pathways (including Wnt , FGF 4848 and PD


TP53 mutations, KRAS amplification, and MYC amplification were significantly associated with entosis in PDAC tissues. From a clinical standpoint entotic CICs were independently associated with a poor prognosis by multivariate Cox regression analysis when considering all cases or primary PDACs specifically. These results provide a contextual basis for understanding entosis in PDAC, a highly aggressive cancer for which molecular insights are needed to improve survival.Entosis is a type of regulated cell death that promotes cancer cell competition. Though several studies have revealed the molecular mechanisms that govern entosis, the clinical and genetic correlates of entosis in human tumors is less well understood. Here we reviewed entotic cell-in-cell (CIC) patterns in a large single institution sequencing cohort  of more than 1600 human pancreatic ductal adenocarcinoma (PDAC) samples to identify the genetic and clinical correlates of this cellular feature. After case selection, 516 conventional PDACs and 21 ASCs entered this study and ~45,000 HPFs (median 80 HPFs per sample) were reviewed; 549 entotic-CICs were detected through our cohort. We observed that entotic-CIC occurred more frequently in liver metastasis compared with primary in PDAC. Moreover, poorly differentiated adenocarcinoma or adenosquamous carcinoma had more entotic-CIC than well or moderately differentiated adenocarcinoma. With respect to genetic features  TP53 mutations [KRAS and Rac signaling have been implicated in establishment of winner cell status and in promoting cell death [Entosis is a type of regulated cell death that originates from actomyosin-dependent cell-in-cell (CIC) internalization and is executed by lysosomes . Entosisll death .TP53 status and/or metabolic related gene expression changes are positively correlated with formation of entotic CIC [TP53 contribute to this phenotypic change. For this reason, we investigated the CIC pattern in PDAC using a large single institution cohort of both primary and metastasis samples for which targeted sequencing data of more than 400 genes are available.While studies in vitro have revealed molecular mechanisms of entosis, translational studies exploring the clinicopathological and genetic correlates of entosis in patients are limited. The available data point to entosis as a correlate of aggressive behavior. In rectal cancer, head and neck squamous cell carcinoma (HNSCC) and in lung cancer, a CIC pattern has been associated with poor prognosis , 7, 8. Iotic CIC . HoweverAll genetic and clinical information and digital whole slide images were obtained from the MSK IMPACT Clinical Sequencing Cohort database through cBioPortal (version2.2.0) . GeneticSummary of case selection was shown in Fig. Entotic CIC structures were defined using H&E images as reported previously  as origiDigital whole slides available for the 537 cases selected for study from the MSK IMPACT Clinical Sequencing Cohort were visualized through Smart Slide Viewer (version 1.3.1583). All evaluable tumor regions submitted to the IMPACT sequencing in digital slides were reviewed by two pathologists (AH and CID). A total ~45,000 HPFs  were reviewed and all potential or suspected entotic-CICs (~1000 unique images) were captured and reviewed with discussion. Entotic-CICs were quantified by calculating the average number per ten HPFs. Based on this information we divided the cohort into two groups as previously reported .The histologic classification of entosis in 15 representative cases with entotic-CICs from the MSK IMPACT Clinical Sequencing Cohort was validated using immunohistochemistry for E-cadherin . Five miUsing cBioportal as a guide the top ten most frequently mutated genes and the top five most frequent copy number alterations in the cohort of 537 patients were included for correlation to entotic-CICs.Matched primary and metastatic tissues from three patients in the Last Wish Program at Memorial Sloan Kettering Cancer Center and 21 patients from the Gastrointestinal Cancer Rapid Medical Donation Program at Johns Hopkins Hospital were used for the validation autopsy cohort. All slides were reviewed by two gastrointestinal pathologists (AH and CID). At least two slides  and 50 HPFs  for each primary and metastasis site were evaluated for entotic-CICs. Only metastases from the liver, lung, and/or peritoneal cavity were included.This study was approved by the Review Boards of Johns Hopkins School of Medicine and Memorial Sloan Kettering Cancer Center.Results or Figure Legends. Statistical significance was considered if P value is <0.05.All statistics and graphs were performed and made using GraphPad Prism (version 8.2.1) and/or XLSTAT (version 2019.4.2). Each analysis method was described in We identified 539 entotic-CICs within 537 PDAC  cases in the MSK IMPACT Clinical Sequencing Cohort. Entotic-CICs per ten HPFs ranged from 0 to 3.06 (median 0.12) per neoplasm Fig. . Per ourP < 0.0001, the two-sided Fisher’s exact test)    Table . For prit) Table .Table 1CP = 0.023, the Wilcoxon matched-pair signed rank test, two-sided)  Fig. . To deteed) Fig. .Fig. 2HeTP53 mutations  and KRAS and MYC amplification   nor did we find a difference in the proportion of truncating and non-truncating TP53 mutations between the two groups .        Fig.  or primast) Fig. . Cox pros Tables  and 6. E3) Table  and multWe find that entosis in PDAC is a correlate of aggressive biology and an independent prognostic factor by multivariate analysis. Moreover, metastases appear to have a greater number of entotic-CICs than primary tumors. These findings not only validate prior reports in rectal, head, and neck and breast cancer and but provide a deeper understanding of the clinicopathologic relevance of entosis in PDAC.TP53 mutations with entotic-CIC parallels prior reports in lung cancer [TP53 hotspot mutations. While the mechanisms by which TP53 alterations contribute to entosis is unknown, one putative molecular mechanism is that loss of TP53 or delta-133TP53 expression increases extracellular ATP release and the consequent activation of purinergic P2Y2 receptors, which induces cell engulfment [Our finding that entotic-CICs are more prevalent in poorly differentiated PDAC parallels prior observations in breast cancer . We extegulfment . Alternagulfment .TP53 inactivation and KRAS amplification are more frequent in metastasis [KRAS amplified samples, six were liver metastases and only two were primary carcinomas . While PDAC metastases does not appear to have a genetic basis [The reason for an increased prevalence of entotic-CICs in metastatic PDAC, specifically liver metastases, also remains to be determined. However, we expect that the mechanisms are multifactorial. For example, a recent report showed tastasis . Our datic basis , subclonic basis . Thus, iPoor prognosis in entotic-CIC high tumors that we report here have been previously reported in lung adenocarcinoma , HNSCC , 8, rectSupplementary Information 1Supplemenatry Information 2Supplementary Information 3


The Hippo signaling pathway, an evolutionarily conserved protein kinase cascade, plays a critical role in controlling organ size, cancer development, and tissue regeneration. Recently, mounting evidence has suggested that Hippo signaling also has an important role in regulating immunity, including innate and adaptive immune activation. In the neuronal system, Our laboratory results, together with those from other studies, demonstrate that the Hippo signaling pathway is involved in neuroinflammation, neuronal cell differentiation, and neuronal death. In the present review, we summarize the recent findings pertaining to the function and regulatory mechanism of Hippo signaling components in the neuronal system, implicating the potential of Hippo signaling as a therapeutic target for the treatment of neuronal system diseases. Drosophila, plays a critical role in regulating cell contact inhibition, proliferation, differentiation, and apoptosis. As such, this pathway is closely associated with the control of organ size, cancer development, and autoimmune diseases (Drosophila]) and their adaptor protein, Sav family WW domain containing protein 1 . The phosphorylation of MST1/2 activates large tumor suppressor 1/2 [LATS 1/2 (orthologs of Warts in Drosophila)], a downstream protein, which in turn phosphorylates the downstream Yes-associated protein (YAP [ortholog of Yki in Drosophila]) or a transcriptional coactivator with PDZ-binding motif (TAZ); this results in the prevention of nuclear translocation by interacting with cytosolic protein 14-3-3. Meanwhile, unphosphorylated YAP or TAZ are relatively enriched in the nucleus and bind to their key transcriptional factors, the TEA domain transcription factor (TEAD) family [TEAD 1–4 (orthologs of Sd in Drosophila)]; thus regulating the expression of many genes that enhance cell proliferation, differentiation, and survival.The Hippo signaling pathway, originally identified in diseases –6. ImporDrosophila, the Hippo signaling pathway in mammals also plays critical role in the control of organ size, cancer development, and tissue regeneration. Recently, accumulating evidence has suggested that Hippo signaling is important in regulating cancer immunity, and for innate and adaptive immunity  of ic death ; howeverand MST2 . Consistnd heart –5, 14, sRecently, mounting evidence has shown that the Hippo signaling pathway also plays a critical role in the neuronal system. Using an RNA-Seq transcriptome and splicing database of glia, neurons, and vascular cells of the cerebral cortex , we analIn the vertebrate brain, neural stem cells (NSCs) are self-renewing, multipotent cells that generate neurons and glial cells during embryonic development . It is nYAP, using Nestin-cre or GFAP-cre, decreases the number of neocortical astrocytes and impairs astrocytic proliferation through the BMP2-YAP-SMAD1 pathway  cause massive apoptosis through the induction of YAP/TAZ activation, and upregulating a series of genes associated with cell growth and proliferation . Additio pathway . Furtheriescence .It has been established that Hippo signaling is involved in the control of organ size and tumor development. Hippo inhibition results in higher activity of YAP and leads to tumorigenesis; however, its activation plays a role in neurodegeneration by mediating oxidative stress-induced neuronal death. Oxidative stress activates MST1 and then induces either YAP-dependent or YAP-independent cell proliferation and cell death , 25.The mammalian fork-head transcription factors of the O class (FOXOs) are well-characterized substrates of MST1. By stimulating oxidative stress, we found that MST1 phosphorylates FOXO proteins, disrupts their interaction with protein 14-3-3, and promotes FOXO nuclear translocation, thereby inducing cell death in neurons , 27. ApaInterestingly, there is a functional interaction between Hippo-YAP signaling and FOXO1 in treatments that induce oxidative stress. YAP acts as a nuclear co-factor of FOXO1, which modulates the FOXO1-mediated antioxidant response. Activation of Hippo antagonizes YAP-FOXO1, leading to increased ischemia/reperfusion (I/R)-induced cell death through downregulation of catalase and MnSOD . These rMST1 and MST2 KO mice, MST2—but not MST1—was shown to be a critical regulator of caspase-mediated photoreceptor cell death in a mouse model of retinal detachment (RD). Mechanically, KO of MST2 decreases caspase-mediated photoreceptor cell death and proinflammatory cytokines, such as monocyte chemoattractant protein 1 and interleukin (IL)-6 during the early phase of RD -mediated neurodegeneration in the adult stage. Importantly, phosphorylated MST1—the active form of MST1—was reported to be significantly increased in the post-mortem cortex of patients with Huntington's disease (HD). Meanwhile, YAP nuclear localization was decreased in both HD post-mortem cortex and neuronal stem cells derived from HD patients  damage, and neurobehavioral impairment  hyperimmunoglobulin E syndrome. Hence, these results strongly indicate that MST1 plays a critical role in the immune system.Apart from the important role of Hippo signaling in controlling organ size and cancer development, its role in immunity activation has recently been extensively studied. The key component of the Hippo signaling pathway, MST1, is highly expressed in lymphoid tissues.  T cells . In 2012trophils . In the stations . Subsequstations ; these fDrosophila immune organ) results in a decreased antimicrobial response and increase vulnerability to infection by Gram-positive bacteria. Mechanically, Gram-positive bacteria could activate Hippo-Yki signaling through Toll-Myd88 signaling, in which Yki directly regulates the transcriptional activity of Cactus, the Drosophila IκB homolog (MST1/2 deficient bone-marrow-derived macrophages exhibited higher toll-like receptor 4-mediated nuclear factor (NF)-κB activation, resulting in increased levels of some pro-inflammatory cytokines, such as IL-6, tumor necrosis factor-alpha, and IL-1β  in the lens led to cataracts in mice (YAP cKO reduced proliferation of epithelial cells, delayed fiber cell denucleation, and increased cellular senescence in the lens; the inflammation levels were also significantly altered in YAP cKO mice. Collectively, these results suggest that Hippo signaling is also important in neuroinflammation in the neuronal system, which may not be consistent with its functions in the peripheral immune system; thus, warranting further investigations.The functions of Hippo signaling in the neuronal system have recently been elucidated. We found that KO of icroglia . Consisticroglia . These r in mice . Mechani in mice . MechaniThe Hippo signaling pathway is not only critical in controlling organ size, cancer development and tissue regeneration, but it is also important in regulating immunity, including the activation of the innate and adaptive immune systems. Recently, multiple studies have shown that Hippo signaling components paly critical role in the neuronal system, including the regulation neural stem cell proliferation and differentiation, oxidative stress-induced neuronal cell death, and in neuroinflammation; thus implicating a potential therapeutic target for the treatment of neuronal system diseases. However, the role and the regulatory mechanism of Hippo signaling in the neuronal system still requires clarification, especially in different diseases or environmental conditions.JC wrote the manuscript and prepared the figure. SW and YD review the manuscript. JC and ZY conceived the review topic and performed a comprehensive review of the literature.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.


Morganella morganii, in phosphate buffer and tuna meat slurry by high hydrostatic pressure (HHP) alone or in combination with 0.2% lemon essential oil (LEO) treatments were studied using viability measurement and scanning electron microscopy (SEM). HHP alone or in combination with LEO treatments showed first‐order destruction kinetics to M. morganii during pressure holding period. The D values of M. morganii (200 to 600 MPa) in phosphate buffer ranged from 16.4 to 0.08 min, whereas those in tuna meat slurry ranged from 51.0 to 0.10 min, respectively. M. morganii in tuna meat slurry had higher D values and were more resistant to HHP treatments than in phosphate buffer. In addition, the D values of HHP in combination with LEO treatment were lower than those of HHP treatment alone at <400 MPa of pressure, indicating that it is more effective to inactivate M. morganii under the same pressure. The results showed the M. morganii at HHP in combination with LEO treatment was more susceptible to pressure treatment alone. HHP with or without LEO treatments can be used to inactivate M. morganii by causing disruption to bacterial cell membrane and cell wall as demonstrated by SEM micrographs.The inactivation and damage of histamine‐forming bacterium,  Morganella morganii was more resistant to HHP and LEO in fish slurry than phosphate buffer. With LEO treatment, M. morganii was more susceptible to pressure than HHP alone. SEM show HHP and LEO cause cell wall and membrane damage. The peel pieces were vacuum‐freeze dried and then ground into powder. One hundred gram of powder was placed into the supercritical CO2 extractor, designed by Dr. Shane‐Rong Sheu at Far East University, Tainan, Taiwan. The extraction parameters are as follows: 1.5 L capacity, temperature, 323 K; pressure, 10 MPa; flow rate of CO2, 3.5 kg/h; time, 90 min. Components of the extracted essential oil were analyzed according to the previous method  tube (5 ml) and incubated at 35°C for 12 hr; then, 100 μL aliquot of the bacterial culture was added to 100 ml sterile TSB medium at 35°C for 24 hr. The cultured broth was centrifuged at 8,000 x g for 15 min at 4°C, and the bacterial pellet was washed and re‐suspended in 0.1 M phosphate buffer (pH 6.8). The bacterial suspension was then adjusted to a concentration of 109 CFU/mL.One loopful of M. morganii inoculum (109 CFU/mL) to get a final bacterial population of 107 CFU/mL. In LEO treatments, the phosphate buffer or tuna meat slurry was added and mixed with LEO solution to get at 0.2% LEO concentration before M. morganii inoculation. The test samples were added to sterile vacuum bags in 10 ml portions, vacuum packaged and heat‐sealed, and then subject to HHP treatments immediately.Fresh tuna flesh was purchased from a local market in Kaohsiung City, Taiwan, and transported in ice to the laboratory immediately. After washing with a 75% ethanol solution for 1 min and rinsing with sterile water, the flesh was ground to mince in a sterile food homogenizer. The fish mince was then blended with 0.1% peptone water  for 2 min in a blender . Both the sterile phosphate buffer  and the tuna meat slurry (99 ml) were inoculated with 1 ml of 2.3SEM analyses.Test bags in triplicate were treated with a laboratory model of high pressure processing system  at 200 to 600 MPa for 0 to 15 min at room temperature (25°C). This high pressure processing system having a 6.2‐L chamber can be operated at up to 600 MPa at a pressure increase rate of approximately 300 MPa/min and the release times of less than 20 s at all pressures. Water was used as a pressure transmission medium in this study, and the reported pressurization times did not include the time for pressure increase or release. An untreated bag placed in ice water at ambient pressure (0.1 MPa) served as a control. Samples subject to pressure treatment were set in ice water and immediately processed for bacterial counting and 2.4The HHP‐treated, HHP in combination with LEO‐treated and nontreated bacterial suspensions in phosphate buffer or fish slurry were 10‐fold serially diluted in sterile phosphate buffer . With regard to pour plate counting, aliquots (1.0 ml) of the diluents were mixed in petri dishes with 15 ml TSA (Difco) at 45–50°C. After the agar medium was solidified in a laminar flow hood, the plates were transferred to an incubator and incubated at 30°C for 24–48 hr. Bacterial colonies numbers on the plates were counted. The detection limit of bacterial count was 1.0 log CFU/mL. Data from triplicate samples were presented as mean ± standard deviation.M. morganii during the pressure–hold time phase with log numbers of survivors.0N is the initial number of M. morganii in untreated samples, N is the surviving number of M. morganii after pressure treatment for time t (min). The D value or decimal reduction time is the treatment time at any given pressure causing 90% reduction of the M. morganii population, that is,. resulting in one logarithm reduction of the microbial population. D value was obtained by the negative reciprocal slope of the log (0N/N) versus time.The linear first‐order reaction was used as follows to determine the pressure destruction kinetics of The decimal logarithm of D values versus pressure was plotted to determine the pressure sensitivity of the D values, and the pressure z‐value (Zp) was determined as the negative reciprocal of the slope. The Zp is the increase of pressure needed to change the D value by 90%.2.5Morganella morganii cells in 0.1 M phosphate buffer (pH 6.8) were harvested from pressure‐treated (500 MPa for 10 min), LEO pressure‐treated (500 MPa for 10 min), and nontreated suspensions via centrifugation at 5,000 rpm for 20 min. After two washes with phosphate buffer, the pellets were re‐suspended in 1 ml of phosphate buffer and then filtered through Millipore membranes . Cells on the filters were fixed with 10 ml of 1.5% glutaraldehyde/0.1 M phosphate buffer (pH 7.3) and left overnight for drying at 4°C. After the cells on the membranes were washed three times with phosphate buffer for 10 min, they were postfixed for 90 min in 1% osmium tetroxide (OsO4) and then rinsed with phosphate buffer twice (10 min per rinse). The cells on the membranes were then dehydrated in a series of 10 ml ethanol solutions , immersed in isopentyl acetate and finally in carbon dioxide medium for critical point drying using a critical point dryer . The dried membranes were then mounted on scanning electron microscope stubs, sputter‐coated with a thin film of gold‐palladium and then observed by the SEM  operating at 15 kV voltage. SEM photomicrographs were taken from different regions of the same dried specimen.2.6p < .05 was used to indicate significant deviation.One‐way analysis of variance (ANOVA) was carried out on the linearized survival slopes at each pressure time calculation for phosphate buffer or tuna meat slurry and was performed using the Statistical Product and Service Solutions, SPSS Version 16.0 for windows (SPSS Inc). Tukey's pairwise comparisons tests were performed within the confidence interval of 95% and value of 33.1M. morganii in phosphate buffer following HHP treatment at 200–600 MPa for up to 15 min with or without 0.2% LEO. The treatment pressure, the LEO addition, and the holding time influenced the destruction of the bacteria. The survival curves at higher pressures were steeper than those at lower pressures indicated that the destruction rate was higher at higher pressures. The first‐order model fits the destruction kinetics of HHP treatment on M. morganii during the hold period, indicating that pressure destruction of M. morganii complied with the semi‐logarithmic model. From the survival curves, the D values could be calculated and used for comparison of microbial resistance to HHP treatments with or without LEO or the effectiveness of such treatment on microbial destruction. The D values of M. morganii (200 to 600 MPa) in phosphate buffer ranged from 16.4 to 0.08 min , and therefore more resistant, than HHP in combination with LEO treatments  (p < .05) when treated with HHP at 200, 300, and 400 MPa (Table p > .05) between HHP treatments alone and HHP in combination with LEO treatments under the same pressure  than HHP in combination with LEO treatments  (p < .05) (Table p > .05) between HHP treatments alone and HHP in combination with LEO treatments under the same pressure (Table The inactivation kinetics during HHP treatment (200–600 MPa for 0–15 min) with or without 0.2% LEO of M. morganii in HHP treatments alone had a higher D value than HHP in combination with LEO treatments in tuna meat slurry, because it was more susceptible to pressure in combination with LEO, which might have accelerated the inactivation impact. In tuna meat slurry, the Zp value of M. morganii at HHP treatments alone was 146 MPa as compared with 158 MPa at HHP in combination with LEO treatments. Analysis of Zp values indicated that sensitivity of M. morganii to pressure changes; therefore, the destruction rate of HHP treatments alone is more sensitive to changes in pressure than HHP in combination with LEO treatments.The HHP decimal reduction time curves for both HHP treatments alone and HHP in combination with LEO treatments showed overlapping pressure region at 500–600 MPa Figure . Thus, ML. monocytogenes  was found to be significantly inactivated effects on E. coli. In this study, the D values of HHP in combination with LEO treatment were lower than those of HHP treatment alone at <400 MPa of pressure, indicating that HHP with LEO treatment is more effective to inactivate M. morganii under the same pressure.The combination of HHP and carvacrol was found to inactivate nd Sheen . Recentlnd Sheen  reportedM. morganii in tuna meat slurry had higher D values than those in phosphate buffer for all the pressure treatment conditions (Table M. morganii was more resistant to pressure treatment in tuna slurry than in phosphate buffer. Many intrinsic and environmental parameters, especially the nature of the suspension medium, influence the resistance of microorganisms to pressure treatment. Simpson and Gilmour (M. morganii cell in tuna meat slurry are more protected against HHP treatment due to protein and lipid contents.In this study, the ns Table , indicat Gilmour  reported Gilmour . Fish ma Gilmour . Patters Gilmour  also sta3.3SEM micrographs of M. morganii in phosphate buffer following HHP treatment at 500 MPa for 10 min with or without 0.2% LEO. Compared to the untreated cells (Figure M. morganii after HHP treatment alone and in combination with LEO (Figure Listeria cells at 400 MPa for 10 min and V. parahaemolyticus at 300 MPa for 10 min (Wang et al., Figure s Figure , damagesO Figure . SimilarSEM to show the presence of bud scars on cellular surface after pressure treatment of L. monocytogenes, and the loss of membrane integrity in most of the bacterial cells. Mackey, Forestiere, Isaacs, Stenning, and Brooker (Saccharmyces cerevisiae in apple juice after HHP treatment at 600 MPa for 7 min. Recently, Wang et al. (M. morganii by HHP treatment could be the cell membrane or cell wall, and membrane‐damaged cells may exhibit sensitivity to LEO.Ritz et al.  employed Brooker , by usin Brooker  showed pg et al.  indicateg et al. . The res4M. morganii using HHP alone and with LEO treatments, showed that HHP can be applied to inactivate histamine‐forming bacterium M. morganii by damaging cell wall and cell membrane. The results showed that M. morganii in tuna meat slurry were more resistant to HHP treatment than in phosphate buffer. With LEO treatment, M. morganii was more susceptible to pressure treatment than HHP treatment alone.This study, aiming of investigating the inactivation of The authors declare no conflict of interest in the publication of this article.This study does not involve any human or animal testing.


Calmodulin binding is a nearly universal property of gap junction proteins, imparting a calcium-dependent uncoupling behavior that can serve in an emergency to decouple a stressed cell from its neighbors. However, gap junctions that function as electrical synapses within networks of neurons routinely encounter large fluctuations in local cytoplasmic calcium concentration; frequent uncoupling would be impractical and counterproductive. We have studied the properties and functional consequences of calmodulin binding to the electrical synapse protein Connexin 35 (Cx35 or gjd2b), homologous to mammalian Connexin 36 (Cx36 or gjd2). We find that specializations in Cx35 calmodulin binding sites make it relatively impervious to moderately high levels of cytoplasmic calcium. Calmodulin binding to a site in the C-terminus causes uncoupling when calcium reaches low micromolar concentrations, a behavior prevented by mutations that eliminate calmodulin binding. However, milder stimuli promote calcium/calmodulin-dependent protein kinase II activity that potentiates coupling without interference from calmodulin binding. A second calmodulin binding site in the end of the Cx35 cytoplasmic loop, homologous to a calmodulin binding site present in many connexins, binds calmodulin with very low affinity and stoichiometry. Together, the calmodulin binding sites cause Cx35 to uncouple only at extreme levels of intracellular calcium. Cell–cell communication is an essential feature of tissue organization in multicellular organisms. Nearly all tissues engage in one form of cell–cell communication via gap junctions, direct cell to cell channels that permit limited diffusion of ions and small molecules between contacting cells . Gap junEvolution has settled on calcium as a central signaling molecule. A number of robust transport mechanisms pump calcium out of the cell and into intracellular membrane-bound compartments so that the free calcium concentration in the cytoplasm is typically in the tens of nanomolar, many orders of magnitude smaller than outside the cell. In concert, a wide variety of calcium-dependent signaling pathways have evolved that respond to nanomolar to micromolar increases in intracellular calcium . This seNot surprisingly, among the orderly mechanisms of cell death is the shutting down of cell–cell communication so as not to endanger the neighbors. It is a nearly universal property of gap junctions that high levels of intracellular calcium trigger their uncoupling ,6. It isA caveat to the dependence on calcium signaling to decouple a stressed cell from its neighbors is the problematic implementation of such a function in excitable cells. Neurons use quite large transient increases in calcium to trigger chemical synaptic transmission, and post-synaptic responses may include substantial calcium influx. Gap junctions that function as electrical synapses in such cells may be subjected to these large changes in calcium. Do they shut down with every synaptic impulse? This would seem to be impractical, and would limit the efficacy of electrical synaptic transmission.In this study, we have examined the properties and functional outcomes of calmodulin binding to Connexin 35 (Cx35 or gjd2b), homologous to mammalian Cx36 (or gjd2). Cx35/36 belongs to the small, evolutionarily distinct delta subgroup of the connexin family ,11. Its ½ for calcium around 3 micromolar [Calcium signaling is very important for the plasticity of electrical synapses composed of Cx36 and its non-mammalian orthologue Cx35 and related connexins. We previously identified a calmodulin binding site in the C-terminus of Cx36 that is conserved in all related connexins , and whicromolar , suggestcromolar . We wishcromolar  to analycromolar  (blue dacromolar  in mammacromolar  had studn = 6), while Cx35-IL had a first order Kd of 6.29 µM and Bmax of 0.32 moles calmodulin/mole connexin (n = 3). Because of the relatively low stoichiometry of binding and the assessment that a portion of the putative intracellular loop binding site may be inaccessible due to its position within a transmembrane domain, we opted not to study its properties further.To examine calmodulin binding to Cx35, we developed GST-Sumo fusion constructs of perch Cx35 intracellular loop (Cx35-IL) and C-terminal (Cx35-CT) domains. The intracellular loop construct contained amino acids 101–192, encompassing the full intracellular loop and portions of the third transmembrane domain including the potential calmodulin binding site; the C-terminal construct contained the full C-terminal domain from amino acids 251–304. We performed surface plasmon resonance (SPR) binding measurements with the Cx35 fusion proteins immobilized on the chip surface and calmodulin in analyte solution. The GST-sumo carrier protein served as a reference. To better understand the physical properties of the C-terminal calmodulin binding site and to develop mutants that could be used to study the functional significance of calmodulin binding, we undertook a mutagenesis study of this site. Calmodulin binds to target proteins containing a variety of target motifs characterized by bulky hydrophobic residues with certain spacings. Some common calcium-dependent calmodulin binding sites have bulky hydrophobic anchor residues at positions 1 and 10 or 1, 5 and 10 (termed 1–10 motifs), or at positions 1 and 14 (1–14 motifs), with variants having additional anchors at positions 5 and/or 8; basic residues throughout the site can also be important ,26. Anot2+. Binding parameters, shown in n = 6 experiments. A deletion of 4 amino acids, R261–K264 (Cx35 RKIKdel), encompassing the predicted number 1 hydrophobic anchor residue and surrounding basic residues largely abolished calmodulin binding (n = 5), which was not statistically different than wild type Cx35-CT , which was significantly lower than Cx35 wild type . This indicated that the basic residues, the hydrophobic residue I263, or the 3-dimensional structure of the beginning of the C-terminus were critical for calmodulin binding to Cx35.To study which of these residues might be involved in calmodulin binding, we made a series of mutants in the C-terminal domain fusion protein, targeting residues marked in red in  binding C. The can = 3; p = 0.737) and Bmax was reduced to 0.055 ± 0.046 moles calmodulin/mole Cx35  and decreased Bmax to 0.31 ± 0.05 moles calmodulin/mole Cx35 . Mutation of the basic residue R268A slightly but significantly increased Kd for calmodulin to 2.46 ± 0.25 µM , but did not change Bmax . The position 15 mutation I277A did not significantly change Kd for calmodulin , but did slightly reduce Bmax to 0.33 ± 0.04 moles calmodulin/mole Cx35 . These experiments suggest an important role for V267 in position 5 of the putative calmodulin binding site, but less critical roles for the basic residue R268 or the potential position 15 anchor residue I277.We then examined the number 5 hydrophobic anchor residue V267, its adjacent basic residue R628, and the potentially anomalous position 15 residue I277, each with alanine mutations E–G. The n = 6; p = 0.161) or the Bmax . These results suggest that Cx35 does not use an IQ motif for calmodulin binding and that position 8 of the conventional motif is not critical for binding.Finally, we examined the potential role of valine 270 in position 8 of the conventional motif and potentially in position 1 of an IQ motif in a combined mutant converting both V270 and Q271 to alanine. Comparing the connexin-calmodulin interactions determined structurally by Siu et al.  with int2+-induced uncoupling, we used ionomycin treatments of HeLa cells stably-transfected with Cx35. 2+ measurement in HeLa cells loaded with Fura-2-AM in zero Ca2+ Ringer solution and returned to normal Ringer containing 2.5 mM extracellular Ca2+. Addition of 5 µM ionomycin resulted in a rapid rise in intracellular Ca2+, with a slow, continued rise over 3 min to approximately 1 µM free Ca2+ before the extracellular solution was replaced with ionomycin-free normal Ringer. This level of free Ca2+ is a bit below the K1/2 for Ca2+ (about 3 µM) of the C-terminal calmodulin binding site [2+ resulted in potent uncoupling of the cells . Both 10 µM and 100 µM W7 treatments significantly increased coupling above the level in ionomycin . These experiments suggest that calmodulin binding uncouples Cx35, in contradiction to a report that calmodulin binding enhances Cx36 coupling [2+-induced uncoupling of gap junctions in Cx35-transfected HeLa cells, it is not entirely clear to what extent Cx35 itself was uncoupled.The best-documented role of calmodulin in regulation of gap junction functions is its role in chemical gating to induce uncoupling of gap junctions at high intracellular calcium . To inveing site , but shohe cells B,C. Treahe cells D, suggescoupling . HoweverTo refine our studies of the functional effects of calmodulin binding to Cx35, we employed mutations in the C-terminal calmodulin binding site that we identified in the in vitro binding studies . The mutp < 0.0001, n = 3 experiments). Cells transfected with each of the Cx35 constructs supported a similar amount of tracer coupling, and each was significantly uncoupled by 5 µM ionomycin . In contrast, coupling was reduced only 37% in the K262E, I263A mutant that eliminates calmodulin binding to the C-terminal site , and the ionomycin-treated coupling was 105% higher than that of ionomycin-treated Cx35 wild type . In the V270A, Q271A mutant that had a very modest effect on calmodulin binding, ionomycin reduced coupling by 52%  to a level that was not statistically different than that of Cx35 wild type . Thus, while it is likely that some of the uncoupling observed in Cx35-transfected HeLa cells is due to uncoupling of the remaining endogenous connexins, loss of calmodulin binding to Cx35 prevented a portion of the uncoupling that can be attributed to Cx35, demonstrating that calmodulin binding to the C-terminal site uncouples Cx35.We also made efforts to eliminate the connexin background in HeLa cells in order to perform functional experiments. We used a HeLa cell line that we have recently developed in which the endogenous Cx45 alleles have been mutated by CRISPR, suppressing its expression . In addionomycin . However2+ achieved in these cells is not known, Moore et al. [2+ in the immediate vicinity of the gap junctions, were about 15% of the peak response to 5 µM ionomycin. This suggests that 100 µM glutamate stimulation should raise Ca2+ to very low hundreds of nanomolar. We would predict that this level of free Ca2+ has little ability to occupy the Cx35 C-terminal calmodulin binding site unless interactions with nearby proteins or other calmodulin binding sites alter the calcium-dependence of the interaction. We used this stimulation to examine coupling of Cx35 and its calmodulin-binding mutants.Calmodulin-dependent protein kinase II (CaMKII) has been demonstrated to phosphorylate Cx36  at a site et al.  found thp = 0.999, n = 5 experiments), while coupling in wild type Cx35-transfected cells increased 118% . In the K262E, I263A mutant that prevented calmodulin binding, glutamate increased tracer coupling by 97% . Neither the control level of coupling nor the glutamate-stimulated level of coupling differed in the K262E, I263A mutant from Cx35 wild type . In the V270A, Q271A mutant that had little effect on calmodulin binding, glutamate stimulation increased tracer coupling by 90% , and again neither control nor glutamate-stimulated coupling differed from wild type Cx35 . These results suggest that calmodulin binding to the C-terminal calmodulin binding site does not contribute to CaMKII-mediated potentiation of coupling during glutamate stimulation.p = 0.626, n = 3 experiments).A calmodulin binding site in the C-terminus of Cx32 has been demonstrated to be important for the proper oligomerization of Cx32 monomers into connexons  and calmn = 15: Cx35 mean rank 51.10, K262E, I263A mean rank 20.13, p < 0.0001; Cx35 mean rank 51.10, V270A, Q270A mean rank 14.70, p < 0.0001). While its mean length was smaller, Cx35 mutant I277A did not reach statistical significance in a non-parametric test . Mean gap junction volume . Thus, disruption of the Cx35 C-terminal calmodulin binding site reduced the average size of Cx35 gap junctions regardless of the effect of the mutation on calmodulin binding.Because gap junctions made by the calmodulin binding site mutants appeared to be smaller than wild type, we measured length and volume of 15 arbitrarily-selected gap junctions of each type from three separate experiments. n volume G was sig2+ should routinely rise transiently to a level that drives some occupancy of the C-terminal binding site by calmodulin. Thus, the role of calmodulin binding in functional regulation of the electrical synapses is potentially of some importance.Calmodulin binding has emerged to be a nearly universal characteristic of gap junction proteins . The mem2+ up to approximately 1 µM, a level below Cx35′s K1/2 for calcium and suitable to drive about 1/3 occupancy of the C-terminal calmodulin binding site [Our experiments in this study showed that ionomycin treatment in HeLa cells caused uncoupling of Cx35 gap junctions, an effect suppressed in the K262E, I263A mutant that essentially eliminates calmodulin binding to the C-terminal site. The experiments also showed residual uncoupling in cells transfected with the K262E, I263A mutant. We believe this to be the response of the remaining endogenous connexins in the cells. The treatment we used increased intracellular free Caing site  if the cn = 1 experiment with 4–8 measurements per condition). We interpret this finding to indicate that, during the 10-min pre-incubation plus 10 min of post-scrape diffusion, the active calcium extrusion mechanisms in HeLa cells . This may be one aspect of specialization to function in the environment of the electrical synapse, and particularly in mixed synapses [Curiously, while our experiments employed 5 µM ionomycin with 2.5 mM extracellular Calar Ca2+ ,42,43,44lar Ca2+ . Indeed,La cells . Thus, C10 motif   reduced the incidence of gap junctions, but not their size . While wBrown et al.  have rec2+ can cause these domains to dissociate from the membrane, further contributing to regulating protein functions [It is quite striking how many functionally important interactions converge on the small portion of the beginning of the C-terminus of Cx35, its orthologues and related connexin isoforms. These interactions include calmodulin binding ,21, tubuunctions .The cytoplasmic end of the connexin 4th transmembrane domain is positioned on the outer rim of the connexon cytoplasmic face in the Connexin 26 crystal structure . Here thThis study correlates in vitro calmodulin binding data to in vivo functional data. While the fusion proteins containing the full cytoplasmic intracellular loop and C-terminal domains are more likely to reflect natural structural properties of connexin cytoplasmic domains than do small synthetic peptides often used for such studies, they still lack the context within an intact protein with 4 transmembrane domains. It is possible that calmodulin binding to Cx35 is influenced by the structural organization of the connexin and the surrounding plasma membrane in ways not captured in the in vitro studies, or that interactions involving both binding sites may change the stability or calcium dependence of the binding interactions. We believe that the experiments with intact Cx35 with mutations that eliminate C-terminal calmodulin binding provide significant insight into the true functional importance of calmodulin binding. Further studies closely examining the relationship of intracellular calcium concentration to coupling will provide additional insight.Escherichia coli strain BL21(DE3). In order to minimize degradation of fusion proteins in bacteria and enhance proper folding, we initially cloned cytoplasmic fragments of Cx35 as fusion proteins with human Sumo3. The full perch Cx35 C-terminus containing amino acids 251–304 and the full cytoplasmic loop plus a portion of transmembrane domain 3, consisting of amino acids 101 to 192 were amplified by PCR using Pfu polymerase  from PCx35-pcDNA [Connexin 35 cytoplasmic domains were expressed as fusion proteins in 35-pcDNA   in frame with Glutathione S-transferase. The resulting GST-Sumo fusion proteins were used for subsequent binding experiments. A control clone was generated by transferring Sumo3 from the parent vector into pET42c in the same way.The full-length perch Cx35 cDNA (PCx35-pcDNA)  was usedE. coli using glutathione sepharose 4B  according to the manufacturer’s protocols. Surface Plasmon Resonance (SPR) studies were performed on a Bioacore 2000  4-channel instrument. Anti-GST antibodies  were immobilized on CM5 SPR chips (Biacore) by amine coupling using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide. The GST-Sumo fusion proteins were then captured in each flowcell as previously described [GST-Sumo fusion proteins of the Cx35 cytoplasmic domains and the control GST-Sumo clone were purified from 2+ and 0.5 mM free Mg2+ with the following components: 140 mM KCl, 10 mM MOPS, pH 7.2, 5 mM HEDTA, 5 mM NTA, 9.54 mM CaCl2, 0.906 mM MgCl2, 0.05% Tween 20 [2+ was used between runs to strip any bound calmodulin off the chip; this solution contained 140 mM KCl, 10 mM MOPS, pH 7.2, 5 mM HEDTA, 5 mM EGTA, 4.62 mM MgCl2, 0.05% Tween 20 [Purified calmodulin  was a gehttps://www.simfit.org.uk). The first order binding constants are reported, as additional terms gave only small improvements of the fits.Data were analyzed with BIAEvaluation software (Biacore) and further processed in Microsoft Excel . Binding data were fit to first and second order ligand binding models with Simfit (GJC1) were mutated with CRISPR [GJA1; Cat. # TRCN0000059773, Sigma-Aldrich, St. Louis, MO, USA) using GenePorter 2 transfection reagent  and cultured for 48 h. Prior to use in experiments, cells were rinsed twice with MEM.HeLa cells  were used between passages 7 and 20 relative to the original cell line obtained from ATCC. A derivative of this cell line in which 5 endogenous alleles of Cx45  were added to each dish and the cells scraped with a 26-gauge needle. Scraped cells were incubated a further 10 min to allow for loading and tracer diffusion, rinsed and fixed with 4% formaldehyde in 0.1 M phosphate buffer. For experiments examining calcium-dependent uncoupling, cells were incubated with 10 µM or 100 µM calmodulin inhibitor W7 , or control medium containing vehicle (0.1% DMSO) for 8 min. The solutions were exchanged for solutions of the same composition plus 0.1% Neurobiotin and 5 µM ionomycin , incubated for 2 min, and then scraped, allowed to incubate for 10 min of diffusion, rinsed and fixed as before. Neurobiotin in fixed cells was visualized with Cy3-strepavidin .Tracer coupling was performed as previously described ,31. BrieLabeled coverslips were imaged at 40× magnification with a digital camera using HCImage software  and intensity data captured in 2 µm circular regions of interest centered within each cell of a labeled cluster. Intensity and position data were fit to a linear compartmental diffusion model ,40 using2+-free Ringer solution  for 45 min in the dark at 32 °C. Ratiometric measurements were made from single loaded cells using a photodiode with an adjustable aperture to limit the emission field using a Zeiss  Axiovert 100 microscope with a 100×/1.25 NA water immersion objective. Alternating excitation at 360 nm and 388 nm was provided by a computer-controlled monochromator-based system [2+ concentration was calculated from the ratio of emitted fluorescence at the two wavelengths [2+ concentration [HeLa cells stably-transfected with perch Cx35 were grown and plated as described above. Cells were loaded with 5 µM Fura-2, AM (Molecular Probes/ThermoFisher) in Cad system ,66. Intrelengths  using cantration .HeLa cells were transiently transfected with perch Cx35 or its calmodulin binding site mutants as described above and cultured for 48 h. The cells were fed with fresh medium and two hours later were fixed with 4% formaldehyde in 0.1 M phosphate buffer, pH 7.5 for 30 min, washed with 0.1 M phosphate buffer, and blocked 1 h in 10% normal donkey serum in 0.1 M phosphate buffer, 0.3% Triton X100. Cells were then immunolabeled using anti-Cx35/36 , 1:500 in phosphate-buffered saline + 0.3% Triton X100, 0.1% sodium azide (PBSTA). Washed coverslips were probed with Cy3-donkey anti-mouse secondary antibodies  and mounted with Vectashield with DAPI (Vector Laboratories). Coverslips were imaged with a Zeiss LSM800 confocal microscope with 63×/1.4 NA objective.Statistical analyses were performed in Prism software . Calmodulin binding parameters from 5 to 6 experiments were analyzed with One-way ANOVA. In the case of Cx35 RKIKdel and Cx35 K262E, I263A mutants, Kd was fit very poorly and varied over a wide range; in 2 of 5 experiments, no Kd could be fit to Cx35 K262E, I263A binding data. For this reason, we adopted a Brown-Forsythe ANOVA to account for differences in standard deviation of the binding parameters of these mutants compared to wild type and other mutants; Dunnett’s multiple comparison tests were used.Tracer coupling data were analyzed with ordinary One-way ANOVA with Sidak’s multiple comparisons (for experiments comparing drug treatments of wild-type Cx35) or ordinary Two-way ANOVA with Tukey’s multiple comparisons (for comparisons of ionomycin or glutamate responses of wild type Cx35 and Cx35 mutants).Analyses of the frequency of gap junctions between pairs of expressing cells employed One-way ANOVA with Dunnett’s multiple comparisons. Analyses of gap junction size parameters employed non-parametric tests because the distribution of sizes were skewed, depending on the abundance of very large plaques. A Kruskal–Wallis test with Dunn’s multiple comparisons was used.


High fertility remains one of the most important public health issues hampering the health and welfare of mothers and the survival of their children in developing nations. In Ethiopia, the high fertility rate has been seen for a long historical period with some pocket areas of high fertility still showing poor improvement. Hence, this study was aimed at determining the magnitude of high fertility status  and associated factors among women of the reproductive age group in Wonago district. p value ≤ 0.05.  A community-based cross-sectional study was conducted on randomly selected 512 women in Wonago district. Data were collected using a pretested structured interviewer administered questionnaire. Data was entered into EpiData version 3.1 and then analyzed by SPSS version 25. Logistic regression was used to analyze the data, and the adjusted odds ratio with the 95% confidence interval was computed, and a significant association was declared at Conclusion and Recommendation. The substantial number of women in the study area has high fertility status far away from the country's costed implementation plan of reducing the total fertility rate to 3.0. Considering these, much is needed to be done among poor, rural residents, who have not yet attained their desired number of children, and on enhancing the knowledge of mothers towards contraceptive methods. This study revealed that 354 (69.1%) of the respondents have high fertility. High fertility is independently associated with residing in rural area , desire for children , history of under-five child mortality , poor knowledge of contraception , and low wealth tertile . On the other hand, women with age at first birth above 18 years  and those with birth interval ≥ 24 months  were less likely to have high fertility.  Based on the demographic concept, fertility is the product or output of reproduction and not the ability to have children. This is to mean that fertility is the actual reproductive performance of a couple . The popWorld population has increased by 2 billion people over the past 25 years, from 5.3 billion in 1990 to 7.3 billion in 2015, and globally, the total fertility rate is 2.5 children per woman Africa has the highest rate of population growth among the main areas of high fertility where the population growth rate is growing at 2.55% annually in 2015 [The United Nations (UN) has projected the median world population for the year 2100 to be 11 billion, and the median projected for Mid-African Countries (MAC) is more than 3.97 billion , 7. HighUncontrolled fertility has adverse influences on socioeconomic, demographic, and environmental development of a country . Women wSub-Saharan African countries particularly have population growth rates that are outpacing their economic growth. Fertility still remains far above the replacement level in a significant number of countries. Each African mother is replacing herself with nearly two daughters with replacement level fertility of 2.1, which leads to fast population growth .Though there is a significant decrement in fertility in the world since the previous decades, it is still unacceptably high in most of the Sub-Saharan African countries. Despite the substantial decline in the total fertility rate in Ethiopia, the country is still characterized by high fertility and very young population structure. In addition, the pattern of decline in fertility is not evenly shared among different societal characteristics and geographical areas . There iIn Ethiopia, the high and persistent fertility rate has been seen for a long period. The TFR has slightly declined from 5.5 in 2000 to 5.4 children per woman in 2005 to 4.8 children per woman in 2011 and to 4.6 children per woman in 2016. Even though a declining trend is shown, like many African countries, the fertility rate of Ethiopia is still high as compared to developed nations –17.In Ethiopia, the levels of maternal mortality and morbidity are also among the highest in the world. Unspaced birth with low maternal health service access seriously affects the health of mothers and children , 19. RecFrom the Ethiopian population, about 83.5% of people live in rural areas, whereas the high fertility and rapid population growth rate especially in the rural area is unacceptably high with the total fertility rate of above 6 children per woman. Having many children is still considered to be a prestige and gift of God in most Ethiopian rural communities . ParticuTaking into consideration these realities and scenarios, this study was aimed at visualizing the current fertility status and factors contributing to uncontrolled high fertility in the study area. This is important to forward the implication of current activities of the government for the achievement of the desired fertility status, particularly the achievement of the costed implementation plan of reducing total fertility of Ethiopian women to 3.0. Moreover, this study will bring about the where and how to focus on such pocket areas of high fertility.A community-based cross-sectional study was conducted among women of reproductive age residing in Wonago district, Gedeo zone, Southern Ethiopia, located 377 km to the south of Addis Ababa. The district is well known for being the most densely populated area in the country and having the highest total fertility rate. The district has 21 kebeles , of which 17 kebeles are rural administration and 4 are semiurban. The dominant ethnic group in the study area is the Gedeo ethnic group. This study was conducted from 05 December 2017 to 18 January 2018.The study population was all women of the reproductive age group from 25 to 49 years. This group of women was taken for this particular study by considering the fact that women in the study area who have given birth at an early age have the possibility of delivering four and above children before they celebrate their twenty-fifth birthday. In this study, women with the number of children ever born alive greater than four were considered having high fertility.p) 28% [Zα/2 of 1.96, and margin of error (d) of 0.05. With this, the calculated sample size wasThe sample size of this study was calculated using the formula for estimation of single population proportion based on the proportion of women with a high fertility rate (p) 28% , 95% levConsidering the design effect (DEFF) of 1.5, the sample size becomes 310∗1.5 = 465. Finally, by adding the 10% nonresponse rate to increase the precision by reducing the sampling error, the final sample size was 512.To select the study participants, the study area was stratified into semiurban and rural, after which two  kebeles A validated structured tool adapted from the survey tools developed by EDHS Survey  and WorlThe questionnaire prepared originally in English was then translated to Amharic and the local language (Gedeo-uffa). To check for consistency of the meaning of each question, the questionnaire was further back translated from the local language to Amharic and English by different translators. The data was collected by trained diploma-holder health professionals who are fluent in Gedeo-uffa  and Amharic. Before data collection, the data collectors were trained thoroughly. The principal investigator strictly supervised the overall activities of data collection.p value less than 0.25 on bivariate analysis were further included in the multivariable analysis model. The adjusted odds ratio with their corresponding confidence interval was used as a measure of association, and p values < 0.05 were taken as statistically significant.For data analysis, the data was entered into the EpiData version 3.1 statistical software package and later exported into SPSS version 25 . DescripThe model goodness of fit was tested by the Hosmer and Lemeshow test. Multicollinearity was checked at the cutoff point of VIF > 10 to test if the independent variables were related.The outcome variable was fertility status measured by the number of children ever born (CEB) alive. It is categorized as low fertility when CEB alive is <5 and high fertility when CEB alive is ≥5. The cutoff point of 5 is taken because the medical and obstetric risk for mothers with the number of CEB greater or equal to 5 is significantly higher compared with that of less than five . It is aCEB represents the number of all children ever born alive to a particular woman in her lifetime fertility experience by the time of the survey.Maternal knowledge towards contraception was assessed by asking six knowledge-related questions. A score of 1 was given if the mother answered the given questions correctly and 0 if not. The sum of each question was computed. Taking into account the distribution of the sum score which was normally distributed, the mean was used to categorize the overall score of each participant into two groups: greater than the mean as good knowledge and less than or equal to the mean as poor knowledge.Household wealth status was estimated by principal component analysis based on fourteen household variables  that measure household assets. SPSS version 25 software was used to perform principal component analysis (PCA). Household wealth status was estimated with 3 components that explained a total variance of 62.5% and measure of sampling adequacy of 0.72. Finally, wealth status can be categorized into three groups and ranked as low, middle, and high as it was given by the factor analysis result.Ethical clearance was obtained from Dilla University College of Health Sciences and Medicine Institutional Review Board. Verbal consent was obtained from each study participant after detailed explanation of the main purpose of the study.Wide ranges of variables which can potentially affect fertility status were included in the analysis. The mean age of mothers was 35 years (SD = 5.7), and one-third of the mothers  were within the age range of 35-39 years. Majority of the study participants are Gedeo in ethnicity . Most of the children's parents  were married and live together. The residence of 383 (74.8%) mothers is rural setting, and more than half of the mothers  were illiterate. Occupationally, 268 (52.5%) mothers were housewives. About 210 (41%) of the participants in the study area were in the lowest wealth tertile .Three hundred fifty-four (69.1%) of mothers were found to have “high fertility.” About 17.8% of the participants' husbands in the study area have two or more wives. One hundred fifty-six (30.5%) of the women in the study area have ever aborted or had still birth, and 186 (36.3%) had history of under-five child mortality. The mean number of children ever born (CEB) alive for women (age 25-49 years) in this study is 6.00 with (SD = 2.1), and the parity ranges from 1 to 12. The mean age at first childbirth for the participated women is 17 years with (SD = 1.9) .Interviewers collected information regarding knowledge of contraceptive methods, and the result revealed that 397 (77.5%) of women have commendable knowledge about contraceptive methods and 115 (22.5%) of women did have poor knowledge of contraceptive methods.After controlling for the effects of possible confounders, current age category, place of residence, mother's education, wealth index, age at first birth, birth interval, knowledge of contraceptive, desire for children, and history of under-five mortality were significantly associated with fertility status . As it iRegarding the place of residence, women living in rural areas were about 5 times  more likely to have high fertility as compared to those living in semiurban settings. Women who have given their first birth at 18 years and above are 65.9%  more likely to have high fertility compared to women who have given their first birth before 18 years. Similarly, women with average birth spacing ≥ 2 years were about 74%  less likely to have high fertility compared to those who have a shorter birth interval (less than 2 years).Women who have poor knowledge of contraceptive methods were nearly 3 times  more likely to have higher fertility as compared to those who have good knowledge. Mothers within the low wealth tertile were also 2 times  more likely to have higher fertility as compared to those with high wealth tertile. Women who express their desired number of children as God given were nearly 7 times  more likely to have high fertility than those who desire to have less than five children. On the other hand, women with history of under-five child mortality were 5 times  more likely to have higher fertility than women who did not have history of under-five child mortality .The intention of this study was to determine the magnitude and factors associated with high fertility among women of the reproductive age group aged 25-49 years. The finding revealed that 69% (95% CI: 65%-73%) of the 512 respondents had high fertility. This figure is also significantly higher compared to other findings from studies conducted in different parts of Ethiopia, which include a study from Enderta district (51%)  and ButaDifferent sociodemographic characteristics have been indicated to be associated with high fertility in these studies. Regarding the household wealth tertile, women within the lower wealth tertile were more likely to have high fertility than those within the high wealth tertile. This could be due to poor access to media and health care services including family planning and low educational status by the poor. This finding is also similar to the facts in EDHS 2016  and findResidency also showed a strong association to high fertility in the current study. The fertility levels in the urban and rural areas tend to be different; women who live in the rural area have more children as compared to those living in the urban area. It is generally true that women residing in the urban area stay longer in school, thereby delaying the time for marital engagement. Moreover, the demand for human power in agrarian living condition and the notion of considering the family with a large number of children as a blessed family in the rural area in the district may drive couples to have an increased number of children. This is consistent with previous studies done in the case of Butajira , Ghana , and NepEarly childbearing is a leading sociodemographic characteristic that indicates the exposure of women to teenage pregnancy. In the area of the current study, early marriage is found to be a common phenomenon as also suggested by the result showing that more than half of the women that participated in this study had their first birth before 18 years of age. Women who gave their first birth at the age of 18 year and above were more likely to have fewer children than those who gave their first birth at an earlier age. For women with their first pregnancy at an earlier age, the period of fertility is obviously longer, resulting in higher fertility. This phenomenon is particularly serious in this study area where a large number of the women have poor knowledge of contraceptive methods. This finding is similar with previous studies done in Kersa district , EndertaAnother interesting factor in this study was a desire for compensation or replacement for loss of under-five children. In this study, women who have history of under-five mortality have higher probability of having a large number of children. This finding is consistent with previous studies done in Kersa district , EndertaKnowledge of contraception is found to be another determinant factor where women with poor knowledge on contraception were at higher risk of having higher fertility as compared to those who have good knowledge. Similar studies have been found in other parts of the country including Addis Ababa , Bale ZoWomen of the reproductive age group having an average birth interval of less than 2 years were at higher risk of having high fertility as compared to those with a birth interval more than or equal to two years. This finding is also consistent with a study conducted in Arbaminch district  and in DThis study considered a wide range of fertility variables to see high fertility differentials among women in the study, and it used a reasonable sample of respondents selected with probable sampling technique. However, there are some important limitations in using the finding of this study. As the study is quantitative in nature, there are some hidden factors that need to be addressed by qualitative study. Though the assessment was carefully done to reduce the recall bias, estimation of some sociodemographic variables like age at first birth might have been mistaken.The finding of this study implies that fertility status in the study area was alarmingly high, contesting the effort of the country to achieve the lower total fertility rate as planned on the costed implementation plan of 2020. Moreover, the finding implies that the women in the study area have a substantial unmet need for family planning that needs public health attention. Among the impressive factors influencing fertility status were the following: loss of under-five children, low wealth tertile, residing in rural areas, age at first birth, desire for children, and poor knowledge of contraception. Targeted works on knowledge about contraception, the negative effect of early childbearing/marriage, and correcting the desire for children among women in the study area are urgent public health concerns needing due consideration.