To report surgical site infections (SSI) after Tibial Plateau Leveling Osteotomy (TPLO), treatment course, associated risk factors, bacterial isolates and antimicrobial resistance.Retrospective clinical cohort study.Six hundred and twenty seven dogs and 769 TPLO procedures.Data from electronic medical records of dogs undergoing TPLO between 2005 and 2015 at a single institution have been retrospectively reviewed. A generalized mixed logistic regression was used to determine possible risk factors. The Chi-Square test of independence was used to examine the relationship between the isolation of multidrug-resistant (MDR) bacteria and the development of major infections undergoing additional surgical treatment. To assess the correlation between number of SSI and number MDR isolate per year, Pearson's correlation coefficient was calculated.n = 149). SSI was most frequent with 8.5% (n = 65). Major SSI occurred in 6.8% (n = 52) TPLO (80.0% SSI). Staphylococcus (S.) pseudintermedius (n = 37) and S. aureus (n = 10) were most frequently isolated. Multidrug-resistant bacteria were identified in 2.7% (n = 21) TPLO (32.3% SSI) but were not associated with major SSI (p = 0.426). There was a strong positive correlation between number of MDR isolates per year and number of SSI per year . Factors associated with SSI were previous TPLO in the contralateral stifle  and German Shepherd dogs . The use of non-locking implants was found to be protective .The overall complication rate was 19.3% (Infection with multidrug-resistant bacteria is an emerging problem in veterinary practice and treatment is challenging. The incidence of major SSI was found to be high but was not associated with the isolation of MDR bacteria. Tibial Plateau Leveling Osteotomy (TPLO) is one of the most commonly performed surgical procedures for the treatment of cranial cruciate ligament (CCL) disease in dogs , 2. Post–40% multidrug-resistant Staphylococcus (S.) aureus and S. pseudintermedius isolates among dogs with SSI after TPLO  bacteria is widely acknowledged and poses a major therapeutic challenge in both human and veterinary medicine , 19. Litter TPLO , 20, 21.ter TPLO . In veteter TPLO . Consequnfection . For thiThe aim of this retrospective study was firstly to analyze risk factors for SSI after TPLO and report bacterial isolates and their antimicrobial resistance pattern. Secondly, we sought to investigate the influence of occurrence of multidrug-resistant bacteria on treatment of SSI.A retrospective cohort study was conducted. Medical records of dogs that underwent stifle arthroscopy and TPLO in a veterinary teaching hospital between January 2005 and December 2015 were reviewed. The minimum duration between data collection and surgical procedure was set to 6 months. Procedures were excluded, if medical records were incomplete or other orthopedic procedures were simultaneously performed on the same stifle apart from arthroscopic treatment of meniscal injury. Medical records were considered incomplete, if information on signalment, documentation of surgical procedure and anesthesia, or postoperative care was missing. Available follow up information was extracted from medical records in the form of documentation of in-house rechecks or communication logs. If no postoperative complications were recorded, the recovery was considered uneventful. Dogs undergoing bilateral staged TPLO were considered independent cases and the statistical model was designed to account for the dependence on multiple observations of a single animal by modeling a random intercept for each animal. Data collection included breed, age, sex, body weight, history of dermatitis, use of locking or non-locking implant, duration of anesthesia, pre-, intra-, and postoperative antimicrobial administration, and if previous contralateral TPLO had been performed (during or before the study period). In cases of postoperative complications, the following data was collected: Type and timepoint of complication, type of therapy and outcome. Results of bacteriological culture and antimicrobial susceptibility testing were reviewed.Arthroscopy was used for inspection of the stifle joint before every TPLO. Remnants of ruptured CCL were debrided, and meniscal damage treated with a combination of meniscal knife, motorized shaver, and arthroscopic punch forceps. TPLO as described by Slocum and Slocum  was perfCriteria for diagnosis of SSI were based on those published by the United States Centers for Disease Control and Prevention (CDC) , with miBacterial cultures and antimicrobial susceptibility testing (AST) were performed at the Institute of Veterinary Bacteriology (IVB) at the Vetsuisse Faculty, University of Bern. Bacterial cultivation of swab/aspirate and implant samples followed standard diagnostic procedures. Identification of colonies were performed by Vitek™ 2 Compact technology  until 2010. Since 2011, isolates were identified by Matrix-Assisted-Laser-Desorption/Ionization-Time-of-Flight-Mass-Spectroscopy (MALDI-TOF MS) .Pasteurella spp. The above-mentioned methods were not applicable, therefore detection of beta-lactamase enzymatic activity using BBL™ DrySlide™ Nitrocefin (BD Becton Dickinson) was used. Bacteria were evaluated for multidrug resistance (MDR) according to criteria published by the European Center for Disease Control (ECDC) and Centers of Disease Control (CDC)   and interpretation of test results followed manufacturers' instructions. Since August 2010, AST was performed with the Vitek™ 2 Compact technology (BioMérieux). Isolates were classified as susceptible or resistant, according to clinical breakpoints published by the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. If no EUCAST breakpoints were available, veterinary clinical breakpoints of the Clinical and Laboratory Standards Institute (CLSI) were used. For scribed) .p < 0.05. Pearson's Chi-Square test was used to test for independence between the presence of MDR bacteria and the presence of major infection. To assess the correlation between number of SSI and number MDR isolate per year, pearson's correlation coefficient was calculated.Descriptive data were reported as mean ± standard deviation or median (range) as appropriate. Normality of data was assessed using Shapiro-Wilk Test. Statistical analyses were performed by use of statistical software . A generalized mixed logistic regression was fitted to the data with SSI as the response variable and sex, age, body weight, ASA (American Society of Anaesthesiologists Classification) grading, pre- and postoperative antimicrobial therapy, previous contralateral TPLO, duration of anesthesia, implant (locking or non-locking) and breed included as fixed predictors. Breed was defined as a categorical variable with a separate category for the most represented breeds . The remaining breeds were summarized in one category (other). The variance inflation index was investigated and was found to be between 1 and 2, hence no problematic correlations among independent variables were identified. The identifier of an animal (ID) was included as a random intercept to account for the dependence on multiple observations of a single animal . Significance was set as n = 28) procedures were excluded due to incomplete medical records. A total of 627 dogs were included in the study population, 142 (22.5%) of which underwent staged bilateral TPLO, resulting in total of 769 TPLO procedures. In addition to the bilateral staged procedures performed within the study population, four  cases were found to have undergone TPLO before the study period, leading to a total of 146 (19.0%) cases with a history of previous TPLO surgery on the contralateral stifle. The study population consisted of 402 (52.3%) neutered and 83 (10.8%) intact females, and 187 (24.3%) neutered and 97 (12.6%) entire males. The mean age was 5.9 ± 2.7 years, and median body weight 35.8 kg (range 10–87 kg). The most common breeds were mixed breed , Labrador Retriever , Golden Retriever , Bernese Mountain Dog , Boxer , Rottweiler , Great Dane  and German Shepherd . Only seven (0.9%) dogs had ongoing antimicrobial therapy at the time of surgery for reasons unrelated to TPLO surgery. Follow up information was available after 494/769 (64.2%) procedures. Mean time to last follow up was 247 days . A summary of patient's demographics with and without SSI can be found in During the study period, 797 TPLO procedures were recorded. Twenty-eight (n = 427) of procedures, TPLO was completed on the left stifle, and 44.5% (n = 342) on the right. All surgeries were performed by, or under the supervision of, an experienced board-certified surgeon. A complete rupture of the CCL was diagnosed during 472 (61.5%) procedures, a partial rupture during 297 (38.6%). Partial meniscectomy was necessary in 298 stifles (38.5%), of which 284 (95.0%) affected the medial, 12 (4.0%) the lateral and three (1.0%) both menisci. Total meniscectomy was performed on the medial meniscus in three cases and medial meniscal release procedure in seven. Mean duration of anesthesia was 255 ± 49 min. In 71 (9.2%) cases, antibiotic treatment was continued postoperatively. The rational of continued post-operative antibiotic treatment of these cases was found to be history of or suspected ongoing local dermatitis in 13/71 (18.3%) cases, continuation of pre-operative antibiotic treatment in 4/71 (5.6%), minor breach of sterility during the surgical procedure in 2/71 (2.8%) and unknown in 52/71 (73.2) cases. Non-locking implants were used in 124/769 (16.1%) cases and locking in 645/769 (83.9%). The distribution of usage of locking and non-locking implants over time is presented in In 55.5% (n = 65). Median duration between surgery and diagnosis of SSI was 14 days (range 4–855). Catastrophic SSI occurred after one TPLO . Major SSI was diagnosed in 52/65 (80.0%) SSI. Surgical implants were removed in 42/65 (64.6%). Median duration between surgery and implant removal was 132 days . Antimicrobial therapy was initiated or continued after 15/42 explantation procedures. Overall, after 57/65 (87.7%) procedures with SSI antimicrobial therapy was prescribed, consisting of cefalexin (20 mg/kg BID po), amoxicillin/clavulanic acid (12.5 mg/kg BID po), clindamycin (5.5 mg/kg BID po), enrofloxacin (10 mg/kg SID po) or a combination of these. Median duration of antimicrobial treatment was 22 days (range: 5–52 days). According to our data, dogs that had previous TPLO on the contralateral stifle  and German Shepherd Dogs  were more likely to develop SSI. Furthermore, use of non-locking-implants was associated with lower odds of infection . Postoperative antibiotic treatment was not associated with development of SSI (p = 0.61).An overview of encountered postoperative complications can be found in n = 21/769) of TPLO or 32.3% (n = 21/65) of SSI. One catastrophic complication occurred; A dog was euthanized upon owner's decision seven days post-surgery after being diagnosed with multiple complications including SSI with isolation of MDR bacteria and tuberositas tibia fracture. Major infection occurred after 18/21 (85.7%). Implants were removed in 14/21 (66.7%) cases, seven of which had been treated with adequate antimicrobials according to AST (p = 0.426). Absolute and relative number of SSI and SSI involving MDR bacteria is shown in r(9) = 0.79, p = 0.004].Incidence of SSI involving MDR bacteria was found to be 2.7% , antimicrobial therapy with cefalexin 20 mg/kg for 7–10 days , whilst 11 (64.7%) dogs underwent implant removal. Samples from removed implants for bacteriological culture were collected from 6/11 (54.5%), none of which yielded bacterial growth.After 17 (2.2%) TPLO procedures, persistent lameness >50 days after TPLO without any evident cause was diagnosed. Six of seventeen dogs (35.3%) underwent conservative treatment consisting of non-steroidal anti-inflammatory drugs and restricted activity (n = 63/91) of the samples bacterial growth was detected. Sixteen different bacterial species (S. pseudintermedius being by far most frequent (n = 37) followed by S. aureus (n = 10).A total of 91 bacteriological culture results collected from 80 patients were available for analysis. In 69 (86.3%) dogs, a single culture was performed, and paired cultures in 11 (13.8%) dogs. Overall, in 69.2% ( species  were ison = 50/69) of the samples. In 11 dogs, bacterial culture was performed when SSI was first suspected as well as during implant removal, which allowed pairing and comparison of results  or during removal of TPLO implants . Bacterial growth was detected in 72% ( results . Paired S. pseudintermedius and five of ten (50.0%) S. aureus were classified as MDR. Other MDR isolates included Acinetobacter baumanii (n = 2), Klebsiella pneumoniae (n = 1) and Escherichia coli (n = 1) (S. aureus and A. baumanii) were isolated from the same surgical site.A total of 22 MDR bacteria were isolated after 21/769 TPLO . Thirteen of 37 (35.1%)  (n = 1) . After oThe purpose of this study was to investigate SSI after TPLO, associated risk factors, isolated bacteria and respective antimicrobial resistance pattern, as well as the course of treatment. Postoperative SSI rate was found to be 8.5%, which is in line with previously published data , 29, 30.The incidence of major SSI of 80.0% in the subpopulation of patients diagnosed with a SSI is higher than previously reported , 9, 31. The rate of SSI subjectively shows a trend toward a higher incidence in the latter study period . SimilarStaphylococcus spp. was dominant among isolates from SSI following TPLO, is in accordance with previously reported study results . In comparison, the prevalence of methicillin-resistance amongst isolated Staphylococci from infection sites after TPLO reported in literature varies between 20% and 40%  or S. aureus (n = 1) spp. was isolated; a genus of bacteria known to form biofilm that impedes host response and curative effect of antimicrobials  non-locking implants were used in 2005 and 2006, postoperative infection rates were found to only 3.5% vs. 9.6% during the later study period  patients evaluated for ongoing lameness after TPLO were classified as having persistent lameness of unknown origin. Even though bacterial samples from implants of 6/11 (54.4%) were collected and did not yield bacterial growth, it is possible that the infection rate could have been underestimated due to a misclassification bias. On the other hand, lameness could have been caused be factors not directly related to TPLO surgery, such as progression of degenerative joint disease of the stifle. This applies mainly to cases, where the diagnosis was made several months after the surgery.In total, 2.2% (There are several limitations of this study. In respect to its retrospective nature, we wholly depended on completeness and accuracy of data collected from electronic medical records. Furthermore, subsequent patient information was not always available, due to owners electing to perform rechecks in a different practice. Additionally, the definition of SSI used was very narrow and would not include cases of early-stage SSI which only show signs of inflammation. For this reason, complication rates may have been underestimated.We can conclude that incidence of major SSI was found to be high in the study population but was not associated to isolation of MDR bacteria. In contrast, isolation of MDR bacteria was correlated with SSI rates in general. Additional research is needed to investigate a possible link between previous hospital stay and use of non-locking vs. locking implants on risk for SSI after TPLO in general and SSI involving MDR bacteria in particular.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.BH, UR, GO, and FF designed the study. BH and GO collected the data. GO provided data on bacteriological examination of the SSI cases discussed. BH was responsible for the statistical analysis of the collected data. UR supervised data collection and analysis. BH, UR, and GO drafted the manuscript. All authors critically revised the manuscript and approved the final version.


Mitogen-activated protein kinase (MAPK) p38 is a central regulator of intracellular signaling, driving physiological and pathological pathways. With over 150 downstream targets, it is predicted that spatial positioning and the availability of cofactors and substrates determines kinase signaling specificity. The subcellular localization of p38 is highly dynamic to facilitate the selective activation of spatially restricted substrates. However, the spatial dynamics of atypical p38 inflammatory signaling are understudied. We utilized subcellular targeted fluorescence resonance energy transfer (FRET) p38 activity biosensors to map the spatial profile of kinase activity. Through comparative analysis of plasma membrane, cytosolic, nuclear, and endosomal compartments, we confirm a characteristic profile of nuclear bias for mitogen-activated kinase kinase 3/6 (MKK3/6) dependent p38 activation. Conversely, atypical p38 activation via thrombin-mediated protease-activated receptor 1 (PAR1) activity led to enhanced p38 activity at the endosome and cytosol, limiting nuclear p38 activity, a profile conserved for prostaglandin E2 activation of p38. Conversely, perturbation of receptor endocytosis led to spatiotemporal switching of thrombin signaling, reducing endosomal and cytosolic p38 activity and increasing nuclear activity. The data presented reveal the spatiotemporal dynamics of p38 activity and provide critical insight into how atypical p38 signaling drives differential signaling responses through spatial sequestration of kinase activity. The diverse physiological functions and the conserved mechanism of activation by MKK3/6 dictates that intracellular signaling is regulated through substrate availability and intracellular localization of p38.The highly conserved subfamily of MAPK p38  are essential regulators of intracellular stress responses. Dual phosphorylation of p38 at the conserved Threonine-Glycine-Tyrosine (TGY) motif (T180 and Y182) by the upstream MAP kinase kinase (MKK3/6) is essential for classical p38 activation. MKK3/6 can activate all four family members, regulating many physiological and pathological processes, including cell growth, proliferation, wound healing, and inflammation2. Conversely, p38 also has critical roles in the cytosol, activating cytosolic transcription factors to induce their nuclear translocation and activating other cytosolic kinases3.Cellular stress induces p38 activation and intracellular redistribution, targeting p38 to selective pools of substrates. In a resting state, p38 is typically distributed between the cytosol and nucleus. However, after activation, all p38 isoforms are rapidly translocated to the nucleus, a step that is critical for nuclear resident transcription factor activation3. The first selectively occurs in T-cells, utilizing the tyrosine kinase ZAP70 to phosphorylate p38 at Tyr323. Phospho-Tyr323 triggers a conformational change driving p38 autophosphorylation at Thr180 in trans between homo or heterodimers of p38α and p38β5. The second atypical p38 pathway is mediated by the direct binding of p38 to the adaptor protein transforming growth factor beta-activated-kinase-1-binding protein-1 (TAB1)6. The c-terminal peptide of TAB1 selectively binds only the p38α isoform at two distinct binding sites, inducing a conformational change and enabling p38α autophosphorylation9. Critically, TAB1-dependent atypical p38 activity has been shown to regulate key pathological processes in a widening array of diseases, including cardiac damage, vascular inflammation, viral and parasite infections, recently reviewed3.In addition to the classical MKK3/6 activation pathway Fig. Ai, p38 c11 6. Atypical p38 signaling must therefore exhort differential cellular responses through an alternative regulatory process.Atypical p38 is initiated through several mechanisms, including hypoxia, oxidative stress, and G protein-coupled receptors (GPCRs). Stimulation of multiple inflammatory GPCRs can trigger activation of an E3 ubiquitin ligase, NEDD4.2, through a conserved tyrosine phosphorylation switch mechanism, inducing GPCR ubiquitination and scaffolding of the atypical p38 signaling complex through TAB2 binding to ubiquitinated GPCRs and recruitment of TAB116. Advances in genetically encoded Fluorescence Resonance Energy Transfer (FRET)-biosensors have enabled detailed spatial studies of ERK1/2 activity using the EKAR4 ERK activity biosensors17. Despite the development of a comparable p38 activity FRET biosensor18 there have been no studies into the spatial control of atypical p38 MAPK.Recent studies have highlighted the spatiotemporal control of G-proteins and MAPKs at the endosome, Golgi, and ER, driving selective and physiologically critical signaling responses where the subcellular location of the kinases or receptors induces selective cellular responsesThis study aimed to develop a platform of subcellular targeted genetically encoded FRET biosensors to spatially map p38 activity. Using live-cell imaging of cells transfected with p38 FRET sensors targeted to the plasma membrane, cytosol, nucleus, and endosome, Fig. B, we com18. The FRET biosensor is a single molecule, with an n-terminal YFP and c-terminal SECFP, linked by a flexible peptide containing a p38 binding and phosphorylation motif and a WW domain  domains from hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) to target the endosome via binding to the membrane lipid phosphatidylinositol 3-phosphate, PtdIns3P which are enriched in early endosomes21, a nuclear export sequence  to target the cytosol22, and nuclear localization sequence  to target the nucleus23 ; Biotracker™ orange = plasma membrane, r = 0.466 ± 0.131; GAPDH-647 = cytosol, r = 0.351 ± 0.107; RedDotTM2-far-red nuclear stain = nucleus, r = 0.436 ± 0.132;   indicated that 2xFYVE targeting of the biosensor colocalized with Rab5 positive early endosomes  was rapidly achieved with a FRETT½  within 30 s in all but the cytosol, in which individual cells had a greater distribution of FRETT½ values over 2–4 min with an average of 2 min  and 1.14 (red) over baseline, respectively o4A Fig. A11. ThisMAX values from Fig. MAX checkered bars). NaCl activity analysis of the FRETMAX for all sensors revealed no significant change MAX at ~ 7 min. We have previously shown that PGE2 activation induces atypical p38 activity, and our data demonstrates that atypical p38 signaling drives a comparable spatial bias for PGE2-induced p38 signaling by the human EP2 and EP4 receptors . Further studies will be required to explore how conserved this bias is across GPCRs and non-GPCR-induced atypical p38 signaling.The cytosolic redistribution was conserved with the prostaglandin receptors EP2 and EP4 but not seen for NaCl treatment which displayed a two-phase increase in p38 activity, an initial rapid activation, followed by a slower rise to FRETOf note, NaCl stimulation induced rapid endosomal signaling, suggesting that the endosome may also play a role in the initial phases of osmotic stress-mediated activation of p38.20 early endosomal targeted FRET sensor, we also included an alternative endosomal targeting strategy using the full-length wt-Rab5-FRET sensor. Rab5 is an early endosome GTPase that plays a critical role in endosomal trafficking and is classically used as a marker for the endosome42, Rab5 binds to and colocalizes with EEA1 at PI3P lipid membranes20. Differing from the FYVE targeting, Rab5-FRET displayed a rapid peak in p38 activation after thrombin signaling before dipping and rising again. However, NaCl failed to activate Rab5-FRET biosensor responses. As Rab5 is being used as a targeting motif and the GTPase activity is not explicitly being measured, it is possible that the full-length Rab5 could be acting as a steric hindrance displacing the FRET sensors too far from the endosome to detect NaCl-dependent signaling. Conversely, atypical p38 is scaffolded by GPCR-ubiquitin chains that drive the formation of the TAB2-TAB1 signaling complex, and this additional structure may position p38 activity to ideally activate the Rab5-FRET complex. Additional studies will be required to explore this in more detail.In addition to the PI(3)P binding 2xFYVE43, follicle-stimulating hormone (FSH)44, and the beta2-adrenergic receptor (β2-AR)-dependent endosomal signaling16. Each case alters the specific interactions with molecular adaptors that drive kinase activity. While Dyngo4A treatment didn’t induce the predicted rapid increase in PM p38 signaling, inhibition of CME significantly reduced thrombin-dependent FRETMAX of both endosomal and cytosolic p38 activity . Furthermore, differential signaling by p38 has been demonstrated by the duration/strength of p38 activity, where transient activity leads to the proliferation of fibroblasts, but sustained signaling drives cell cycle arrest46. Our data clearly demonstrates that atypical p38 drives a spatiotemporal bias and so perturbing access to essential adaptors and transcription factors in the nucleus. Importantly, the p38 biosensor used in this platform detects spatial kinase activity and substrate availability. However, FRET responses are also a measure of phosphatase activity to control p38 activation and dephosphorylation of the sensor to terminate FRET signaling. While NaCl activity led to transient biosensor activation, demonstrating that FRET activation is reversible, thrombin activity was sustained for 30 min post-stimulation. Critically, SB203580 addition at 30 min demonstrated that the FRET sensor was not locked into an active conformation, triggering a decaying FRET response that confirms the presence of active phosphatases, and suggesting that p38 kinase activity is sustained to maintain a FRET response to 30 min. A specific phosphatase has not yet been identified for atypical p38 activity, and studies are ongoing to determine how atypical p38 signaling is terminated physiologically. Additional studies are also required to characterize the kinetics of dephosphorylation of activity reporters in models of endogenous GPCR expression. Defining temporal changes in phosphorylation and transcriptional control in the nucleus and cytosol will be essential for understanding the pathological triggers regulated by atypical p38 in vascular inflammation and cardiac damage.The substrate and adaptor accessibility of p38 dynamically controls cellular functions, including the regulation of transcription factors10. Indeed, p38-dependent phosphorylation of TAB1 has also been suggested to spatially bias TAB1 away from the nucleus into the cytosol36, and that over-expression of TAB1 can antagonize MKK3/6 activation, altering p38 subcellular localization47. Spatial restriction is an essential regulatory mechanism for many kinases. Our current data shed new light on these dynamics, demonstrating that although p38 activation by both MKK3/6 or TAB1 leads to the identical dual phosphorylation of p38, differential functional outcomes of atypical p38 are likely driven by limiting access to specific adaptors and transcription factors in the nucleus. Indeed TAB1-p38 activation is known to play essential roles in inflammation, including endothelial barrier homeostasis and activation of cytokine expression49. Spatial sequestration in the cytosol likely enhances access to these critical elements driving pathological signaling. However, further studies are required to define the specific downstream targets of atypical p38 and the specific mechanism that TAB1-p38 harness to regulate pathological signaling. Furthermore, Dyngo4A-dependent blockade of CME flipped the temporal dynamics of atypical p38 signaling, shifting the focus from endosomal and cytosolic to nuclear signaling responses. The current studies provide a framework by which to explore the spatiotemporal dynamics of atypical p38 signaling in GPCR-dependent and -independent mechanisms and screen for molecular regulators that could be therapeutically targeted. Future studies should focus on determining how the endosome selectively controls the spatial bias in atypical p38 activity and identifying the spatially restricted targets that drive pathological atypical p38 responses.In summary, we have successfully demonstrated a distinctive profile for spatiotemporal atypical p38 signaling by GPCRs. After the formation of the TAB1-p38 complex, TAB1 remains bound to p38, which is thought to spatially bias p38 from entering the nucleus or slow its translocation2.HeLa cells  were used for all experiments. Dulbecco’s modification of Eagle’s medium  supplemented with 10% (v/v) fetal bovine serum  was used for cell maintenance. Cells were incubated at 37 °C in humidified conditions with 95% air and 5% CO9. PTGER 2 and 4 (Prostaglandin E receptor 2 and 4) were a gift from Bryan Roth  biosensor was a kind gift from Dr. Saito  and has been previously characterized18. The FRET sensor was subcloned into pcDNA3.1 with spatial targeting motifs PCR-cloned onto the C-terminus of SECFP. The targeting motifs were (1) the plasma membrane, using a C-terminal lipid modification, Kras motif19, (2) the early endosome using 2xFVYE PI3P binding motif from mouse Hrs20, or the full-length wild-type Rab525, (3) the nucleus using a nuclear localization signal motif23 and (4) diffuse cytosolic with nuclear export sequence18. mRFP-Rab5 was generated for this study using PCR-directed cloning. Briefly, mRFP  was cloned onto the n-terminus of pcDNA3.1 Rab5 wt and Q79L52 using 5′ BamHI and 3′ EcoRI, sequences were verified using Sanger sequencing.pcDNA3.1 FLAG-PAR1 was as previously described2  with DMEM (10% FBS) in a humidified incubator for 24 or 48 h.Cells were transfected in suspension with polyethyleneimine  incubated with plasmid DNA in Gibco OptiMem Reduced Serum media  before plating in 35 mm microscopy dishes  coated with human fibronectin, 1.5 µg/cm2, 2 g/L d-glucose  0.23 mM Sodium Pyruvate  in Gibco Hanks Modified Balanced Salt Solution , pH 7.4. SB203580, , Dynamin Dyngo 4A . Rabbit anti-EEA1 , rabbit-anti-GAPDH-Alexa647 , rabbit Alexa fluor-594 (Cat No. A11012. Fisher Scientific). Rabbit anti-total p38 , Rabbit anti-phospho-p38 , HRP-conjugated goat–anti-rabbit . RedDotTM2-far-red nuclear stain .Starvation medium composed of 0.1% bovine serum albumin  20 mM HEPES  1 mM CaCl  in phenol-red free DMEM  and filtered through a nylon 0.45uM filter, was applied to transfected cells for 24 h before live-cell imaging and exchanged to FRET imaging buffer  was calculated. FRET/SECFP channel light intensity was adjusted prior to data collection to achieve a baseline ratio average of 1.0. Typically, between thirty and fifty individual whole-cell ROIs were taken per experiment. In addition, ROIs were mapped across the experiment to follow cell movement. Cell exclusion criteria were limited to cells with a saturated SECFP or FRET intensity, cells morphologically indicative of death/division (rounded up or blebbing), or if cells were not wholly in the imaging frame. Images were collected using epifluorescence microscopy and presented as a ratiometric analysis of the emission from cyan (SECFP) and yellow (YFP/FRET). Fluorescence intensities were background-corrected by subtracting the background fluorescence intensity of a cell-free region from the emission intensities of biosensor-expressing cells. As such the FRET ratio from a small area versus the whole cell is comparable as the FRET response is normalized to the total CFP in the ROI rather than a fraction of the total area. As such an endosome/PM versus the whole cell ROI yields comparable dynamic responses , Colibri LED, Axiocam 506 mono, and Plan-Apochromat 20x/0.8 M27 objective, with a heated environment stage 37 °C in humidified conditions with 95% air and 5% COr) was calculated within Zen Blue 3.0 software from whole cell ROIs.For high-resolution 63× confocal imaging, live-cell images were collected with a 0.4 µm z-section. FRET sensor expressing cells were co-labeled with RedDot2 (1:200) at 37 °C for 20 min, media was exchanged with phenol red free DMEM before live-cell imaging. For PM biotracker orange, cells were placed at 4 °C and stained with 5 µl per ml for 15 min. Cells were then rinsed with phenol red free DMEM before being fixed with 4% paraformaldehyde, washed with PBS and immediately imaged, as slide mounting disrupted PM labelling. For co-labeling with EEA1 or GAPDH-647, transfected cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% in triton X100, then incubated overnight at 4 °C with a primary rabbit anti-EEA1 (1:250) antibody and secondary anti-rabbit Alexa-596 (1:500), or GAPDH-647 (1:250) mounted and imaged with a plan-apochromat 63×/1.4 oil DIC M27 objective. Pearson Correlation Coefficient , as indicated.All data were analyzed using Prism 9.0 software  to unbiasedly calculate FRET max and T1/2 values. Prism was also used to calculate statistical significance using Student’s Supplementary Figures.


In this article, we analyse the relationship between union instability and cumulated fertility among ever-partnered women in several regions across Europe and the Americas with different patterns of demographic behaviour in terms of fertility levels, union instability and fertility across partnerships. We hypothesise that the relationship between union dissolution and fertility might be less negative in contexts where repartnering is more prevalent. The analysis is performed on a large dataset of 25 countries, combining information from the Harmonised Histories of the Generation and Gender Programme with our own harmonisation of survey data from three Latin American countries. This allows for the inclusion of countries with differing prevalence of union instability as measured by (a) the proportion of women who separated by age 40, and (b) the proportion who repartnered by age 40. We first examine the prevalence of separation and repartnering during reproductive ages across regions, and we estimate the proportion of cumulated fertility attributable to unions of different ranks using a decomposition method. We then analyse the links between union instability and the number of children born by age 40 among ever-partnered and ever-repartnered women, using Poisson regression. Despite observing a high degree of heterogeneity in the proportions of births occurring in the context of repartnering both within and between regions, we find a pattern where a greater prevalence of repartnering by age 40 is accompanied by higher cumulated fertility in second or subsequent unions. Our multivariate findings reveal a negative statistical relationship between separation and cumulated fertility that is partially offset by repartnering in some contexts, and that the time spent in a union during the reproductive lifespan is a key determinant of cumulated fertility, regardless of national context and independently from age at union formation and union rank. In a context of increasing separation and divorce rates, the relationship between union dissolution, repartnering and fertility outcomes becomes a central piece in the analysis of demographic dynamics and the projection of future fertility trends. However, analysing the effect of separation and repartnering on achieved fertility is a challenging task, mainly because the aggregate observed effects are the result of counteracting mechanisms. On the one hand, unstable partnership trajectories can result in fewer children through the consequent reduction of the time spent in union. On the other hand, the formation of second or higher order unions can result in larger family sizes than would have been achieved in the absence of interruptions if sharing biological children is seen as a way to consolidate new partnerships. In other words, whether the association between union instability and fertility is negative or positive depends, among other things, on whether additional birth(s) associated with repartnering outweigh the reduction of time spent in union caused by separation.The links between union instability and fertility could be different across several European regions, North and Latin America, since these regions have historically differed in terms of the timing of the fertility transition –resulting in differing current fertility levels and timing of births– and have also experienced the changes associated with the second demographic transition at different paces and starting points.The demographic literature has been concerned with the links between union instability and fertility since the early 1970s in the context of an increasing deinstitutionalisation of conjugal life, rising divorce rates and the displacement of marriage by cohabitation. The evidence emerging from a large number of studies conducted until now in Europe and North America is unambiguous: union dissolution, in the absence of repartnering, is negatively associated with the number of children by the end of the reproductive trajectory. Even though repartnering may help to partially compensate for this loss, the completed fertility of women who experience repartnering tends to be lower than the completed fertility of women that reach the end of their reproductive trajectories without experiencing separation or divorce. These results seem to be independent of the geographical context or time period partnering behaviour. Several studies show that the presence, number and age of children from previous unions are also important determinants of childbearing in a new partnership after first union breakup  the experience of union dissolution, in and of itself; b) the experience of union dissolution in the absence of repartnering, compared to the experience of a separation followed by repartnering during reproductive ages; c) the total time spent in a conjugal union during the reproductive lifespan, which is the result of the timing of the events of union formation, dissolution and repartnering.Several demographic behaviours related to union instability and fertility exhibit geographical patterns across regions.Completed cohort fertility is overall higher in the United States than in Europe Sobotka, . In the 1Although fertility postponement is characteristic of the fertility decline across regions Sobotka, , the patThe timing of childbearing also varies by regional context; across Europe, age at first birth is lowest in Eastern Europe, followed by Central Europe, and it is the highest in Western-Northern and Southern Europe  might be less pronounced in national contexts and regions where union dissolution and repartnering are more prevalent and socially accepted. Two mechanisms underlie this assumption. First, a higher prevalence of repartnering during childbearing years creates larger pools of individuals at risk of having children in new unions. Secondly, similarly to the literature on the relative risks of dissolution of marriages and cohabiting unions  and achieved fertility.To do so, we harmonised partnership and fertility trajectories information from national surveys in Colombia, Uruguay and Mexico.We perform our analysis on a sample of women aged 40 or older at the time of the survey,In order to conduct our analysis, we used biographical information on the number of unions, age at union formation and age at separation or divorce for each union episode. This allowed reconstructing conjugal trajectories up to the age of 40. We classed ever-partnered women according to whether, by age 40, they were: still in their first union; separated from a first union (and not repartnered); or in a second or subsequent union (repartnered). A number of descriptive statistics for this data can be found in Table Firstly, we present a descriptive section in which we analyse the geographic patterns of union instability in terms of the prevalence across regions of union dissolution and of repartnering in a second or subsequent union. In order to assess the links between conjugal transitions and fertility, we also perform a descriptive decomposition of cumulated fertility among ever-partnered and among ever-repartnered women by union rank of births. In this analysis, we estimate the number of children and the fraction of cumulated fertility that can be attributed to first unions, to second or subsequent unions, and to out of union births. The procedure for such decomposition entails identifying the conjugal status of the respondent at the time of each birth , using biographical information on union and births histories in a person-year dataset. Once each birth has been associated to a specific conjugal status, respondents are classed according to the different conjugal situations in which they had children. These conjugal situations are: in first union only; in first and in second or subsequent union; in second or subsequent union only; other8We then model the number of children ever born up to age 40 as the outcome of different characteristics of the partnership history, using Poisson regression for each country and survey iteration. Since our goal is to measure the result of the experience and the timing of conjugal transitions in the life course, the central variable of interest indicates the conjugal status by age 40; the categories of this variable vary in each model so as to allow focusing on different aspects of the process of union dissolution.In the first three models, we estimate cumulated fertility by age 40 among ever-partnered women.In our first model estimates, the main independent variable indicates ever having experienced a separation by age 40, as opposed to remaining in the first union (reference category). In the second model, we explore whether the links between union dissolution and fertility are different depending on whether the separation was followed or not by repartnering during reproductive ages. Thus, the categories of the main independent variable indicate: (a) experiencing separation and not forming a new union by age 40, or, (b) experiencing repartnering by age 40 (compared to still being in a first union as the reference category). In a third model, we use the same main independent variable as in model 2, but we add a measure of the total time spent in any union by age 40, as a way of disentangling the effects of the events of separation and repartnering themselves from the reduction of the time spent in a union on the coefficients for completed fertility. Our analytical approach entails including the total time spent in unionFinally, we estimate a fourth model for cumulated fertility by age 40, this time only among ever-repartnered women. Here, the central variables of interest are the time spent in a first union and the time spent in a second or subsequent union,All our models control for age, type of first union (marriage or not) and whether the respondent has attained a higher educational attainment level (ISCED levels 5 or 6) at the time of the survey.Southern Europe: Italy and Spain;Central Europe: Czech Republic, Estonia, Hungary, Lithuania and Poland;Eastern Europe: Bulgaria, Romania Belarus, Georgia, and Russia;Western and Northern Europe: Austria, Belgium, France, Germany, Netherlands, Norway, Sweden, and the United Kingdom;North America: Canada and the United States;Latin America: Colombia, Mexico, and Uruguay.We incorporate a geographic perspective throughout the analysis as a way to a) examine whether countries that have been less explored in the previous literature stand out with respect to the link between union dissolution, repartnering and fertility, and; b) to understand whether the observed results are context dependent. To classify the countries into regions, we follow the classifications proposed by Zeman et al.  and by SOur classification of European countries slightly differs from that of Zeman et al.  and thatAcross regions, ever-partnered women’s most common conjugal status by age 40 was to be in their first union. However, union instability levels, both defined as the percentage separated and the percentage repartnered by age 40, exhibited great variation between regions, and in some cases also within.Overall, Southern and Eastern Europe stand out as the regions with the lowest prevalence of separated women by age 40, with Spaniards and Italians exhibiting the lowest shares of separated women (3% to 6%) of all observed countries Fig. , as wellA high degree of heterogeneity in the prevalence of separation by age 40 is observed among women in Western and Northern European countries, ranging from 8% of ever-partnered women in Belgium (2005) to 13% in Austria. In North and Latin America, this percentage oscillates between 10% (Uruguay) and 15% (Colombia), the second to highest proportion of separated women in the sample , Georgia and Italy between 1% and 2%), followed by women in Bulgaria, Spain (2018) and Romania (between 4% and 5%). Again, repartnering is, however, much more common in Russia –where almost one in five ever-partnered women have repartnered by age 40 (19%)– than in the rest of Eastern Europe  of ever-partnered women had repartnered by age 40  and France to more than a quarter of ever-partnered women in Belgium, the German-speaking countries (Austria and Germany) and Sweden   , at 1.4 children per woman.Cumulated fertility is higher for North American women, those in Nordic countries (Norway and Sweden) and some Western European countries  as well as in Poland and Georgia. The highest fertility levels in our sample are found in Latin American countries, particularly in Colombia and Mexico where the average number of children per woman is close to three , where about 5% of births are attributable to higher order unions, to somewhat higher shares in United Kingdom, Norway and the Netherlands (2013), and much higher shares in Belgium, Germany and Sweden, the country with the largest share of repartnered women by age 40 among the European countries; 20% of ever-partnered Swedish women’s cumulated fertility is attributable to births in higher order unions, the highest observed in any region  and 14% and 16% of births in the United States (1995 and 2007). A great degree of variability is also observed among women in the Latin American countries, where the share of cumulated fertility attributable to second or higher order unions in Colombia (11%) is about twice the share observed in Mexico and Uruguay –despite overall cumulated fertility being the highest among all observed countries both in Mexico and in Colombia , in the Central European countries and in Belarus. In the Southern European countries, this share is between one fifth and one quarter of all repartnered women. Repartnered women in Northern and Western Europe stand out, on the contrary, for having the highest shares of births only in second or subsequent unions; in countries like Belgium, Germany and Sweden, close to half the births to repartnered women happened in the context of higher order unions Fig . .The trajectories of repartnered women in Central and Eastern Europe are characterized by the experience of births both in their first and in their second or subsequent unions; between a quarter and a third of them had this type of childbearing trajectory by the time they were 40 years old. This type of trajectory is also more frequent among ever-repartnered Latin American women, a quarter of Colombians and Uruguayans having had children both in the first union and in second or subsequent unions. This share is slightly lower in Mexico (22%) , where childbearing within the first union is less common than in second or subsequent unions Fig. .1313The analysis of the share of births by conjugal setting focusing only on ever-repartnered women sheds light on the fact that childbearing within repartnering is relatively common in some regions where union instability is the least prevalent, such as in Southern Europe. We also observe that childbearing both in the first union and in subsequent unions is more prevalent in certain regions, particularly among repartnered women in Latin America, Central and Eastern Europe.Our first model examines the overall impact of experiencing union dissolution on cumulated fertility by age 40 among ever-partnered women. As expected, in almost all regions and national contexts-periods we observe lower fertility among women who experienced separation by age 40, as denoted by the exponentiated coefficients below 1. Among women in Southern and Eastern Europe and in North America, having separated by age 40 is consistently associated with lower cumulated fertility. This is also the case for women in Central Europe, except for the Czech Republic, where the coefficient is negative but not statistically significant, as denoted by confidence intervals touching the horizontal black line Fig. .14 In WeLatin America is the only region in which none of the countries show a significant negative coefficient for the relationship between the experience of union dissolution by age 40 and fertility.Another relevant result for model 1 . This means that, across regions, the relationship between being in a second or subsequent union and cumulated fertility by age 40 is now either positive or not statistically distinguishable from continuing in an intact first union by age 40  the coefficients both for time spent in a first union and for time spent in second or subsequent unions are positive and significant. In Central and Eastern Europe, the coefficients for time spent in a second or subsequent union are positive and, in some cases, significant . Most coefficients for the first union in these two regions are not statistically significant despite being positive.Model 4 introduced controls for the timing of the first union and also for the timing of the second or subsequent union formation , in Western-Northern European countries, in North America, and in Colombia among the Latin American countries. However, a higher prevalence of union dissolution by age 40 is not necessarily met with higher levels of repartnering among Central European women . Repartnering before age 40 is much more common in the German-speaking European countries (Austria and Germany), in Sweden, the United States and Colombia. Albeit at lower levels, second or subsequent union formation was also not rare in the other Western and Northern European countries, and in Russia.Although most childbearing among ever-partnered women happens in first unions, there is a large degree of heterogeneity across national contexts in the proportion of births that take place in second or subsequent unions. One of our goals was to gain an understanding into whether a higher prevalence of repartnering during childbearing years was associated with fertility outcomes, across a wide range of cultural and socioeconomic contexts. The descriptive analysis revealed a pattern where the higher the share of repartnered women, the higher the proportion of cumulated fertility in second or subsequent unions. In this sense, women in regions such as Southern, Eastern and some of Central Europe repartner very infrequently , notably the roles played by (a) experiencing union dissolution before age 40; (b) experiencing repartnering before age 40; and (c) the total time spent in a conjugal union before age 40.As expected, and in line with previous findings, experiencing union dissolution –rather than remaining in the first union during the prime childbearing years– is mostly associated with lower cumulated fertility. However, such a negative relationship was not found in the Czech Republic, Belgium, the United Kingdom, and the three Latin American countries, Uruguay, Mexico and Colombia.Once we made the distinction between experiencing union dissolution and remaining single, compared to repartnering following the demise of the first union, we observed a compensating effect of forming a new union on cumulated fertility. In a few countries, the relationship between repartnering and cumulated fertility is even positive, meaning that repartnered women tend to have higher fertility than those in intact unions. This was the case in some of the same countries where the separation itself was not statistically associated with cumulated fertility .timing of union formation and dissolution, showed a positive association with cumulated fertility in most national contexts. Moreover, once this control was added to the models, we identified changes in the previously observed associations between union instability and fertility. Most notably, experiencing separation – and remaining single – no longer has an association with cumulated fertility in all countries, except in some where the association is now actually positive . This suggests that the negative association between union dissolution and cumulated fertility is not only offset by repartnering, but also mediated by the time spent in union.The total time spent in union before age 40, a key variable linked to the The association between repartnering and fertility is also strongly influenced by the amount of time spent in a conjugal union during the prime reproductive years: once this variable is controlled for, repartnering becomes either no longer associated with cumulated fertility, or positively associated. Among women in Southern Europe, the conjugal trajectory and cumulated fertility are not statistically linked once the time in first union is taken into consideration; this finding is consistent with their much higher ages at first union formation and also at repartnering, among the highest observed in the sample. On the other hand, the relationship between repartnering and fertility is clearly positive in the Czech Republic, Estonia and Lithuania in Central Europe, Romania and Belarus in Eastern Europe, the United Kingdom and Sweden in Western-Northern Europe, and Mexico and Colombia in Latin America.timing of dissolution and of new union formation are not protracted during the prime reproductive ages.These results shed light on the fact that union instability and repartnering in and of themselves might be neutral or even positive for fertility, as long as the The one exception to this overall pattern is found in the United States (2007), where repartnering is negatively associated with cumulated fertility. This result is puzzling given that women in the United States exhibit the highest share of multiple-partner fertility among industrialized nations . This analysis revealed that, in most settings, time spent in a union any of those unions so that fertility goals can be achieved during the reproductive ages.The positive association observed in most countries between the amount of time spent in a conjugal union and cumulated fertility by age 40 confirms previous findings by Thomson et al.  for FranEven though we found geographical patterns in the levels of union instability, the levels of repartnering, and in the importance of births in second or subsequent unions during childbearing ages, we did not find clear-cut geographical patterns in the relationship between union instability and fertility in the multivariate models linking the conjugal trajectory and cumulated fertility, once we estimated this relationship net of other confounders that vary greatly across regions (age at union formation and time spent in union among others). The fact that a positive association between repartnering and fertility is found in several countries despite their differing levels of union instability and overall fertility suggests that the experience of multiple partnerships might become a propelling factor for fertility -or at least an attenuating factor for decreasing fertility- in some sociocultural contexts. However, this can only have a visible global impact on fertility levels if repartnering becomes more prevalent following union dissolution: even a positive relationship between repartnering and cumulated fertility would have a negligible aggregate effect in regions where second union prevalence is very low such as in Southern, Central and Eastern Europe.Finally, the main limitation of our study is that we were unable to incorporate factors like the presence, number, and age of children from previous unions for women and their partners (Beaujouan & Solaz, Future work should focus on the relationship between union and fertility dynamics in a wider range of Latin American countries or sub-regions, representing diverging stages of the fertility transition.


However, increased intake of nutrients, including glucose, fructose, and saturated fat, induces hepaticCaenorhabditis elegans. Chronic exposure to environmental stress disrupts lipid metabolism in worms, and the role of IRE-1 and HSP-4 in energy homeostasis inC.elegans has been reportedThe most conserved signaling pathway in the UPR is IRE1α/XBP1s. Numerous studies have suggested that IRE1α/XBP1s regulates nutrient metabolism in the liver. The role of IRE1α/XBP1s in lipid metabolism has been established using many mammalian models ,3. MoreC.elegans contains two analog proteins,trf-1 andtrf-2. According to sequencing analysis,trf-1 is the gene that is most analogous toTRAF2 in humans andTraf2 in mice. Previous studies have demonstrated that tunicamycin (TM)- or thapsigargin (TG)-induced ER stress increases the degradation of TRAF2 in the L929 cell lineXBP1 toXBP1s, regulates IRE1α-dependent decay using the RNase domain, and recruits TRAF2 by IRE1α and initiates the JNK/NF-κB pathway.Traf2 andXbp1s play crucial roles in metabolic responses in mouse models  and lipid metabolism signaling   H. These trf-1 RNAi in transgenic worms, which expressed the ER stress reporter HSP4::GFP, induced hyperactivated ER stress signaling (xbp-1 dependent) upon dithiothreitol (DTT) treatment. Expression of the HSP4 promoter-driven reporter presented by GFP fluorescence (white) inC .elegans was increased bytrf-1 knockdown. Additionally,xbp-1 knockdown inhibited the expression of the reporter, which was mediated bytrf-1 RNAi (xbp-1 also decreased lipid accumulation inC.elegans (H.sapiens andC.elegans.TRAF2 is a RING domain E3 ligase. We overexpressed TRAF2 and XBP1s in HEK293T cells to further investigate the crosstalk between TRAF2 and IRE1α/XBP1s signaling. Our results revealed that TRAF2 dramatically increased the ubiquitination of XBP1s A. XBP1s -1 RNAi E. Knockdelegans F. These TRAF2 is regarded as the most significant adaptor in NF-κB signaling and can be activated by ER stress. Numerous studies have focused on the IRE1α/XBP1s pathway in the metabolism of nutrients, including glucose and lipids. It is well known that the regulation of ER stress signaling is complex. However, how TRAF2 and XBP1s, which are downstream of IRE1α, communicate with each other is unclear. Our results revealed a crosstalk between TRAF2 and XBP1s, the two arms of the IRE1α signaling pathway. The results of our study demonstrated that TRAF2 can ubiquitinate and inhibit the transcriptional activity of XBP1s. Although there is insufficient evidence indicating that TRAF2 is the direct E3 ligase of XBP1s, it may be valuable to study this further. The crosstalk between TRAF2 and XBP1s may regulate lipid metabolism in the liver. In conclusion, we revealed the IRE1α-TRAF2-XBP1s integrated signaling, which may be targeted to develop metabolism-related disease therapy.


Hepatozoon spp. are apicomplexan parasites known to cause musculoskeletal disease in a variety of animals. Two species are known to infect wild and domestic canids in the US: Hepatozoon canis and H. americanum.Hepatozoon. Histology slides of heart and skeletal muscle were assessed for Hepatozoon cysts and associated inflammation when fresh tissue was available (n = 96).In this study, blood, heart, and/or spleen samples were collected from 278 wild canids  in the eastern US and tested via PCR for Hepatozoon spp. were found in 24.2% (59/278) of individuals, with Hepatozoon canis in 14.0% (34/278) and H. americanum in 10.7% (26/278). One coyote was positive for both H. canis and H. americanum. Foxes were more likely to be positive for H. canis than coyotes , while only coyotes were positive for H. americanum. Of the eight sampled states, H. canis was present in six  while H. americanum was found in two southern states (South Carolina and Louisiana). Infection status was positively correlated with myositis and myocarditis, and heart or muscle cysts were found in 83% (5/6) of H. americanum-positive coyotes.H. canis and H. americanum in states where the parasite was previously unrecorded including South Carolina and Pennsylvania.This survey showed a moderate prevalence of The online version contains supplementary material available at 10.1186/s13071-023-05968-x. Hepatozoon is a genus of apicomplexan parasites known to cause musculoskeletal disease in numerous terrestrial vertebrate species  of coyotes). Hepatozoon canis was found in all tested states except for Georgia and Maryland  as the outgroup, resulted in a 488-bp alignment of which 390 were invariant and 32 of the 98 variable characters were parsimony informative. The top match in GenBank for each unique sequence was included in the tree for comparison. Sequence analysis of the Hepatozoon sequences revealed 34 unique sequences. Cloning was performed on 11 PCR products, with up to four unique sequences present within an individual sample. One coyote was co-infected with both H. americanum and H. canis.Phylogenetic alignment of the 18S rRNA gene performed using one sequence for each unique sequence group, along with related organisms and Hepatozoon americanum sequences were between 94.2 and 100% similar to each other, with 46% (21/45) of H. americanum sequences > 99.5% similar to several sequences from dogs (e.g. EU146062 and AY864676) and coyotes (e.g. JX415170-JX415174) in the southern US. We found a second group of sequences (n = 11) that aligned more closely with H. americanum than H. canis but did not fully resolve into the H. americanum clade. These sequences most closely aligned with several sequences in GenBank including a South American gray fox  from Argentina (MK049949) and a Pampas fox  from Uruguay (MZ230033). We found minimal genetic diversity in the H. canis sequences, with sequences 98.2–100% similar to each other and 79% (27/34) of sequences aligning > 99.5% with several sequences from around the world including foxes from France (MK673844-MK673850), gray wolves from Germany (MN791089), and a dog from Cuba (MN393911). We found another distinct genotype of H. canis in five canids from Virginia, Tennessee, and North Carolina (OQ592106-OQ592110), which aligned 99.78% with several sequences from around the world including dogs from Thailand (MK830996), Algeria (MK645969), Nigeria (OP837324), and India (JN584477). Two Louisiana canids (OQ592117 and OQ592118) aligned 100% with a coyote from Oklahoma (JX415165). Unlike H. americanum, we did not find any evidence of paralogs or coinfection with multiple genotypes of H. canis.H. americanum on PCR , with myocarditis present in 57% (4/7) of positive cases compared to 4.5% (4/89) of negative cases. However, two myocarditis cases occurred in animals suffering from sepsis (the fox with sarcoptic mange and the coyote with osteomyelitis). Therefore, we re-assessed the statistical correlation between myocarditis and Hepatozoon status with those two cases removed, and the correlation remained . Likewise, myositis was found in 50% (3/6) of positive cases and only 8% (7/87) of negative cases . Skeletal muscle was not available from the sarcoptic fox or from two of the Tennessee coyotes. Both myositis and myocarditis were typically mild to moderate apart from the two septic animals  of samples, and myositis was present in 10.8% (10/93) , only three samples were collected from Georgia and only two from Maryland, making critical interpretation of prevalence in those states impossible.Delaware . Both foHepatozoon spp. prevalence in the US with which to compare our findings. A 2011 study published Hepatozoon spp. sequences from a variety of mammals in the southern US, including five coyotes with H. americanum from Oklahoma and Texas and one gray fox with H. canis from Georgia [H. americanum. There are only two other surveys assessing prevalence in coyotes, both of which focused on the south-central US. One survey reported Hepatozoon spp. prevalence of 40% in 20 coyotes in Oklahoma via histology, and a second study reported a prevalence of 80% in 44 coyotes in Oklahoma and Texas using a combination of histology and PCR [H. americanum emerged in the east alongside this migration, further research is necessary to determine if the eastern migration of coyotes has impacted H. americanum prevalence.There are limited studies on  and PCR , 17. Pre and PCR . AlthougHepatozoon in the US included samples from 614 domestic dogs with suspected hepatozoonosis, 455 of which were from the southeast [H. americanum in 13 of 28 states, and 5 states had both H. americanum and H. canis. Unlike in our study, Hepatozoon was not found in Tennessee or Pennsylvania, and samples from South Carolina were not obtained. Positive cases were primarily found in the southeastern US, though outliers with unknown travel history in Vermont, Washington, and California were detected [A 2008 survey of H. canis than the domestic dog survey [H. canis compared to the 12.4% (34/278) prevalence we found in wild canids. The relative percentages of each Hepatozoon species were also markedly different between domestic dogs and our study. Only 14.4% of Hepatozoon cases in domestic dogs were H. canis or mixed infections, while 57% of cases in wild canids from our study were H. canis or mixed infections. H. canis infections were rare in the Texas and Oklahoma coyote study as well, with only 2% (1/44) of coyotes positive [Hepatozoon canis in our study is likely explained by our inclusion of foxes, which had a significantly higher prevalence than dogs or coyotes. Whether H. canis is more common in the east than the central US remains to be seen, and future work assessing foxes in that area may clarify the question. Though the domestic dog study found a higher prevalence of H. americanum than we did (29.4% vs. 10.4%), a direct comparison cannot be made since the dog survey targeted suspected positive dogs, while ours sampled all individuals [Our study found a higher rate of g survey . Only 4.positive . The higHepatozoon spp. infection is commonly associated with co-infections that may be the true cause of inflammation [Despite the severe clinical signs of ACH in domestic dogs, infection did not appear to cause significant lesions in coyotes. However, infection status was positively correlated with risk of myocarditis and myositis. Myositis was six times more likely in positive individuals, while myocarditis was over ten times more likely. However, with only seven positive cases processed for histopathology, two of which had confounding factors, further research is necessary to determine the strength of this correlation. Even if the correlation persists in larger surveys, a causal connection could not be established from surveys alone given that ammation –25.H. americanum-positive coyotes had limb abnormalities, this is not likely a result of ACH. ACH is known to cause osteoproliferation and musculoskeletal pain, but loss of limbs or severe limb deformities are not known symptoms. In addition, the coyote with the missing hind limb survived at least 1.5 years after initial sampling and was in good body condition at the time of necropsy. It is possible that Hepatozoon infection can be exacerbated by immunosuppression. Domestic dogs typically only develop clinical signs from H. canis if they are immunosuppressed or co-infected with other diseases like Babesia, Leishmania, or Toxoplasma gondii [Hepatozoon cysts compared to all other positives, suggesting that replication may increase during times of stress or illness. The H. canis-positive red fox with sarcoptic mange unfortunately only had heart, skin, and kidney processed for histopathology, none of which are the typical locations to find meronts. Therefore, though we did not find meronts, we cannot assess the extent of its H. canis infection.Though two of the six a gondii . The coyH. americanum-positive coyotes. Forty-two percent of the positive coyotes (11/26) had evidence of multiple different sequences of H. americanum present. Previous work on Hepatozoon in coyotes suggested these different sequences were evidence of coinfection with multiple related Hepatozoon species, but intraspecific variation of the 18S gene  have been documented in H. canis and numerous other apicomplexan species [H. americanum clade, a subset fell in between H. canis and H. americanum  in the  species , 20, 26.H. canis sequences compared to H. americanum, with most sequences differing by fewer than three base pairs. This limited diversity has been documented in previous work, which classified H. canis 18S rRNA sequences into five genotypes, with each genotype differing by five base pairs or fewer [H. canis worldwide found 76 total haplotypes [H. canis genotypes were documented in southern states while only one genotype was documented in the north. This suggests diversity may be higher in the south, although further research is necessary to verify this assessment.We found lower genetic diversity (98.2–100%) in our or fewer . A largeplotypes . HoweverHepatozoon spp. in the eastern US. Wild canids appear to play a significant role in maintaining the sylvatic life cycle of Hepatozoon. A high prevalence in wildlife implies a high percentage of infected ticks in the area and can be used as a proxy for domestic dog risk. In addition, Hepatozoon prevalence can provide a better understanding of its vectors’ prevalence in the area. The tick vectors of Hepatozoon may carry other pathogens including Ehrlichia canis (R. sanguineus), Babesia canis vogeli (R. sanguineus), Francisella tularensis (A. maculatum), Rickettsia parkeri (A. maculatum), and Rickettsia rickettsii (R. sanguineus) [. These pathogens may lead to severe disease in dogs on their own or contribute to the severity of coinfections, and both Rickettsia spp. pathogens can cause life-threatening disease in people [This study provided a clearer understanding of the geographic distribution of guineus) , 29–32. Hepatozoon spp. prevalence ideally would always include both histology and PCR. Further research is necessary to assess this disparity, though PCR of whole blood may be a reasonable, less invasive alternative diagnostic tool in cases where obtaining a muscle biopsy is not practical. Finally, phylogenetic analysis using only a small section of the 18S rRNA gene may miss significant variation found elsewhere in the gene. Future studies can improve our understanding of canid Hepatozoon genotypes by using longer sequences or full genome sequencing.Limitations of this study include the opportunistic nature of collection, the limited sample size in certain states, and the variability of available tissue samples from individual canids. Future work can help fill in the geographic gaps of this survey to better understand the distribution and impact of this pathogen. In addition, future studies should strive to use whole carcasses for analysis whenever possible to give a clearer picture of tissue tropisms and tissue responses. Though our study found a high agreement between histology and PCR results, a previous study showed disagreement between the two methods, and the gold standard for diagnosis is still considered muscle biopsy , 16. TheHepatozoon is a widespread parasite infecting wild canids throughout the eastern US. Hepatozoon canis is far more common, particularly in foxes, than previously realized. Mild inflammation in both the skeletal muscle and the heart was more likely in infected individuals (Additional file Additional file 1: Table S1. List of species, capture locality and PCR results. Number of cysts listed for positive cases with available histopathology.


The 5-year survival rate of childhood cancer exceeds 80%, however, many survivors develop late effects including infertility. The aim of this study was to evaluate the current status of oncofertility care at Vilnius University Hospital Santaros Klinikos (VULSK) within the framework of the EU-Horizon 2020 TREL project.All parents or patients aged 12–17.9 years treated from July 1, 2021 until July 1, 2022 were invited to complete an oncofertility-care-evaluation questionnaire. After completing the questionnaire, patients were triaged to low-risk (LR) or high-risk (HR) of gonadal damage using a risk stratification tool (triage). Data was assessed using descriptive statistics.n = 6) agreed the risk for fertility impairment had been mentioned as compared to 49.1% (n = 27) of uncounseled. All counseled respondents agreed they knew enough about fertility (vs. 42%).Questionnaires were completed by 48 parents and 13 children triaged as 36 (59%) LR and 25 (41%) HR patients. Most HR respondents  were not counseled by a fertility specialist. Six boys  were counseled, none of the girls was counseled. Three HR boys underwent sperm cryopreservation. Only 17  respondents correctly estimated their risk. All counseled boys (Respondents counseled by a fertility specialist were provided more information on fertility than uncounseled. HR patients were not sufficiently counseled by a fertility specialist. Based on the current experience oncofertility care at VULSK will be improved. Currently, the 5-year survival rate of patients with childhood cancer exceeds 80%, leading to an increase in the number of childhood cancer survivors (CCS) , 2. HoweThe International Late Effects of Childhood Cancer Guideline Harmonization Group (IGHG) recommends informing all patients with childhood cancer about their gonadal damage risk and offering fertility counseling to those at risk –14. Howehttps://siope.eu/TREL-project). A previous cross-sectional study revealed that a majority of adult CCS treated at VULSK had limited knowledge about reproductive health and had not received sufficient information regarding fertility  through a twinning exercise with the Princess Máxima Center within an EU-Horizon 2020-funded project “Twinning in Research and Education to Improve Survival in Childhood Solid Tumors in Lithuania (TREL).” The project aims to foster research on different aspects of pediatric cancer including survivorship . We pursued to assess the awareness of patients with childhood cancer or their caregivers of their infertility risk and its compliance with the individual gonadal damage risk assessed by the triage. The results of this study will serve as a baseline for future research.First, the oncofertility-care-evaluation questionnaire developed by the Princess Máxima Center was adapted for Lithuanian patients . Two queSecond, the original gonadal damage risk-stratification tool (triage) used in the Netherlands was amended by the replacement of the treatment protocols that differed between the two institutions . All pro2) and HGDR  for girls (2) or HGDR  for boys  or patients aged 12–17.9 years treated for pediatric cancer (ICD-10-AM C00-96) between July 1, 2021, and July 1, 2022, were invited to participate in the study. Patients were identified in the institutional database. All patients diagnosed within the evaluation period and those diagnosed previously but still undergoing treatment were invited.Baseline characteristics including the age at diagnosis, time from the date of diagnosis to enrollment, malignancy type, and treatment (protocol and arm) were retrieved from the medical records. As recorded in the patient files, the date of diagnosis was defined as the date of communicating the cancer diagnosis by a pediatric oncologist to the family.Participants were divided into two groups: a group counseled by a fertility specialist and a group informed by a pediatric oncologist. Before the start of treatment, every patient received written information on potential risk for fertility impairment  during the provision of information on chemotherapy and its potential side effects. Thus, all participants not counseled by a fertility specialist were considered informed about the risk for infertility by a pediatric oncologist.Fertility counseling and fertility preservation were both provided before chemotherapy to all patients at the Santaros Fertility Center, a specialized unit at VULSK. Pubescent boys were counseled by an embryologist based on the results of the semen analysis. During the evaluation period, fertility preservation was only allowed for children aged ≥14 years according to the Lithuanian Law for Assisted Reproduction . TherefoA cross-sectional survey within the previously specified timeframe was performed. All participants completed the oncofertility-care-evaluation questionnaire. All patients were already receiving treatment when the questionnaire was handed in, no questionnaires were given between diagnosis and treatment initiation. The questionnaire was completed during the inpatient stay or the scheduled outpatient visit. Questionnaires were completed separately by patients or their parents, and no parent–child combinations were included. As mentioned above, two questionnaires were used: one for patients counseled by a fertility specialist (counseled group), and another for those informed by a pediatric oncologist alone (uncounseled group). The responses were evaluated in July 2022. The responses of the counseled group opposed those of the uncounseled group. The responses to each question were analyzed, and the frequencies of the responses were calculated.The respondents were also asked to self-estimate their specific risk for infertility. Thereafter, each response was compared with the infertility risk assessment provided by the study team using the above-mentioned risk-stratification tool.After completing the questionnaires, the respondents were retrospectively triaged to LGDR or HGDR using the amended version of the gonadal damage risk-stratification tool. Data were analyzed in September 2022.Descriptive statistics were used for all quantitative analyses. The normality of the distribution of continuous variables was evaluated using the Shapiro–Wilk test and expressed as medians with interquartile (IQR) and minimal–maximal ranges. SPSS Statistics version 17  was used for all quantitative analyses.n = 61) completed the oncofertility-care-evaluation questionnaire. More than half of the invited patients  refused to participate without specifying the reason for refusal. Therefore, their data were not analyzed.In total, 126 patients were invited to participate in the study that evaluated the existing oncofertility . Of the n = 16, 64%) of all HGDR patients were boys and 9 (36%) were girls. Of all 61 respondents, 6 (9.8%) boys were assigned to the counseled group  were <14 years old; therefore, fertility preservation was not an option as per the legal framework enforced at the time of the study. However, fertility preservation was not offered to six HGDR respondents aged >14 years. Two of them were diagnosed before this study, and three HGDR boys and one HGDR girl aged ≥14 years were diagnosed during the evaluation period.In the uncounseled group, 55 respondents (48 parents and 7 children) completed the questionnaire . Of thesThe median age of the respondents at the time of diagnosis was 8 (IQR 4–11) years, which ranged from 3 months to 17 years . The medn = 6, 4 HGDR, 2 LGDR) indicated more often that they were provided more information on fertility than the uncounseled group  (n = 27). The counseled group reported that they now know enough about fertility  and knew options for fertility preservation . Only 10/55 (19.2%) of uncounseled respondents received supportive material on fertility . All responses of the uncounseled and counseled groups are provided in The counseled group (34 LGDR) . All sixQuite a few  respondents commented to the open question, “What time do you think is the best time to have a conversation about fertility?” Most respondents  answered that fertility should be discussed before initiating cancer treatment. Four uncounseled respondents  thought the best time would be after the treatment. Some  uncounseled respondents stated they never had a conversation about fertility.The majority  of respondents could not correctly self-estimate their specific gonadal damage risk in comparison with the risk allocated by the study team . In totaThe increasing number of CCS raises the need for focusing the research on the late effects of cancer treatment and the well-being of CCS. Gonadal damage risk and fertility counseling have beneficial effects on the quality of life of CCS regardless of the decision on fertility preservation , 28. ThuTo achieve an essential breakthrough in oncofertility and survivorship care at VULSK, research collaboration with Princess Máxima Center was initiated as part of the EU-funded HORIZON 2020 twinning project TREL. As previously mentioned, a five-step oncofertility care plan launched at the Princess Máxima Center in 2019 served as a model . Until r2) leads to HGDR for boys compared with girls  boys were counseled by a fertility specialist . As mentn = 6) shared that they know enough about fertility  of the respondents did not know their specific gonadal damage risk . All couertility ; howeverertility . Resultsertility . Approxiertility , 41. In ertility . Consideertility . Thus, iAccording to the IGHG guidelines, a pediatric oncologist, endocrinologist, fertility specialist, or a specialized nurse may conduct fertility counseling . Our resThe low total number of respondents and counseled respondents could be a major study limitation. The number of study participants reflects the annual patient volume at VULSK, i.e., approximately 50–60 new patients with childhood cancer are diagnosed and treated annually (there are two pediatric oncology centers in Lithuania for the 2.8 million population) . ConsideThe results of this study revealed that fertility care for patients with childhood cancer can be quite difficult in clinical practice. Results revealed a need for daily identification, coordination, and documentation of newly diagnosed, timely triaged, and informed patients. Therefore, the oncofertility care plan was implemented at VULSK using an adapted risk-based oncofertility care system, and its systematic evaluation was implemented at the Princess Máxima Center. Therefore, the experience of fertility care from the points of view of the patients and parents could be compared between the two centers.This study contributed to the awareness of pediatric oncologists of the gonadal damage risk and facilitated referral to a fertility specialist. The study provides insight to further oncofertility care practice, and the results will contribute to the enhancement of fertility care and possibly aid in improving the quality of life of CCS. The oncofertility care at VULSK will be reevaluated after the implementation of the oncofertility care plan. The twinning activities between the two institutions contributed to the reduction of disparities in oncofertility care and research.The existing system of informing patients on gonadal damage risk, along with all other potential side effects of chemotherapy, must be improved. Counseling of children at HGDR by a fertility specialist is important and appears more efficient than informing by a pediatric oncologist, only providing adequate information. The absence of systematic gonadal damage risk stratification before treatment initiation and former restrictions in the Lithuanian legal framework jeopardized sufficient patient empowerment regarding their fertility-preservation options. These baseline results will be used to compare oncofertility care before and after the implementation of the oncofertility care plan. The oncofertility care plan established by the Princess Máxima Center was adopted and is in the process of implementation at VULSK as a part of the twinning project TREL, which will contribute to the reduction of disparities in oncofertility care and research.


A Delta-Notch signaling component, Notch1, is involved in the normal development and multiple disorders of the kidney. Although the increase in Notch1 signaling is crucial to these pathogeneses, the basal signaling level in ‘healthy’ mature kidneys is still unclear. To address this question, we used an artificial Notch1 receptor fused with Gal4/UAS components in addition to the Cre/loxP system and fluorescent proteins in mice. This transgenic reporter mouse system enabled labeling of past and ongoing Notch1 signaling with tdsRed or Cre recombinase, respectively.We confirmed that our transgenic reporter mouse system mimicked the previously reported Notch1 signaling pattern. Using this successful system, we infrequently observed cells with ongoing Notch1 signaling only in Bowman’s capsule and tubules. We consider that Notch1 activation in several lines of disease model mice was pathologically significant itself.The online version contains supplementary material available at 10.1186/s13104-023-06326-x. The Delta-Notch signaling pathway is a ligand‒receptor signaling pathway that mediates cell‒cell communication in developing organs . Upon biROSA26 locus following a floxed EGFP-STOP sequence (R26GRR mouse) [Here, we examined ongoing Notch1 signaling in mature kidneys using our original reporter mouse system that consisted of three transgenic mouse lines. The first mouse line expresses the artificial Notch1 receptor, the intracellular domain of which was replaced with the yeast transcription factor Gal4VP16  . The secR mouse) . TherefoTdsRed protein was detected in proximal tubular epithelial cells, suggesting that our reporter mouse system worked. Using this working system, we examined ongoing Notch1 signaling in the ‘healthy’ mature kidney.Animal experiments were carried out in accordance with the Regulation for Animal Experiments in our university and Fundamental Guidelines for Proper Conduct of Animal Experiment and Related Activities in Academic Research Institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology. Approval was obtained from the Institutional Animal Care and Use Committee and the DNA Experiment Committee of the University of Tsukuba  . UAS-Cre mice are available from RIKEN through the National BioResource Project of Japan: Crl:CD1(ICR)-Tg(UAS-cre/T2A/miRFP670)216Staka (No. RBRC11716). In addition, R26GRR mice are also available from RIKEN through the National BioResource Project of Japan: C57BL/6 N-Gt(ROSA)26Sor < tm1(CAG-EGFP/tDsRed)Utr>/Rbrc (No. RBRC04874).We used three N1-Gal4VP16; UAS-Cre; R26GRR mice (10–13 weeks old), one UAS-Cre; R26GRR mouse (6 weeks old) and one Non-Tg mouse (5 weeks old) for the analysis. To reduce the number of mice used, we used post-weaning single mouse per group for the negative control . Prior to sampling the organs, mice were sacrificed by cervical dislocation and perfused with PBS and Mildform 10 N . For fluorescent imaging, 10-µm-thick frozen sections were counterstained with Hoechst 33342 . For immunohistochemistry, 4-µm-thick paraffin sections were sequentially incubated with a rabbit monoclonal anti-Cre recombinase antibody  or a rabbit anti-RFP antibody , Histofine SimpleStain , Histofine DAB Substrate Kit  and counterstained with Mayer’s hematoxylin solution . Images were captured by using BIOREVO-BZ-X810 .To examine whether our reporter mouse system works, we started by visualizing the past Notch1 signal (tdsRed expression) in the ‘healthy’ mature kidney. Fluorescent imaging showed tdsRed expression in the cortex and medulla specifically in N1-Gal4VP16; UAS-Cre; R26GRR mice . Indeed, we never observed Cre-expressing cells in the medulla. In contrast, we occasionally observed these cells in the tubular epithelium Fig. a and Bowhttps://github.com/MasaharuYoshihara/Notch1-Signal-Kidney).Finally, to examine our finding that Notch1 signaling in ‘healthy’ mature kidney is rare, we conducted a single-cell RNA-sequencing analysis using a previously reported dataset (GSE107585) . The metWe confirmed that proximal tubular epithelial cells underwent Notch1 signaling. In contrast, cells with ongoing Notch1 signaling were very rare in ‘healthy’ mature kidneys.We were unable to describe the specific characteristics of Cre-expressing cells, although those cells tended to be located near each other. That was one reason the present study lacks explanation on the mechanism behind ongoing Notch1 signaling in the ‘healthy’ mature kidney.Below is the link to the electronic supplementary material.Supplementary Material 1: Additional file 1: Single-cell RNA-sequencing analysis of healthy mature mouse kidney.


The reprogramming of metabolism is a recognized cancer hallmark. It is well known that different signaling pathways regulate and orchestrate this reprogramming that contributes to cancer initiation and development. However, recent evidence is accumulating, suggesting that several metabolites could play a relevant role in regulating signaling pathways. To assess the potential role of metabolites in the regulation of signaling pathways, both metabolic and signaling pathway activities of Breast invasive Carcinoma (BRCA) have been modeled using mechanistic models. Gaussian Processes, powerful machine learning methods, were used in combination with SHapley Additive exPlanations (SHAP), a recent methodology that conveys causality, to obtain potential causal relationships between the production of metabolites and the regulation of signaling pathways. A total of 317 metabolites were found to have a strong impact on signaling circuits. The results presented here point to the existence of a complex crosstalk between signaling and metabolic pathways more complex than previously was thought. The link between metabolism and cancer has been well known since almost one century ago , with thThe profusion of detailed biological knowledge on signaling and metabolism, as stored in pathway repositories such as KEGG , ReactomHere, a study on the crosstalk between metabolism and signaling has been carried out in BRCA, using mechanistic models to simultaneously infer from gene expression data the production of metabolites ,22 and tGene expression data for BRCA and controls were downloaded from the TCGA portal and processed as described in Materials and Methods R2 score and mean squared error (MSE). As expected, not all tasks (circuit activities) can be predicted with reliability. However, there are no clear signs of overfitting, as can be seen by inspecting the joint distribution plot  show that the variability decreases as the predictive performance increases (less is better), for both the test and train fold sets. As with the R2 score, the error trend is clearly linear and close to the diagonal (identity).As outlined in Methods, a 100-times repeated 5-fold cross-validation was carried out in order to measure the viability of the model in terms of the ion plot , left ofUsing the multi-task Gaussian process, a total of 317 metabolites were found to have a strong impact on the activity of signaling circuits in BRCA . These cR2 score > 0.5) and non-relevant (R2 < 0.5) predictions that have or have not common genes. As a result, signaling circuits predicted and metabolic circuits corresponding to the most predictive metabolite do not show any significant enrichment in common genes . Actually, if the circuits analyzed are expanded to those corresponding to the 20 best predictive metabolites, the scenario is the same . This discards common genes between metabolic and signaling pathways as an explanation for this cross-talk, reinforcing the hypothesis that the metabolites detected are triggers of signaling pathway activity.Since the activity of a number of signaling circuits can be predicted from the previous production of metabolites, it is likely that such metabolites are the ultimate trigger for this cross-talk. However, signaling and metabolic pathways share some genes  but could not be detected in the metabolomic profiles , but not using an experimental metabolomics approach , a protective amino acid against cancer  interval and returns output values in the same interval. Such results are non-dimensional values that, like gene expression values, can be interpreted in the context of a class comparison.c fluxes . The EquThe mechanistic model implemented in Hipathia infers signaling circuit activities from the expression levels of the genes corresponding to the proteins involved in the circuits.Similarly, Metabolica estimates the production activity of the metabolites using the reactions involved in the sub-pathways analyzed . It is it-test for paired samples is used to assess the significance of observed changes in metabolite concentrations or in metabolic production activity when samples of two conditions are compared.Finally, a Student’s A False Discovery Rate (FDR)  is used Relating the production of metabolites with the activity of signaling pathways involves solving several Multi-Task regression problems, one for each signaling pathway and tumor type (for machine learning tasks only tumor samples are used). Here, Gaussian Processes (GP)  were usesn signaling circuit activities) can be expressed as ln latent GPs of the inputs (mn metabolite production activities) mixed by W . The covariance function of the GP is built using a linear combination of the squared exponential  and linear covariance functions. The kernel hyperparameters are forced to be shared across all the inputs , the inducing variables are shared between all the inputs (in= 50 inducing points are used) and W is the identity matrix of dimension ln. Note that Sparse Variational Processes are known to correctly scale across the data and output dimensions and all the computations are possible due to the data generating processes  [The model assumes that the outputs (y input) .R2 scores  and mean squared errors (MSE) were calculated for each circuit to evaluate the performance of each model. Note that since the model hyperparameters are optimized as part of the GP modeling, R2 is indeed measuring the suitability of the model building procedure which is in line with recent recommendations for machine learning use in computational biology [R2 ranges from -infinite to 1 where 0 is the baseline prediction (predicting the mean of each output). The R2 score for a given signaling circuit and its prediction is given by:n is the number of samples, j indexes the signaling circuit, i the samples, and ^ refers to the mean prediction of the gaussian process model. While the mean squared error (MSE) is given by:To assess the quality of the model, a 100-times repeated 5-fold cross-validation schema was performed in order to measure the generalization performance of the model.  biology . Here thShapley values are a game theory approach to estimate the importance of any individual player as a part of a collaborative team , which cR2 score of the training and testing splits.Here, the kernel-SHAP method  (v0.24.0The functionality triggered by signaling circuits is assumed to correspond to the functionality of the effector protein ,33,86. TSince signaling circuits trigger specific functionalities in the cell, metabolites that potentially regulate these signaling circuits can be annotated with the functions corresponding to these circuits. The results presented here reinforce the idea that, beyond the well-known influence of signaling on the reprogramming of metabolism in cancer ,11, a no