Fertility is considered an important economic trait in dairy cattle. Most studies have investigated cow fertility while bull fertility has received much less consideration. The main objective of this study was to perform a comprehensive genomic analysis in order to unravel the genomic architecture underlying sire fertility in Holstein dairy cattle. The analysis included the application of alternative genome-wide association mapping approaches and the subsequent use of diverse gene set enrichment tools.KAT8, CKB, TDRD9 and IGF1R, with functions related to sperm biology, including sperm development, motility and sperm-egg interaction. Moreover, the gene set analyses revealed many significant functional terms, including fertilization, sperm motility, calcium channel regulation, and SNARE proteins. Most of these terms are directly implicated in sperm physiology and male fertility.The association analyses identified at least eight genomic regions strongly associated with bull fertility. Most of these regions harbor genes, such as This study contributes to the identification of genetic variants and biological processes underlying sire fertility. These findings can provide opportunities for improving bull fertility via marker-assisted selection.The online version of this article (doi:10.1186/s12863-016-0454-6) contains supplementary material, which is available to authorized users. Improving reproductive efficiency of dairy cattle has become one of the major challenges of the dairy industry worldwide. The intense selection for production traits in the last decades has led to a decrease in fertility , 2. FertReproduction is a very complex process that involves numerous consecutive events, including gametogenesis, fertilization, and early embryo development, that should be accomplished in a well-orchestrated manner in order to achieve a successful pregnancy. The relative importance of the parental effects on the reproductive success, i.e., maternal versus paternal contribution to the zygote, is still largely unknown . Most stMAP1B and PPP1R11, were significantly associated with male fertility in Holsteins − 1âg, where ŝ is the vector of SNP marker effects, D is a diagonal matrix of weights of SNPs, and âg is the vector of GEBVs , and transmembrane transporter activity  showed an overrepresentation of significant genes. Of particular interest, two closely related terms, SNARE binding  and SNAP receptor activity , which involve a group of membrane-associated proteins that participate in different reproductive events including spermatogenesis and acrosome reaction, were significantly enriched with at least three genes, STX1A, STX1B and STX8, associated with sire conception rate.Several GO terms classified into the Molecular Function domain showed an overrepresentation of genes associated with sire fertility , sperm capacitation (D013075), and sperm motility (D013081). Five genes associated with SCR, namely AKAP3, BSP3, BSP5, NTRK2 and ZP2, were part of these terms. Additionally, two other terms related to fertility, follicle stimulating hormone (D005640) and pregnancy rate (D018873), were also enriched with significant genes, including AKT1, CTTNBP2NL, FSHR and IGF1R. Finally, functional categories involving protein kinases (D017868) and GTPases (D020691) were also detected as significant in the MeSH-informed enrichment analysis.Table There is growing evidence that bull fertility is influenced by genetic factors. The present study was specifically performed to unravel the genomic architecture underlying sire conception rate, an accurate phenotypic measure of dairy sire fertility. Although previous studies have attempted to identify potential genes and pathways related to SCR , 24, thiMany methods have been proposed to detect and localize genes underlying complex traits. Given that there is no method that is clearly superior than the others, it is recommended to combine multiple approaches in order to obtain more reliable findings . As suchCKB and TDRD9 that might be directly involved in sire fertility. Gene CKB encodes the enzyme creatine kinase, and previous studies have reported that elevated levels of creatine kinase in the sperm are associated with severe oligospermia and male infertility [TDRD9 encodes an helicase which plays an important role during spermatogenesis by silencing potential transposable elements, and hence, protecting the integrity of the male germline [CKB and TDRD9 in bull fertility. No less important, the results of ssGBLUP in BTA21 at 8–9 Mb strongly suggest IFGF1 as a candidate gene for sire conception rate. This gene belongs to a family of insulin-like growth factors that has important roles in sex determination, testis development, spermatogenesis and steroidogenesis [IGF1R has been implicated in regulating Sertoli cell proliferation and maturation, testis size, and sperm capacitation [IGF1R and male fertility.The significant region in BTA21 located at 68–71 Mb (see Figs. ertility . In factertility . Similargermline . Hence, ogenesis . Interescitation , 48. TheKAT8 and CCT6A, with potential roles in dairy sire fertility. The gene KAT8, a member of the MYST histone acetyltransferase family, is highly expressed during sperm development [CCT6A encodes a molecular chaperone that mediates the sperm-ooctyte interaction during fertilization [SEPT12 and MGRN1. Indeed, SEPT12 is expressed specifically in the testis and encodes a GTP-binding protein that has been implicated in sperm morphogenesis, sperm motility and male infertility [MGRN1 is widely expressed in the male reproductive system, and recent studies have shown that MGRN1 knockout in mice results in male infertility, with disruption of hormones secretion and impaired sperm motility [Both ssGBLUP and cGWAS identified the region in BTA25 at 26–28 Mb as significantly associated with SCR. This region harbors at least two genes, namely elopment , and it elopment . In addilization . Moreoveertility , 53. Likmotility . It shoumotility . OverallAKAP3, BSP3, BSP5, NTRK2, SLC22A16, and ZP2, were part of these functional categories. Interestingly, the gene AKAP3 is expressed in the spermatozoa and is involved in sperm motility, sperm capacitation, and the acrosome reaction [BSP3 and BSP5 are two binder of sperm proteins implicated in sperm capacitation and fertilization [ZP2 encodes a sperm receptor that mediates gamete recognition during the fertilization [Given that whole-genome scans only detect the most significant regions, and these regions explain only a small fraction of the genetic variance, additional approaches are needed in order to dissect the complex genetic architecture of a quantitative trait. In the present study, different pathway-based approaches, using GO and MeSH databases, were used in order to obtain additional insights regarding the genetic determinants and biological mechanisms underlying sire fertility. Interestingly, some biological processes directly related to male fertility, such as fertilization and sperm motility, were among the most significant functional categories. Further analyses revealed that at least six genes associated with SCR, including reaction . In addilization . The genlization . These fOf special interest, GO molecular function terms related to SNARE proteins showed an overrepresentation of significant genes. SNARE proteins are implicated in membrane fusion events, including several events that occur during spermatogenesis and also the acrosome reaction . In factIn this study, a comprehensive genomic analysis was performed with the purpose of unravelling the genetic architecture underlying sire conception rate in Holstein dairy cattle. Genomic regions in BTA5, BTA9, BTA13, BTA15, BTA21 and BTA25 were associated with sire fertility. Most of these regions harbor genes with known roles in sperm biology, including sperm maturation, motility and fertilization. Moreover, gene set analysis revealed that many of the significant terms, such as reproductive process, calcium ion channels, and SNARE proteins, are implicated in biological processes related to male fertility. Overall, this integrative study sheds light on the genetic variants and mechanisms underlying this complex phenotype in cattle. In addition, these findings can provide opportunities for improving bull fertility via marker-assisted selection.


The multifunctional sphingosine-1-phosphate (S1P) is a lipid signaling molecule and central regulator in the development of several cancer types. In recent years, intriguing information has become available regarding the role of S1P in the progression of Glioblastoma multiforme (GBM), the most aggressive and common brain tumor in adults. S1P modulates numerous cellular processes in GBM, such as oncogenesis, proliferation and survival, invasion, migration, metastasis and stem cell behavior. These processes are regulated via a family of five G-protein-coupled S1P receptors (S1PR1-5) and may involve mainly unknown intracellular targets. Distinct expression patterns and multiple intracellular signaling pathways of each S1PR subtype enable S1P to exert its pleiotropic cellular actions. Several studies have demonstrated alterations in S1P levels, the involvement of S1PRs and S1P metabolizing enzymes in GBM pathophysiology. While the tumorigenic actions of S1P involve the activation of several kinases and transcription factors, the specific G-protein -coupled signaling pathways and downstream mediated effects in GBM remain to be elucidated in detail. This review summarizes the recent findings concerning the role of S1P and its receptors in GBM. We further highlight the current insights into the signaling pathways considered fundamental for regulating the cellular processes in GMB and ultimately patient prognosis. The lipid mediator sphingosine 1-phosphate (S1P) regulates a variety of cellular processes including inflammation, oncogenesis, metastasis, survival, stem cell behavior and the formation of microvascular networks, which provides nourishment to cancerous cells . The S1POver the last two decades or so, a large amount of information has become available regarding the role of S1P-mediated cellular processes in cancer development. Several studies have associated alteration in S1P levels, and the involvement of its receptors and metabolite enzymes in many types of cancer pathophysiology. In an animal model of intestinal tumorigenesis, increased S1P levels associated with reduced S1P lyase (SPL) expression and enzyme activity were observed compared to other local tissues . In humade novo from serine, palmitoyl-CoA and fatty acids or from the breakdown of membrane-resident sphingomyelin [In vitro and in silico analyses showed that sphingosine metabolized in lysosomes was preferentially utilized for the generation of ceramide in astrocytes. In contrast, in glioma cells, sphingosine was primarily used for the production of survival-promoting S1P [Sphingolipid and particularly S1P synthesis are tightly controlled by the metabolism of ceramide. Ceramide biosynthesis occurs either gomyelin . Ceramidgomyelin . S1P deggomyelin  . In the following section, we attempt to elaborate how the individual or simultaneous S1P-mediated signaling cascades participate in cellular responses in GBM progression. In GBM, S1P activates multiple signaling pathways in parallel including mitogen-activated extracellular signal-regulated kinase (MAPK/ERK), protein kinase C (PKC), Ca2+ system and PLD [The activation of the mitogen-activated extracellular signal-regulated kinase cascade occurs via the stimulation of several growth factors, including the epidermal growth factor, the platelet-derived growth factor and the vascular endothelial growth factor . In a co and PLD .Some small adapter proteins also play an additive role in the activation and inactivation of ERK signaling. For example, GluR2 (glutamate receptor 2) overexpression in U87-MG cells inhibits proliferation by inactivating ERK1/2/Src (sarcoma proto-oncogene kinase) phosphorylation, and induces apoptosis and expression of the scaffold protein GRIP (glutamate receptor interacting protein), which is essential for the effect of GluR2 on ERK/Src inactivation . AnotherPI3K/AKT is another signaling pathway involved in GBM cell growth, proliferation, survival, and motility . The serIn our own studies, to find out which pathway is involved in the S1P-stimulated migration of LN18 GBM cells, specific inhibitors of signaling protein molecules known to be activated by S1P, were utilized. Only the blocking of PI3K/AKT1 signaling by LY294002 completely inhibited S1P stimulated LN18 cell migration. This result was underlined by an amplified phosphorylation of AKT1 after stimulation of the cells with S1P. The PI3K/AKT1 pathway is known to be activated by S1PR1, S1PR2, and S1PR3 via the Gi-coupling of these receptors, of which, AKT is considered as a major downstream signaling molecule of S1PR1. For S1PR1 and S1PR3 a PI3K/AKT-dependent stimulation of cell migration was described, whereby S1PR2 uses this signaling cascade only as a side trail, while G12/13-protein-mediated Rho signaling /phosphatase and tensin homolog (PTEN)/PI3K/AKT pathway is mutated or overly activated in the majority of GBM subtypes. Thus, activation of AKT and phospho-AKT levels are elevated in the majority of GBM tumor samples and cell lines, enabling these cells to grow in an uncontrolled manner, evade apoptosis, and enhance tumor invasion . IntriguProtein kinase C isozymes are a ubiquitous group of phospholipid-dependent serine/threonine kinases that function in numerous different cell types including cancerous cells. A broad foundation of data has established the central role of PKC isozymes in multiple signal transduction systems as key regulators of cell function, including differentiation, proliferation, survival and motility . PKC isoThe role of PKC and its isozymes in the oncogenic regulation has been studied in in-vitro and in-vivo models of high-grade gliomas . PKC isoDespite the profound role of various PKC isoforms in GBM, the link between S1P signaling and PKC is only sparely addressed in GBM. It has been demonstrated that PKC regulates SphK1 and increases S1P secretion, thereby allowing its autocrine/paracrine actions . InteresIn recent years, several studies have shown that the activation of phospholipase signaling networks has been involved in cancer growth. It has been shown that S1P increases inositol phosphate levels by activating phospholipase C (PLC) . S1P-indIn most cases, when the GPCRs are linked to PLC, the PTX insensitive part of the enzyme activation is mediated through the G-proteins . Thus, i2+ mobilization, in several types of cells [2+ signals in glioma C6 cells [2+ system and PLD which is responsible for proliferation [2+ system [S1P has been shown to activate PLC, and subsequently Caof cells . SphingoC6 cells . Furtherferation . However+ system . Thus, SPLD enzymes, namely PLD1 and PLD2, hydrolyze membrane phospholipids, such as phosphatidylcholine (PC), to generate phosphatidic acid (PtdOH), which acts as an important lipid second messenger for GPCR. As a result, both PLD and phosphatidic acid contribute to cancer cell proliferation and survival. Multiple cancer categories, including breast, gastric, and renal cancers, show elevated PLD activity compared with normal tissue . FurtherGTPases are molecular knobs that regulate a wide variety of signaling pathways in many cell types including glial cells. The principal function of these GTPases is to control the regulation of the actin cytoskeleton. However, their role in the formation of microtubule dynamics, cell polarity, and transcription factor activity is also imperative . SignaliThe Rho signaling pathway is involved in the inhibition of GBM cell migration through S1PR2, which is linked with the PTEN-independent activation of RhoA and suppression of Rac1 . On the Altogether, evidence from diverse in vitro and in vivo studies indicate that S1P, its metabolizing enzymes, and S1PRs play significant roles in GBM cell fate determination. Numerous studies demonstrated that activation of S1PRs-mediated intracellular signaling pathways establish functional actions of S1P utilized by GBM. Despite the fact that the signaling mechanisms of each S1PR subtype have been fairly well recognized, their functional roles in GBM are still inadequately understood. Further, although convincing implications are available for the role of S1P in GBM proliferation and survival, invasion, migration, and metastasis, there are still some discrepancies about the impact of S1P signaling on the molecular mechanisms of GBMs due to their heterogeneity. An alternative reason is the complex glioblastoma microenvironment which is dynamically regulated by intra- and inter-cellular signaling cascades. The involvement of small proteins like mTORC1, metalloproteases, FGF-2 and NF-kB promotes tumor growth and invasion by enduring glioblastoma stem cells, thereby promoting angiogenesis. Therefore, there is much to be discovered about the benefits and risks of inhibiting the different small molecules and effector proteins involved in multiple tumorigenic pathways. Further clinical studies are essential to finding a promising alternative to treat GBM patients rather than the conventional treatment. Last, but not least, a broader understanding of the mechanisms of S1P signaling in GBM progression and a more sophisticated experimental system are required in order to develop effective treatment strategies.


Soft-tissue sarcomas account for 0.7% of all malignant tumors, with an incidence rate of 3 per 100,000 persons/year. The undifferentiated pleomorphic sarcoma (UPS) with giant cells, a high grade tumor of soft tissue, is very unusual, especially in young adults before the age of 40. Human T-cell lymphotropic virus type 1 (HTLV-1) is a human retrovirus, classified as group 1 human carcinogens by The International Agency for Research on Cancer, that causes an aggressive malignancy known as adult T-cell lymphoma/leukemia and a progressive chronic inflammatory neurological disease named HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-1 causes accumulation of genetic mutations in the host genome that could contribute to cellular transformation, one of the oncogenic features of HTLV-1.We describe a case of a young woman with UPS who suffered from HAM/TSP with 3 years of evolution. In 2013, the patient started with neurological symptoms: weakness in the legs and bladder dysfunction. One year later, the patient developed a mild paraparesis in both extremities, anti-HTLV-1 antibodies were detected in plasma and in cerebrospinal fluid, and HAM/TSP was confirmed. In November 2015, a benign ganglion cyst was first suspected without intervention and by March 2016 a sarcoma was diagnosed. Three weeks after surgical resection, the tumor aroused in deep tissue and behaved aggressively, implicating a curative wide resection of the fibula, joint reconstruction, and soft-tissue graft. Histopathological examination confirmed UPS with giant cells.The unapparent subclinical immunodeficiency state due to HTLV-1 infection deserves to be considered in order to carefully monitor the possibility of developing any type of cancer. Besides, reaching an accurate and timely diagnosis of UPS can be challenging due to the difficulty in diagnosis/classification and delayed consultation. In this particular case, considering the high grade of UPS and the progressive invalidating myelopathy caused by HTLV-1, treatment should be carefully evaluated to positively impact on the patient’s life expectancy. Human T-cell lymphotropic virus type 1 (HTLV-1) is a retrovirus that causes an aggressive malignancy known as adult t-cell lymphoma/leukemia (ATLL) and a progressive chronic inflammatory neurological disease named HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). In 2014, HTLV-1 was classified as group 1 human carcinogens by The International Agency for Research on Cancer (IARC) (KRAS proto-oncogene and in PIK3CA have been recently identified by gene mutation screening (KRAS (located at 12p12.1) is frequently altered with mutations occurring in 17–25% of all cancers, while mutations in PIK3CA have been identified in several human solid tumors including breast, colon, ovarian, liver, and lung cancers (Considering undifferentiated pleomorphic sarcoma (UPS), two missense mutations in the  cancers , 5.®, Inbio, Tandil, Argentina) and analyzed with “in-house” n-PCR and by real-time SYBR Green PCR (qPCR) as previously described, respectively  was indicated with a decreased in PVL in the next months and improvement of neurological symptoms and signs.We present a case of UPS with giant cells in a 46-year-old woman with HAM/TSP from a non-endemic HTLV-1 area of Argentina. In November 2013, the patient started with weakness in the legs and bladder dysfunction. One year later, she presented mild paraparesis in both extremities and hyperreflexia. By February 2015, detection of anti-HTLV-1 antibodies in plasma and cerebrospinal fluid (CSF) by ELISA  and western blot  confirmed HTLV-1 infection. For HTLV-1 molecular confirmation and proviral load (PVL), DNA was extracted from peripheral blood mononuclear cells (PBMCs) by column extraction  that had arisen in the anterior right ankle. A benign ganglion cyst was first suspected without the need of an immediate intervention. However, a consistent mass grew quickly. A computed tomography revealed a mass of indefinite borders, and consequently, a surgery to remove the tumor was performed in March 2016. Based on a biopsy, a sarcoma was diagnosed; metastasis was discarded by positron emission tomography (PET) scans. Three weeks after surgical resection of the primary tumor, a new mass was detected near the primary one and the patient attended a private hospital, where a local recurrence was confirmed. Metastasis was again discarded by PET scans. She had no pain, no symptoms (weight loss and/or fatigue), and the laboratory analysis was normal. In June 2016, after a new laboratory exam and MRI, two possible surgical interventions were discussed in an athenaeum with different specialists: leg amputation considering the rapid progressive spasticity due to HAM/TSP or an excisional biopsy. The latter was indicated considering also the patient’s decision. This time the tumor aroused in deep tissue and behaved aggressively, implicating a complete and extended surgical resection to the fibula, joint reconstruction, and soft-tissue graft. Macroscopic examination showed a tumor with a solid grayish cut surface and no clear envelope. The histopathology exam showed fascicles and sheets of spindle cells in a loose/storiform pattern. Numerous markedly atypical cells and some multinucleated giant cells admixed with tumor pleomorphic cells were observed. Chronic inflammation and abundant atypical mitosis, and necrosis, and hemorrhages areas were also present. This histopathologic pattern defines the lesion as UPS with giant cells . All co-authors had access to all the study data and were responsible for the accuracy of the analysis. They also had authority over manuscript preparation and the decision to submit the manuscript for publication. 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.


Similarly, co-expression of Cx40 Q236H with Cx43 resulted in a significantly lower Gj. Transjunctional voltage-dependent gating (Vj gating) properties were also altered in the GJs formed by Q236H. Reduced GJ function and altered Vj gating may play a role in promoting the Q236H carriers to AF.Atrial fibrillation (AF) is the most common form of cardiac arrhythmia. Recently, four novel heterozygous Cx40 mutations—K107R, L223M, Q236H, and I257L—were identified in 4 of 310 unrelated AF patients and a followup genetic analysis of the mutant carriers’ families showed that the mutants were present in all the affected members. To study possible alterations associated with these Cx40 mutants, including their cellular localization and gap junction (GJ) function, we expressed GFP-tagged and untagged mutants in connexin-deficient model cells. All four Cx40 mutants showed clustered localization at cell–cell junctions similar to that observed of wildtype Cx40. However, cell pairs expressing Cx40 Q236H, but not the other individual mutants, displayed a significantly lower GJ coupling conductance (G Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia affecting millions of people worldwide ,2. With GJ channels are composed of connexins. In humans, there are 21 different connexins and all of them share a similar topological structure of four transmembrane domains (M1–M4), two extracellular domains (E1 and E2), one cytoplasmic loop (CL), and both the amino terminus (NT) and carboxyl terminus (CT) lie within the cytoplasm ,16. Six GJA5) have been linked to AF patients and families [GJA5 in four of 310 unrelated AF patients, resulting in heterozygous missense mutants in Cx40 protein: K107R, L223M, Q236H, and I257L [The importance of Cx40 and Cx43 in the heart has been highlighted in animal models and genetic mutation studies. Mice with an ablation of Cx43 in the heart develop ventricular arrhythmias leading to sudden cardiac death . An in vfamilies ,12,13,14nd I257L . Furthernd I257L . FunctioExpression of AF-linked Cx40 mutants  was used to study their localization in live HeLa cells. As shown in js) of cell pairs expressing each of the Cx40 mutants and wildtype Cx40 are presented (p > 0.05 for each of the mutants). The coupling conductance (Gj) of cell pairs expressing K107R, L223M, or I257L was also not different from that of wildtype Cx40 (j was observed in cell pairs expressing Q236H (p < 0.05).Dual whole-cell patch clamp was used to study the functionality of untagged AF-linked Cx40 mutants in N2A cell pairs. Representative junctional currents (Iresented A. The avype Cx40 C. Howevej gating) of AF-linked Cx40 mutants, we measured Ijs in cell pairs in response to a series of Vj pulses  when Vjs ≥ 40 mV  of each mutant (filled circles) or wildtype Cx40 (open grey circles) was plotted at different Vjs  at each of the tested Vjs , except Q236H GJ that showed consistently lower τs at all tested Vjs (p < 0.05).To analyze Vsted Vjs A. The avjs that were not statistically different from those of wildtype Cx40:Cx43 (j of cell pairs co-expressing Q236H:Cx43 was significantly lower than that of wildtype Cx40:Cx43 (p < 0.05).To investigate if AF-linked Cx40 mutants had a trans-dominant negative effect on wildtype Cx43, each of the mutants was co-expressed with Cx43 (with an untagged DsRed). Cell pairs successfully expressing both connexins were selected for dual whole-cell patch clamp. Cell pairs successfully co-expressing K107R:Cx43, L223M:Cx43, or I257L:Cx43 showed coupling percentages and Gx40:Cx43 B,C. The js than wildtype Cx40 when expressed alone or co-expressed with Cx43. To further test whether Q236H could affect the function of heterotypic Q236H/Cx40 GJs, we mixed cells expressing Q236H (with untagged GFP) with cells expressing Cx40 (with untagged DsRed) and performed dual patch clamp on heterotypic cell pairs  (Propidium iodide (PI) uptake assay was used to investigate the hemichannel function of AF-linked Cx40 mutants as elevated PI uptake was observed in AF-linked Cx40 mutants, including L221I ,39. Figu< 0.001) B. These j) when expressed alone or together with Cx43. The GJs formed by each of these mutants also failed to show any obvious change in the Vj gating properties. In contrast, Q236H GJs exhibited a significantly reduced Gj when expressed alone or together with Cx43. In addition, Q236H GJs also showed altered Vj gating, specifically a reduction in the Vj required to close the channel (V0) and an increase in Vj-gating kinetics. These defects associated with Q236H might play a role in the pathogenesis of AF in the mutant carriers.In this study, we examined morphological and functional characteristics of four recently identified AF-linked Cx40 mutations  in vitro. Our localization experiments showed that YFP-tagged K107R, L223M, Q236H, and I257L were able to form GJ plaque-like structures at the cell–cell interface in HeLa cells, similar to that of wildtype Cx40. PI uptake by each of these mutant (untagged)-expressing cells showed no significant increase from that of Cx40, indicating no increase in hemichannel activity in any of the mutant-expressing cells. Dual patch clamp experiments revealed that the Cx40 mutants  showed no apparent change in coupling conductance (GGJA5 gene (encoding Cx40) have been identified in AF patients with genetic predisposition [j) was observed in G38D, I75F, A96S, and Q236H GJs, probably due to impairment at the GJ channel [j levels of G38D and A96S GJs have been reported [j of heterotypic mutant/wildtype GJs, altered Vj gating properties of homotypic  or heterotypic  Vj gating properties, a substantially reduced open probability without changing unitary channel conductance (I75F), or elevated unitary channel conductance (G38D and M163V) [So far, a total of ten germlines and three somatic mutations in the coding region of the position ,14,37,40position . Similarposition . Functioposition ,41. (2) position ,14,38,39 channel ,42. Some channel ,42. Althreported , the majreported . (3) Detd M163V) . (4) Onld M163V) . (5) Thed M163V) ,45,46,47j) of AF-linked Cx40 mutants due to either an impaired localization or GJ channel function is known to reduce the action potential conduction velocity [j reduction not only in the mutant GJs but also when they are co-expressed with wildtype Cx40 and/or Cx43 [j when expressed alone and together with Cx43. We did not perform co-expression of this mutant with wildtype Cx40 because our untagged Cx40 construct (Cx40-IRES-DsRed) had a very low transfection efficiency. Second, enhanced Vj gating by lower Vjs and faster gating kinetics by Cx40 mutants could also dynamically down-regulate Gj when sufficient junctional delays exist [j gating with a minimum conductance level reaching a quarter of the maximum Gj [0, and faster Vj-dependent deactivation kinetics of Q236H mutant GJs predict an increased Vj gating when sufficient junctional delay exists. It is not clear whether Vj gating of Cx40 or Q236H GJs could dynamically down-regulate Gj as observed for Cx45 GJs [j) in different possible atrial GJs. At present, we cannot rule out other changes, such as biosynthesis, and turnover of the mutant Cx40 protein may also change the abundance and function of Cx40 at the intercalated discs, which may need genetically modified animal models to assess fully.As discussed above, there is a variety of molecular/cellular changes associated with AF-linked Cx40 mutants. Whether these molecular/cellular changes play a role in the pathogenesis of AF is not clear. Several theoretical possibilities exist. First, a reduced macroscopic coupling conductance . It is possible that one or more of these mutants are benign mutants that do not necessarily play a role in promoting AF, especially at the least conserved position, I257. (2) Both of our model cells, HeLa and N2A cells, are convenient model systems to study localization, function of GJs, and hemichannels. They are GJ deficient, easily transfected with connexin mutant constructs, and easily accessible for morphological, dual patch clamp, or dye-uptake experiments. However, these cells are not cardiomyocytes and may not recapitulate all aspects of GJs at the intercalated discs of cardiomyocytes and, therefore, some defects might go undetected. Future studies on cell models that are closer to atrial myocytes will likely help to resolve the role of these Cx40 mutants linking to AF. (3) We have good rationales to focus our study on the morphology and functional changes in GJs and hemichannel activities. However, there are other unconventional functions of connexins, including but not limited to aggregation of protein complexes at the cell–cell junctions, adhesion, cell growth control, and differentiation, etc., that require specific biological assays to evaluate.In our present study, we did not detect any obvious defects in GJ distribution, function, or hemichannel activities in three out of four AF-linked Cx40 mutants . It is not clear how these mutants relate to AF. Here are some possibilities. (1) These mutants are located in the CL (K107R), M4 (L223M), and CT (I257L) domains of Cx40. The CL and CT domains of Cx40 show a lot more residue variation in different vertebrate species than the E1, E2, and M1–4 domains. Following this general trend, the conservation percentage at these residue positions in Cx40 are L223 (in M4 domain) 85%, K107 (in CL domain) 74%, and I257 (in CT domain) 28% across 47 different species  were created as previously described ,59. The K107RForward: 5′ CAGGAGAAGCGCAGGCTACGGGAGGCC 3′Reverse: 5′ GGCCTCCCGTAGCCTGCGCTTCTCCTG 3′L223MForward: 5′ CTCCTCCTTAGCATGGCTGAACTCT 3′Reverse: 5′ AGAGTTCAGCCATGCTAAGGAGG 3′I257LForward: 5′ CCCTCTGTGGGCCTAGTCCAGAGCTGC3′Reverse: 5′ GCAGCTCTGGACTAGGCCCACAGAGGG3′Q236HForward: 5′ GGAAGAAGATCAGACACCGATTTGTCAAACC3′Reverse: 5′ GGTTTGACAAATCGGTGTCTGATCTTCTTCC3′All these Cx40 mutant constructs were sequenced to confirm the accuracy of the nucleotide sequence.2. Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM)  containing 10% fetal bovine serum, 1% penicillin, 1% streptomycin, 4.5 g/L d-(+)-glucose, 584 mg/L l-glutamine, and 110 mg/L sodium pyruvate. Twenty-four hours before cell transfection, N2A or HeLa cells were replated into a 35-mm dish at 60% confluency. Transfection was performed the next day by adding 0.8–1 μg of DNA with 2 μL of the transfection reagent X-tremeGENE HP DNA . To assess the effect of Cx40 mutants on wildtype Cx43, N2A cells were transfected in a 1:1 ratio of Cx40 mutants-IRES-GFP and Cx43-IRES-DsRed. Cell pairs successfully co-expressing both GFP and DsRed were selected for measuring coupling conductance with dual whole-cell patch clamp (see below).Connexin-deficient mouse neuroblastoma (N2A) and human cervical carcinoma (HeLa) cells  were cultured at 37 °C with 5% COHeLa cells were transiently transfected with YFP-tagged Cx40 mutants. One day after transfection, cells were replated on 10 mm glass coverslips and incubated overnight. The number of successfully transfected cell pairs forming GJ plaque-like structures at the cell–cell interface were counted. A confocal microscope (Zeiss LSM800 with Airyscan)  was used to observe mutant-YFP and wildtype Cx40-YFP localizations as described earlier .2, 2 mM CaCl2, 1 mM BaCl2, 2 mM CsCl, 2 mM Na Pyruvate, and 5 mM d-glucose with pH and osmolarity of 7.4 and 310–320 mOsm, respectively. The recording chamber was placed on an upright fluorescent microscope  to visualize reporter (GFP)-positive cell pairs. Patch pipette was filled with intracellular solution (ICS) containing 130 mM CsCl, 10 mM EGTA, 0.5 mM CaCl2, 4 mM Na2ATP, and 10 mM Hepes with pH 7.2 and osmolarity of 290–300 mOsm. Dual whole-cell patch clamp technique was performed on isolated cell pairs expressing the Cx40 mutant. Initially, cell pairs were both voltage clamped at 0 mV. Then, a 20 mV voltage pulse was applied to one of the cell pairs (pulsing cell) while keeping the other clamped at 0 mV (the recording cell). If functional GJ channels exist between the cell pairs then a transjunctional current (Ij) can be measured at the recording cell via MultiClamp 700A  and stored in a PC via an AD/DA interface (Digidata 1322A) and pClamp9.2 software . Gj was calculated (Gj = Ij/Vj). Vj gating properties were studied by applying a series of voltage pulses (±20 to ±100 mV in 20 mV increment) as described in our previous studies [On the experimental day, transfected N2A cells were replated onto glass coverslips and incubated for 1.5 to 3 h. Coverslips with cells were transferred into a recording chamber and bathed in extracellular solution (ECS) containing 135 mM NaCl, 5 mM KCl, 10 mM Hepes, 1 mM MgCl studies ,56.2, 2 mM CaCl2, 10 mM HEPES, and 25 mM d-(+)-glucose adjusted to pH 7.35 and osmolarity of ~298 mOsm. HeLa cells were incubated in a divalent cation-free extracellular solution (DCF-ECS) containing PI (150 μM). Removal of both Ca2+ and Mg2+ ions, as well as including EGTA (2 mM) in the DCF-ECS, were used to facilitate PI uptake via GJ hemichannels. After incubation at 37 °C for 15 min, cells were washed three times with DCC-ECS at room temperature prior to observation with a fluorescent microscope . The number of transfected HeLa cells with or without PI uptake was counted and the percentage of cells with PI uptake was calculated. Only isolated individual HeLa cells were counted to prevent errors caused by GJ channels in cell clusters.AF-linked Cx40 mutants hemichannel function were assessed using propidium iodide (PI) uptake assay. HeLa cells were transiently transfected with each of the Cx40 mutants in an IRES-GFP vector . We usedj) using dual patch clamp, and the percentage of cells displaying PI uptake for the hemichannel study. One-way ANOVA followed by Tukey post-hoc test was used to compare the Boltzmann fitting parameters of each mutant and wildtype Cx40 at the corresponding Vj polarity. Statistical significance is denoted with different levels of significance .Mann–Whitney U test was used to compare each of the mutants against wildtype Cx40 for the percentage of cell pairs with morphological GJ plaques, the coupling percentage and conductance (Gj when expressed alone or with the wildtype Cx43, and altered Vj-gating kinetics and the V0, which might play a role in AF pathogenesis. The other mutants—K107R, L223M, and I257L—did not exhibit any apparent GJ or hemichannel functional impairments in our cell models.In summary, our results indicate that the AF-linked Cx40 Q236H mutation exhibited GJ function impairment by reducing the overall G


Signal transduction is the process through which cells communicate with the external environment, interpret stimuli and respond to them. This mechanism is controlled by signaling cascades, which play the role of intracellular transmitter, being able to transmit biochemical information between cell membrane and nucleus. In theory as well as in practice, it has been shown that a perturbation can propagate upstream (and not only downstream) a cascade, by a mechanism known as retroactivity. This study aims to compare the conditions on biochemical parameters which favor one or the other direction of signaling in such a cascade.n are described by an iterative map of second order, meaning that the cascade tiers are actually coupled three-by-three. We study the influence of the biochemical parameters in the control of the direction of transmission – upstream and/or downstream – along a signaling cascade. A numerical and statistical approach, based on the random scan of parameters describing a 3-tier signaling cascade, provides complementary findings to the analytical study. In particular, computing the likelihood of parameters with respect to various signaling regimes, we identify conditions on biochemical parameters which enhance a specific direction of propagation corresponding to forward or retro-signaling regimes. A compact graphical representation is designed to relay the gist of these conditions.From a mathematical point of view, we show that the steady states of a cascade of arbitrary length The values of biochemical parameters such as kinetic rates, Michaelis-Menten constants, total concentrations of kinases and of phosphatases, determine the propensity of a cascade to favor or impede downstream or upstream signal transmission. We found that generally there is an opposition between parameter sets favoring forward and retro-signaling regimes. Therefore, on one hand our study supports the idea that in most cases, retroactive effects can be neglected when a cascade which is efficient in forward signaling, is perturbed by an external ligand inhibiting the activation at some tier of the cascade. This result is relevant for therapeutic methodologies based on kinase inhibition. On the other hand, our study highlights a less-known part of the parameter space where, although the forward signaling is inefficient, the cascade can interestingly act as a retro-signaling device.The online version of this article (doi:10.1186/s12918-016-0303-2) contains supplementary material, which is available to authorized users. Cell signaling is responsible for the development and functioning of both unicellular and multicellular organisms. Abnormal cell signaling leads to diseases which involve at least one breakdown in cell communication .Signaling pathways control and regulate the flow of biochemical information between cells and their external environment, which is essential for cell signaling. Typically, a stimulus  is detected on the surface of the plasma membrane, activating complex signaling. Covalent modification cycles are one of the major intracellular signaling mechanisms, both in prokaryotic and eukaryotic organisms . Kinase Recent theoretical and experimental studies have demonstrated that kinase cascades exhibit bidirectional signal propagation via a phenomenon termed retroactivity –11. This1In , 11–14 tIn , an arbiIn  it has bIn  the authSimilar conclusions have been experimentally observed on a two-branch MAP-Kinase cascade, allowing to activate responses of JNK and p38MAPK  . Here thIn this paper, we address the question of simultaneous bidirectionality in signaling cascades, and we use both analytical and numerical approaches. Our main goal is to develop a comparative study of parameters affecting forward or retroactive responses in linear signaling cascades Fig. a. In the1uniform distribution of biochemical parameters lying in some predefinite ranges. However, the fact that numerous efficient signaling cascades, and retroactivity effects, have been measured experimentally, suggests that the estimates reported on Fig. 1Evidently, any estimate of such probabilities strongly depends on the assumed distribution of biochemical parameters from which the sampling is performed. Presumably, the actual parameter distribution existing in natural signaling pathways is not uniform. On the other hand, this knowledge is currently out of reach, or would be very hard to access. Therefore in this paper we choose as a reference point, a n of cycles of single covalent modifications, e.g. single phosphorylation-dephosphorylation cycles. We also assume that the last level of the cascade may be altered by a kinase inhibitor, represented by a drug D, that blocks the action of the active protein by sequestering it into an inactive complex  to the stimuli it is subjected to, namely the activator signal (at the top) and the inhibiting drug (at the bottom).Our overall purpose is to investigate the working principles of such a generic cascade in terms of biochemical parameters like reaction rates and total enzyme concentrations. Thus, identify which parameter ranges are associated to specific signaling behaviors, which we call n−1)th to the first tier) and whether this is compatible with an efficient forward signaling relative to the nth tier.Practically, this means to measure two effects simultaneously: the impact of the activator on downstream proteins (dose-response curves) and the effect of the drug on the upstream proteins (drug-response curves). Specifically, we are interested in studying how the retroactive signaling propagates . Therefore, in a second part of the Results we present a statistical investigation based on numerical computations, about all forms of signaling (forward and/or retroactive), for inhomogeneous cascades but with n fixed to 3.In the following, we firstly show analytical results characterizing the most “natural" direction of propagation – the forward signaling – on a generic cascade of n-cycle cascade depicted on Fig. 1n iterative equations  can be rewritten as: s and xn+1.which represents the iterates of a second-order discrete map with boundary conditions given by signal i.e.dT=0, then fn.We remark that, if the drug is absent, xn(s) as follows, provided that all the parameters (even dT) are fixed, except s : given xn∈ function s(xn) to obtain an analytical expression of xn(s), because this requires finding the roots of a high-degree polynomial. Nonetheless, we illustrate how to provide qualitative knowledge of the non-saturating region, and quantitative estimation of the saturation value of the dose-response function.For a cascade of i=1,2,…,n. We say that such a system is a homogeneous cascade, and in the rest of the paper we will omit to write the lower index i, if unnecessary. Some generalizations of our results to inhomogeneous cascades are given in In order to simplify the analytical expressions we assume that the parameters defined in Eq. xn(s) piecewisely by matching analytical quantities (depending on parameters) evaluated at the origin (s=0) with the ones deduced at saturation (s→+∞), as illustrated in Fig. The strategy consists in approximating The non-saturated part of the dose-response function is roughly described by a polynomial function , and the saturated part by a constant function.χ=xn″(0)<0 <0 Fig. a, then: σ<∞ and 0<α<1 are defined as σ=xn′(0) and where 0<χ>0 . Precisely, if xn(s) is convex (χ≤0), the dose-response only depends on its initial slope σ and on its asymptotic value α, while if xn(s) is logistic-like (χ>0), also the value of its initial curvature χ plays a role .Hence, a dose-response function can be sketched by a simple curve depending on the three parameters =α/2 in Eqs. An advantage of our methodology is that, as shown below, s=EC50.Effective Hill coefficients can be subjected to several definitions. A possible estimate may be obtained by computing twice the response coefficient xn(s) is generally speaking intractable. Nevertheless, for a homogeneous cascade, we prove that  characterizing the sketchy dose-response curves .Therefore we have shown how the parameters  inhibiting the activated protein at the nth tier. Our goal is to study the effect of such a perturbation on the upstream levels as a function of some normalized drug concentration dT, assuming that the signal s at the top of the cascade is constant and fixed at a high value.By retroactivity of order k (1≤k≤n−1), the variation of the activated protein at the (n−k)th level as a function of the drug concentration, described by the function xn−k(dT), which we refer to as drug-response function. In particular the highest order of retroactivity in a linear cascade corresponds to the response curve x1(dT).We classify the retroactivity according to its maximal propagation range, so that we call n, the amount of activated protein decreases, at level n−1 it increases, then it decreases at level n−2 and so on, up to the first level. It follows that function x1(dT) is increasing if n is even and decreasing if n is odd. Moreover, retroactivity is overall attenuated in long cascades, but can propagate and amplify its effect for n sufficiently small, e.g. equal to three , thus considering 4 orders of magnitude  in uniform log10 scale. The interpretation of the results depends yet on the choice of the reference unit concentration . For example, if the reference dimensional concentration is chosen as 0.1 μM, this leads to interpreting the scanned intervals as the range , which seems reasonable as intracellular concentrations [In our study, amongst the 18 sampled biochemical parameters, 12 dimensions corresponded to chemical concentrations and 6 dimensions to reaction rates (with dimension of inverse of time). They were all chosen in the interval [10trations . Howeveri.e. no-signaling, forward signaling, retrosignaling, or simultaneous forward and retro-signaling. Obviously, the absolute value of these numbers depend on the arbitrary thresholds on response amplitudes, that we fixed to assess the occurence of these signaling modes. To give evidence of the effect of changing these thresholds on the probabilities of signaling, Table On Fig. 1k1), admitting both forward and retro-signaling of order 2, are the smallest ones, and get smaller and smaller with higher thresholds. Therefore as a whole, these numbers confirm the general tendency of our numerical results: system’s parameters enabling bidirectional signaling correspond to the most unlikely cases.Obviously, the most probable parameters are always the “non-signaling" cases (000), and the most probable non-trivial signaling regime is the pure forward signaling (001). Then the probability of signaling regimes always decreases markedly between the cases of retrosignaling of first and second order. In particular one observes that the probabilities of regimes . Therefore we can conclude that requiring both response amplitudes of direct and retrosignaling to be large leads to an antagonism in the parameter sets achieving both requirements. The overall combination of forward and retro-signaling appears to be still more rare. This conclusion answers one of the principal question addressed by this study.This property can also be characterized in a quantitative way by computing the conditional probability that bidirectional signaling occurs, knowing that the system exhibits at least one regime of signaling (so excluding (000)). This conditional probability is found to be 6 Another question addressed in Therefore the obtained conditions on parameters, as reported in Results, could be useful to distinguish parameter sets optimizing the probability to get the various regimes of signaling. These conditions are graphically represented in Fig. These graphs embody also a method to restrict parameter space in order to enhance the probability of the main modes of signaling, forward, or retroactive, or both. In order to control how the probability of each signaling regime is optimized by choosing parameter values around the likelihood maxima, we divided the ranges of all the 18 parameters in three intervals: high values, medium values, low values. Each parameter was restricted to one of the three intervals depending on where its likelihood was maximized. As there is one restriction for each parameter, the intersection of those restricted intervals could leave, at the end, no more than 0.1 % of the initial number of simulation sets. The new probabilities of each regime were computed and compared with the former simulation sets in order to see how likely is a given regime if we restrict the ranges of the biochemical parameters. The results are summarized on Fig. dose-response of a signaling cascade, we have also considered the bottom-up drug-response that discloses, when it exists, a retroactive capability of the signaling cascade. Thus signaling cascades can be categorized into 4 modes of responses, according to the existence or not of forward or of retroactive responses. An example of this classification with estimated probabilities was given in 1i.e. usual) signaling, and parameters that enable retroactive regimes, i.e. backward signaling.Living organisms rely strongly on biochemistry. Indeed signaling pathways are meant to transduce physical or chemical stimuli of the external world, relevant to living cells, into variations of activated biochemical species. In this paper, in addition to the common Thus our main conclusion is that the parameter sets allowing both modes of responses, forward and retroactive, occur rarely. Actually, signaling cascades are generally studied uniquely for their forward signaling ability. For instance in cancer etiology, attention is focused on over-activation of kinases in signaling pathways involved in cell proliferation, such as Mitogen Activated Protein Kinase cascades (MAPK). When these cascade pathways are deregulated in this manner, this means that their forward signaling properties are very effective. Moreover in this case, cancer therapies are based on kinase inhibition, which is described by the drug binding term in our mathematical modeling. Therefore, our main result conforts the point of view that in using these therapies, retroactive properties of signaling cascade can be neglected most of the time, eventhough rare off-target effects should not be excluded .On the other hand, henceforth our work prompts the study of those signaling pathways that are overlooked a priori, because ineffective for forward signaling. Our analysis opens the perspective that such systems possibly hide some usefulness in controlling pathways, due to their qualities of retrosignaling. These properties could prove interesting in the fields of drug design or synthetic biology. Indeed we showed that in signaling cascades, novel functionalities can appear precisely in conditions where the biochemical system seems inoperant for forward signaling.i≤n): The kinetic description of the system illustrated in Fig. 1We assume that both the activation reaction  and the Y the protein (kinase), C the enzyme-substrate complex, and E the phosphatase, in each cycle. The lower index i=1,…,n states the cascade level; while the upper indices 0 and 1 refer to variables parameters involved, respectively, in the activation and deactivation reactions. Notably, at any level i, protein Yi is found in either its inactivated or activated form, denoted by Yi).We denote i≤n): The system of non-linear ordinary differential equations (ODEs) corresponding to the kinetic reactions in (10) is given by (1≤with i≤n): Steady states are obtained by setting the ODEs to zero and using the conservation laws : Hence, we end with the following system of 2YiT to get In particular, we work out the second Eq. i.e. the normalized active proteins), and introduce the dimensionless parameters ai, bi, ci, and ei defined by Eq. , and in particular calculate its first derivative with respect to variable xn, according to the chain rule: By iterative application, we can write where l system .i≤n, Let us now suppose that for 1<gi:zi↦xi (1≤i≤n) and set and i≤n .Thus,  cf. Eq.  and \docz0′, i.e.It follows that the first component of xn(s) at the origin is simply obtained by inverting expression (α), evaluating it in s=0. Hence, we get The slope of the dose-response function pression  over theLet us note that this formula is derived for an arbitrary cascade with inhomogeneous parameters, describing the contribution of any system’s parameter to the slope of the dose-response function. In particular, for homogeneous parameters, the initial slope is given by Eq. z0(xn) is a twice differentiable function and let Ji be the jacobian matrix and Let us assume that iated to .xn once again, we find J0=I2 (the identity matrix 2 × 2).Deriving expression  with resz0″, denoted s″(xn), we calculate the inverse function through the relation By selecting the first component of xn″(s) at the origin makes the expression simpler, the general formula for arbitrary n still remains cumbersome, and symbolic computations (e.g. with Maple™) are necessary. As an example, for n=3 and homogeneous parameters, the initial curvature is given in Eq. Although the evaluation of xn″(0) depends on the whole parameter set ai,bi,ci, and ei.We remark that, with respect to the initial slope n, the value of the maximum response α for large stimulus s is lower bounded by the strictly positive fixed point x∗ of the equation xi−1=f, whenever it exists, with f being defined in .We firstly rewrite system  as \doco 1 from ) and dT xn<x∗.Let us suppose the claim is false, that is f, one can prove that f is increasing in y for all x, and increasing in x for y=0 or y=x∗.By considering the partial derivatives of a(1−ey/(y+a))+x2>0. For y=0 the proof is immediate. For y=x∗, we consider the fixed point equation x∗−ex∗/(x∗+a)>0 is sufficient and necessary for the positiveness of In fact, from  one calcf<f<f, namely xn−1<x∗. Then, f<f<f, i.e.xn−2<x∗. Eventually it follows 1=x0<x∗. However, from ))cf. (5)). system ).n=3. In general, we remark that the forward signaling is enhanced for dT=0, namely when no fraction of the last active protein gets sequestrated by the drug.We analyze the case for χ, according to the criteria of efficient forward signaling introduced in Results. From (a+b (a+c−1)−1, a+b (a+c−1) and a+c−1.We firstly study the sign of the initial curvature ts. From  we see tχ results to be non-positive if a>1. Furthermore, for the criterion above mentioned, the slope and the fixed point have to be as large as possible. Thus, from <0 (i.e.a/b+a+c−1<0 implying a+b (a+c−1)−1<0 and a+c−1<0 too), which is actually satisfied if a≪b and a,c≪1. Moreover, χ has to be maximized through the term a≫be and b<1. Eventually, all these conditions ensure x∗ to be already maximized.Conversely, the positiveness of jk1), j,k∈{0,1} in cascades with homogeneous parameters. To go further we perform numerical investigations by randomly sampling the full parameter space, and then classify statistically all the parameter sets according to some characteristics of the response functions they give rise to. The objective is to point out the typical values of parameter that favor one of the 7 signaling regimes (jkl)≠(000). The main tool which is considered below is to seek for parameter conditions that maximize the so-called likelihood of the parameters in the various regimes .EiT, total kinases YiT, Michaelis-Menten constants −2 to 102 generating 1.000.000 sets, using Latin hypercube method [without replacement, and with those values, constructing one set of values for the 20 different biochemical parameters we focused on. This method guarantees an exhaustive exploration of the hyperspace.As we want to look for conditions on parameters that can be formulated in terms of dimensionless parameters, we consider ratios of biochemical parameters to be analyzed. On the other hand we first sample the following 20 biochemical parameters: total phosphatases e method , 27. Thii≤n, j∈{0,1}. Finally, with all the dynamic rates, we solved the ODEs for two different scenarios: Stimulus→+∞, Drug=0Stimulus→+∞, Drug→+∞As the Michaelis-Menten constants are parameters which condense information in equilibrium for enzymatic cycles but no dynamic rates, sampling this constants means an extra degree of freedom to choose some of the dynamic rates, in this work we choose Stimulus→+∞ we replaced, for the first cycle, Eq. , we remove the equations for cycle 3 and set Those replacements amount to remove the inactive protein in cycle 1 because, when With all this information, for each set of random parameters we solved the ODEs for the two scenarios, the initial condition of the system being: We remark that for infinite stimulus, there is no After we solved the ODEs, we computed the following dimensionless output variables: These three dimensionless outputs are the second order retroactivity, first order retroactivity, and forward activity, respectively. The numerical simulation also computes slope, curvature, and fixed point using , 9), an, an9), aIn this study we consider two types of parameters, either dimensional or dimensionless. The dimensional set involves total concentrations of kinases and phosphatases, Michaelis-Menten constants and catalytic rates for the enzymatic reactions. The dimensionless set involves combinations of parameters that appear when solving the dynamical equations for the steady-states, and also ratios of parameters with the same units .λ) and for each regime (jkl), we construct the following probability using Bayes theorem: For each dimensionless parameters  being the probability of finding the signaling regime (jkl), given some specific parameter value λ, P(λ|jkl) the probability distribution of parameter λ given a specific signaling regime, P(jkl) the probability to obtain each regime and P(λ) the probability distribution of parameter λ, whatever the regime. The last one is obtained analytically as a sum of 2 (or 4) uniform distributions.with jkl) is fixed, the function P(jkl|λ) is not a probability distribution over λ, but is called the likelihood of λ, for a given regime (jkl), see [Let us note that when (kl), see . One maiP(jkl) is shown).The reason why we normalize by the the maximum is that the maximum probability of each regimes can be quite different (see Table 1jkl) is to look for values which maximize the corresponding likelihood function.Therefore a rational criterion to characterize parameter values that promote a given signaling regime (Then, considering signaling cascades with inhomogeneous parameters, simulations of the dose-response curves and of the drug-response curves were done for 1.000.000 random sets of biochemical parameters. The histograms of parameters were classified according to the 8 different regimes. Then, using Eq. p of being in the i-th bin and 1−p of being in another bin. Then using error propagation formula for Bayes relation (Let us remark that, despite the large number of samplings, we observe that some signaling regimes are rare. Therefore we computed error bars for all the likelihood curves. For each specific signaling regime the error bars were constructed on the parameter histogram by using a binomial probability relation  we obtai


Individuals affected by cancer report a need for information about fertility from health care professionals (HCPs), in order to inform decision making and alleviate anxiety. However, there is evidence that many health professionals do not engage in such discussions.A mixed method design was used to examine the construction and subjective experience of communication with health professionals about fertility in the context of cancer, from the perspective of patients. A survey was completed by 693 women and 185 men, across a range of cancer tumour types and age groups, and in-depth one-to-one interviews conducted with a purposively selected subsample of survey respondents, 61 women and 17 men. The chi square test for independence was used to test for group differences between women and men on closed survey items. Thematic analysis was used to examine the open ended survey responses and interviews.n = 373) than men  (X2(2517) = 6.54, p = .011) reported that they had discussed fertility with a HCP since diagnosis of cancer. Satisfaction with the discussion was reported by 65% (n = 242) of women and 69% (n = 54) (ns) of men. This discussion was reported to have been initiated by the patient or their partner in 44% (n = 165) of women and 47% (n = 37) (ns) of men. In the interviews and open ended surveys three themes were identified: Feeling heard and informed about fertility after cancer: Positive experiences of HCP communication; “I was never given full disclosure”: HCP silence or reticence about discussing fertility after cancer, including the sub-theme “Their primary concern is getting me cancer free”: Constructions of absence of fertility communication by HCPs; and Confusion and lack of compassion: Unsatisfactory information provision about fertility and cancer.Significantly more women (57%, Discussion with a HCP about fertility concerns, and satisfaction with the discussion, was associated with reports of lower patient distress, greater knowledge and understanding of the consequences of cancer on fertility, involvement in the decision making process about fertility preservation, and satisfaction with health care. Satisfaction with the discussion was reported by 65% (n = 242) of women and 69% (n = 54) of men, with discussion initiated by the patient or their partner in 44% (n = 165) of women and 47% (n = 37) of men. Participants who engaged in discussion with a HCP about fertility concerns, and who were satisfied with the discussion, had greater knowledge and understanding of the consequences of cancer on their fertility, felt that they were involved in the decision making process about the timing of treatment, and were able to discuss options for fertility preservation, where appropriate. In combination, this served to increase health literacy associated with cancer related infertility, providing the sense of self-efficacy that is essential in coping, in order to reduce the distress and threat to identity that cancer related infertility can produce. This confirms that discussion of fertility concerns by oncology clinicians is “a crucial aspect of high quality healthcare”.Individuals affected by cancer report a need for information about fertility from health care professionals (HCPs), in order to inform decision making and alleviate anxiety. However, there is evidence that many health professionals do not engage in such discussions. This study examined the experience of communication with HCPs about fertility from the perspective of 693 women and 185 men patients, across a range of cancer tumour types and age groups. Women were more likely than men to report that they had discussed fertility with a HCP since diagnosis of cancer  completed the survey. The average age of survey participants was 42.53 years (SD = 14.21), and average time from diagnosis 6.22 years (SD = 7.01). The sample was drawn across cancer types including breast (56.7%), gynaecological (12.9%), hematologic (12.7%), gastrointestinal (4.8%), neurologic (3.2%), head and neck (2.9%), skin (2.3%), musculoskeletal (2.3%), genitourinary (0.9%) and respiratory (0.7%). Disease diagnosis status ranged between early and advanced stages, with 67% reporting that their cancer was diagnosed at an early stage. The sample was almost exclusively heterosexual (98%), with 71% reporting that they were currently in a relationship. Fifty-seven percent of survey respondents reported that they had a child.M = 45.10), 61 women and 17 men, accepted the invitation to take part in the interviews.256 people living with cancer  indicated they would be willing to participate in a follow up interview. Purposive sampling  was used: ‘Feeling heard and informed about fertility after cancer: Positive experiences of HCP communication’; ‘“I was never given full disclosure”: HCP silence or reticence about discussing fertility after cancer’, including the sub-theme ‘“Their primary concern is getting me cancer free”: Constructions of absence of fertility communication by HCPs’; and ‘Confusion and lack of compassion: Unsatisfactory information provision about fertility and cancer’. In the presentation of results, gender, age and cancer type is indicated for longer quotes.The final themes developed from the analysis of the interviews weren = 373) than men   = 6.54, p = .011) survey respondents reported that they had discussed fertility with a HCP since diagnosis of cancer. Of those who reported discussing fertility with a HCP, 65% (n = 242) of women and 69% (n = 54) (ns) of men were satisfied with the discussion. Explanation for satisfaction was constructed in terms of HCP’s being “proactive” in informing participants about the possible consequences of cancer treatment on fertility, as well as being “informative,” “clear, accurate,” and “explaining everything well.” Information and advice from HCP’s allowed participants to make “informed decisions” regarding their cancer treatment and consequences for fertility. For example, one woman  said that in her experience, HCP discussions “help you understand the medical side of the cancer and how it affects you”. Many participants valued “honesty”, reporting preferences for comprehensive information in instances where treatment would likely result in detrimental fertility outcomes. For example, as one woman said “they always explained everything well, including the possibilities of being infertile in the future – they didn’t try to sugar coat anything” . Another woman said that it was “satisfying to get a clear view of what was possible and not possible” .In survey responses, significantly more women .In a similar vein, a man  reported that “the doctors were great. They treated me with respect and I suppose honesty as well”. These positive experiences with HCP’s were reported to have left participants feeling “understood” or “heard” because they had been given time to voice their feelings and concerns. For example, participants told us that they had “come away from the appointment relieved and less stressed” , and that they valued being “given the opportunity to verbalise how I was feeling” .Accounts of positive interactions with HCPs extended beyond the content of fertility information, to include the manner in which it was delivered. Participants described positive encounters with HCP’s as those where they felt that HCP’s employed “acceptance,” “warmth”, “understanding,” “sensitivity,” “care,” “respect,” “empathy,” “compassion” and had “taken the time to listen.” In these accounts, HCP’s were described as acknowledging how “serious” fertility was and did not “minimise things,” as evidenced by the following account.n = 298) of women and 51% (n = 49) (ns) of men indicated that they had received or accessed information resources regarding fertility and cancer. Examples included being given a “pamphlet,” “booklet” or “book”, with two participants commenting: “they would recommend resources and things to me”  and “I’m sure I was handed a lot of written information” . It was reported that this “made my life easier”, as a man told us: “I mean, I didn’t have to go out and search for it, she knew exactly sort of what things I was looking for and she’d be able to provide those to me” . Another participant commented on the value of written information in supplementing medical consultations saying, “what you take in during a consultation, a face to face consultation, you might miss a lot of things” . Participants also spoke positively about instances in which written material was provided by HCP’s who then “went through everything” with participants.Many participants gave accounts of valuing written information as part of their consultation with HCPs about fertility concerns. In response to the survey, 46% . Participants valued being given the “choice”, “opportunity” and “time” to fully investigate fertility preservation options, enabled through prompt referral by HCP’s, and where possible, timing of cancer treatments to accommodate fertility procedures: “they were more than happy to write referrals to the relevant fertility specialist”  and “I was pleased that the surgeon said that this could be a possibility before I agreed to have surgery” . Some participants described HCP’s as supportive in the decision making process about fertility preservation services, without attempting to influence the outcome. For example, “my GP was fantastic, as were all the staff at our health centre. She never tried to influence us, but said she would support us whichever route we decided on” .Of those participants who had not discussed fertility with HCP’s, 43% of women and 64% of men, the majority constructed the experience in terms of absence of information: “I was never given full disclosure of side effects of treatment” , “there was no expert opinion on what to do” , and “nothing was offered by anybody” . The consequence of absence of information was positioned as lack of knowledge on the part of patients of the fertility consequences of cancer treatment. For example, a number of men said that they did not know that treatment “could make me sterile” , which was a regret as “I honestly didn't know and I wish that I had” . Similarly, a number of women told us “I wasn't aware that the chemo would affect fertility”  and “I didn't realise my womb was going to go. I think he should have told me before it happened” . Participants described the lack of fertility discussion by HCP’s as contributing to psychological distress associated with compromised fertility, which was described as “heartbreaking”, “sad” and a “loss” that led to “grief”. One interviewee  talked about how the lack of fertility information contributed to a more challenging cancer experience, saying, “when you do have all the information, you can then go into something with your eyes open, whereas to find out things after the fact that it - it makes it so much harder to handle.” Another women  told us that she “felt abandoned” by being left in the dark about the impact of cancer on her fertility.Absence of information was associated with lack of access to decision making or interventions to preserve fertility, with participants saying, “we didn’t really get a choice” , or that fertility was “never in question at all and no alternative offered” . Subsequently, many participants reported finding out about their impaired fertility after the conclusion of treatment, when they had diminished options for intervention. As one man commented: “It was sort of like you need this (treatment), and that’s happening, blah, blah, blah and then it wasn’t until afterwards we thought ‘well hang on what about fertility?’” . Another woman  said, “I have been told that I will not be able to naturally produce children, if I was made aware of this when I was first diagnosed I would have stored some eggs.”I would have preferred to get the information from my doctor. To sit down and ask the questions and him tell me all of my options and then give me the leaflets and be like, ‘we’ve discussed what options you’ve got for the future, I’ve gathered a few different resources if you wanted to do a little bit of home research and look into a few of them’.In the absence of fertility information being offered by HCPs, participants positioned themselves as responsible to source information in order to “understand what the hell had gone on with me” , or to “not panic, and work out ‘where do I go to from here?’” . Such information was also described as having helped participants to be more prepared for their interactions with HCP’s, evidenced in the following accounts: “I was relatively aware … I’d been doing my own research” ; “I do believe very strongly in getting a lot of information because then you can ask the right questions as well” . Sources of written information included online websites and blogs, and booklets produced by cancer organisations, supplemented by verbal discussion facilitated by community organisations, such as support groups and workshops. Such information was not positioned as a substitute for discussion of fertility with HCPs, however, with some participants describing written information as “worrying”, “overwhelming”, “not in-depth enough”, or “too general”. The majority of participants wanted written information combined with discussion of their specific concerns with a HCP, illustrated by Louisa’s  account below:n = 165) of women and 47% (n = 37) (ns) of men who had engaged in discussion with HCPs about fertility reported that they or their partner were the one to raise the issue. This was explained in qualitative accounts, with one woman telling us: “I feel as though they [HCPs] are waiting for me to explain how I feel about infertility before they comment” . In many instances, participants described having to “push” HCP’s in order to access information, or to be referred to a fertility specialist: “it’s just that I had to mention it two or three times rather than once” , and “it just felt like I had to push for it sometimes” . Access to fertility preservation was also described as having occurred due the initiative of the patient, rather than the HCP: “it was only because I asked that I ended up getting help and freezing the embryos” . This was reported to have made access to fertility information and interventions more “challenging”, with some participants describing difficulties in finding specialist care or information. As one woman commented: “I struggled to find anyone knowledgeable about fertility preservation in breast cancer cases” .“Their primary concern is getting me cancer free”: Constructions of absence of fertility communication by HCPsIn the absence of HCP communication about fertility, many participants took up a position of responsibility for initiating the discussion. In survey responses, 44% . Another woman was treated for cancer as a child and described fertility as a “taboo,” amongst paediatric HCP’s. She told us that her mother raised fertility concerns at diagnosis “and the doctors were just speechless at the fact that she openly spoke about the issue” .Participants made sense of HCP reticence to discuss fertility in a number of ways. Some participants attributed the absence of information to HCP focus on treatment and survival, with a number of women participants reporting feeling rushed into treatment, rather than having the opportunity to address fertility concerns: “[the] GP was more concerned with initiating treatment” ; “their primary concern is getting me cancer free” . Participants’ also spoke of being treated by HCP’s who appeared to lack knowledge about the ways in which cancer treatment might compromise fertility, or knowledge of the fertility options available. As one woman  said, “we felt none of the medical professionals had a strong understanding of the impact of treatment on fertility and the options that were available”. Another man  said,And I felt like the doctor was talking more to my mum than to me. I mean, I was 21, I was an adult, I’d lived out of home for four or five years and I still felt like they were treating me a little bit as a child.Other women spoke about feeling fertility concerns were overlooked by HCPs because they already had a child, saying: “Oh she’s already got one [baby] so we don’t need to talk about this” , and “perhaps if I hadn’t had any children, then they might have come forward with more support” . Across the board, participants were in agreement about the importance of HCP’s not making assumptions and providing information and fertility preservation options “whatever age they may be,” regardless of current relationship and children.A number of participants, primarily women, gave accounts of feeling that HCP’s made inaccurate assumptions about their need for information about fertility, as one woman said, “sometimes professionals think they know what is best for you without asking you” . It was reported that HCP’s made assumptions based on whether or not a woman currently had a partner, with participants saying, “often people dismiss your feelings because you aren't in a relationship, so to them you mustn’t have been thinking about children anyway” . Participants also reported feeling that HCP’s made assumptions based on their “older” age at the time of diagnosis, for example: “I feel that due to my age and the fact that people seem to feel that I had already made a distinct choice not to have a child earlier (it wasn’t an issue). That's not really the case at all” . Conversely, some young adult participants reported feeling that fertility options were “overlooked” due to their younger age at diagnosis, or that discussion that did take place was directed at their parent, as one woman  told us:n = 129) of women and 31% (n = 24) of men. A number of explanations were provided for unsatisfactory discussions. Many participants reported receiving inaccurate or conflicting information from HCPs regarding their fertility after cancer, leading to “confusion” and feelings of “frustration”. For example, the following comments were made: “I found the process confusing, rushed, bewildering and confronting” , and “it is confusing and concerning to have so many different views” . “Conflicting information” was associated with reports of “difficulty” in understanding the impact of cancer treatment on fertility, and “uncertainty” regarding “how long to wait” and the consequences of treatment for conception and pregnancy; “no-one ever told me clearly how safe or unsafe it would be for me to have another pregnancy ” .A substantial proportion of participants who engaged in discussion with HCPs did not report satisfaction with the discussion, 35% .In addition, some male participants also did not understand fertility preservations options when they were presented, contributing to “confronting” experiences, as exemplified by one participant who said,they just sort of were like, “Okay, yep, well this is the issue, you go do your sperm banking and then we’re done, we’ll, we’ll start treatment the week after that.” Then it’s sort of like, “Whoa, whoa, whoa, wait, why am I sperm banking?” In addition to conflicting information, participants gave accounts of dissatisfaction due to “limited information”, lack of “real discussion,” or conversations that did not acknowledge the “importance” or “priority” of fertility concerns. For example, one woman  said she received information in an “itsy, bitsy kind of way,” and as a “throwaway comment said while we were standing up waiting for something. It wasn’t even at his desk or anything.” Another woman said fertility options were “lightly glossed over” and a man commented: “I was only given a brief run down about having radiation and how you never regain fertility” . Lack of “in depth” communication was reported to have contributed to psychological distress in participant accounts. For example, a brief comment about fertility control left a woman melanoma survivor “distraught” because it led to her realisation that she had a poor prognosis: “It was probably the first time I realised I wasn't expected to live. The doctor said ‘no children for five years’ and I asked ‘why?’ and she said ‘young children needed a mother’” . Participants also described difficulty processing information when fertility was discussed “in passing”, contributing to challenges for decision making. For example, a number of participants gave accounts of infertility only being mentioned immediately prior to treatment commencement. As one women said,Like, it was all quite, you know, they were all just sort of trying to distract us and being quite cheerful. And – but it just felt really invalidating and really painful to be the only person that was aware of what this chemotherapy meant to me. I’d like for everybody who’s involved in these treatments that can affect our fertility, to be aware of the emotional impact of these treatments on us. They should be sensitive .Women described interactions with HCP’s where they felt they should be grateful for survival and nothing more. For example, “the psychologist I saw was unsympathetic - you are alive, why do you care about fertility/attractiveness etc” ’; “I was told that my focus was to survive and anything more than that would be considered a bonus. I was 19” .A further source of dissatisfaction for a number of participants was perception of negative responses from HCP’s when the patient or their partner raised fertility concerns. HCP’s were described as being “dismissive”, “unsympathetic”, or lacking in “compassion” or “empathy” in response to participants requests for information or referral, reporting: “they didn’t seem to care”  and “I felt a lack of personal touch when I was told about our situation” . These dismissive interactions with HCP’s were described as contributing to feelings of psychological distress. As one woman said, her HCP was “really dismissive” and “blunt.” She described feeling there was “no hope presented,” and that “it really did have a big impact on my mental health and emotional health” . Other participants were distressed with the “clinical” nature of fertility information provided by HCPs: “I appreciated the honesty, but the trauma of the whole cancer experience was still very real and the clinical, cold answers were a bit upsetting” . Many participants told us that they felt that the emotional gravity of fertility was not recognised by HCP’s involved in their cancer care. As one woman said,Many participants acknowledged that the focus of HCP’s on survival was understandable, given the emphasis within the health system on cancer cure. However, it was also argued that other aspects of the person, including fertility, should also be considered. As one participant commented, “cancer doctors should be more prepared that they are treating a human being with hopes and ambitions, not just a disease” .The findings of study support previous research which identified information about the impact of cancer on fertility as a priority for patients who are AYA or of reproductive age, serving to alleviate distress and improve quality of life , 25, 30.Increased knowledge and confidence in discussing health concerns through information provision is known to be associated with positive health outcomes . More spParticipants who received information and advice about fertility from HCPs in a manner that was honest and respectful were more likely to report satisfaction with health care, and with the outcome of fertility preservation discussions, as a result of feeling that their concerns were acknowledged and questions addressed. It is widely recognised that positive and empathic doctor-patient communication is a vital part of cancer care, which can result in greater satisfaction with treatment , 61. TreHCPs are known to avoid discussing fertility with patients who have a poor prognosis, or for whom they believe parenthood is not a concern, due to factors such as relationship status, sexual orientation, or age , 24, 63,It has been reported that men are more likely than women to report discussion of fertility with a health professional, and are also more likely to report satisfaction with the discussion , 32–35. These findings have a number of implications for clinical practice and policy. Reports of absence of discussion of fertility concerns by a substantial proportion of clinicians suggest a deficit in institutional policy and practice guidelines to assist HCPs with such discussions . It has As the psychosocial experience of infertility has been reported to be different across genders , 77, witThis study had a number of strengths and limitations. The strengths were the use of a survey of a relatively large sample of men and women, across cancer types and age groups, and qualitative interviews to examine subjective accounts of infertility in depth. The limitations include the fact that the sample was not nationally representative, and that participants were recruited as part of research study examining experiences of fertility after cancer, which may have resulted in a greater focus on infertility within the accounts, and the greater proportion of women in the sample. The predominance of women and heterosexual cisgender individuals in the sample is also a limitation. Further research using a more equal sized sample of women and men, including a greater proportion of LGBTI individuals, is needed to examine the experience and impact of communication about fertility with HCPs following diagnosis of cancer.In conclusion, compromised fertility following diagnosis and treatment for cancer can have a negative impact on psychological wellbeing, quality of life and identity , 32, in