In total knee arthroplasty (TKA) using the modified gap technique, the soft-tissue balance is measured after osteotomy of the distal femur and proximal tibia . However, after osteotomy, the flexion gap size during 90° knee flexion may be larger than that observed after implantation. The tension of the lateral compartment during 90° flexion may also be reduced after osteotomy of the distal femur. We manufactured a distal femoral trial component to reproduce the condition after implantation and prior to posterior condyle osteotomy. This study aimed to evaluate the effect of the trial component on the flexion gap.This prospective study included 21 consecutive patients aged 78 years with medial osteoarthritis who underwent cruciate-retaining TKA between February 2017 and March 2018. The postoperative flexion gap size and inclination during 90° flexion were compared between cases with and without the trial component.The mean joint gap size with the trial component (13.4 ± 0.80 mm) was significantly smaller than that without the trial component (14.7 ± 0.84 mm). The mean gap inclination angle with the trial component (3.7° ± 0.62°) was significantly smaller than that without the trial component (5.5° ± 0.78°).In the present study, the joint gap size and medial tension were significantly reduced after the trial component had been set. Accurate measurement of the soft-tissue balance is an important factor in the modified gap technique, and this method using a distal femoral trial component can offer better outcomes than those achieved with conventional methods. In total knee arthroplasty (TKA) using the modified gap technique, the soft-tissue balance in extension and flexion is measured after osteotomy of the distal femur and proximal tibia, after which the rotation angle of the femur and the extent of bone resection from the posterior condyle are conventionally determined  –3.With the modified gap technique TKA, the basic concept is to achieve equal balance in both extension and 90°flexion. Intraoperative soft tissue balance has been reported to affect postoperative patient satisfaction and range of motion, and, therefore, it is important to measure the gap accurately , 4, 5. HTherefore, we manufactured a distal femoral trial component  to reproduce the condition after implantation and prior to posterior condyle osteotomy. This study aimed to evaluate the effect of the trial component on the flexion gap. We hypothesized that the flexion gap and joint gap inclination with the trial component would be smaller than those observed without the component.This prospective study included consecutive patients with medial osteoarthritis who underwent Cruciate Retaining (CR)-TKA (Zimmer-Biomet’s Persona CR) using measured resection techniques between February 2017 and March 2018. Informed consent was obtained from all participants. Cases in which the gap was not measured were excluded. The patella was not resurfaced in all knees. A medial parapatellar approach was used in all cases. The distal femoral cut and proximal tibia cut were performed perpendicular to the mechanical axis. The trial component was equal to an implant (thickness: 9 mm)       Fig. a or pateIn TKA using the modified gap technique, the soft-tissue balance is typically measured after osteotomy of the distal femur and proximal tibia. The implant position, rotation angle of the femoral implant, and extent of bone resection from the posterior condyle are then determined –9. This et al. reported that the flexion gap was 2 mm smaller than the extension gap prior to PCL resection, and 1.7 mm larger afterwards [et al. reported that the joint component gap during 90° flexion was positively correlated with the tibial slope in posterior stabilized (PS) TKA [et al. showed that  changing the tibial slope by 5° altered the flexion gap by approximately 2 mm with CR-TKA and 1 mm with PS-TKA [et al. reported that the patellar height influenced the soft-tissue balance and was positively correlated with the joint component gap at greater flexion angles (90° and 135°) in PS-TKA, but the PCL was an important factor influencing the flexion soft-tissue balance in CR-TKA [et al. [It has been reported that posterior cruciate ligament (PCL) resection, the posterior tibial slope, the patellar height, and the setting of a trial component affect the knee flexion gap in TKA –19. Kadoterwards . Oka et (PS) TKA . Okazakih PS-TKA . Nishizan CR-TKA . In the  [et al. , we did et al. reported that the intraoperative gap difference between flexion and extension was significantly greater with the use of a trial component than without the component. This difference was attributed to the surrounding soft-tissue tightness or elasticity [et al. reported that the intraoperative component gap kinematics was affected by the tightness of the posterior structure during extension and the tightness of the anterior structure during deep knee flexion [Hananouchi asticity . Hayashi flexion . The resIn conventional TKA using the modified gap technique, the flexion bone gap is typically measured during a state of relaxed forward tension, after which the posterior condyle is cut with reference to the bone gap. Therefore, the quantity of bone resected in the osteotomy is smaller than necessary, and a larger component is used. Consequently, the posterior condyle of the component tightens the posterior structures, and the joint gap during extension is reduced , 20. TheIn the present study, by using a distal femoral trial component, the joint gap size and medial tension was significantly reduced after the trial component had been set. Accurate measurement of the soft-tissue balance is an important factor in the modified gap technique, and this method using a distal femoral trial component may yield better outcomes than those attained with conventional methods.The limitations of this study were that perioperative measurements were not reflected in the surgical procedure. The current study evaluated the intraoperative gap difference with and without a distal femoral trial component. The femoral component was placed based on these results and the feel of the spacer block insertion. Therefore, the joint gap observed with the use of the distal femoral trial component could not be compared to the component gap.In the present study, the joint gap size and medial tension were significantly reduced after the trial component had been set. Accurate measurement of the soft-tissue balance is an important factor in the modified gap technique, and we can accomplish better outcomes by using a distal femoral trial component than those obtained with conventional methods. In future, we will further evaluate the gap difference between PS-TKA with and without a distal trial component, and will compare the joint gap observed with the use of the distal femoral trial component to the component gap in the cases in which the surgery is performed with reference to the joint gap with the distal femoral trial component.


Effective mentoring is a key mechanism propelling successful research and academic careers, particularly for early career scholars. Most mentoring programs focus on models pairing senior and early career researchers, with limited focus on peer mentoring. Peer mentoring may be especially advantageous within emerging areas such as implementation science (IS) where challenges to traditional mentoring may be more prevalent. This special communication highlights the value of peer mentoring by describing a case study of an early career IS peer mentoring group. We delineate our curriculum and structure; support and processes; and products and outcomes. We highlight important group member characteristics to consider during group formation and continuation. The group’s long-term (6 years) success was attributed to the balance of similarities and differences among group members. Members were in a similar career phase and used similar methodologies but studied different health topics at different institutions. Group members gave and received instrumental and psychosocial support and shared resources and knowledge. Peer mentoring can serve an important function to provide emotional, logistical, and professional development support for early career scholars. Our case study highlights strategies to foster peer mentoring groups that provide a generalizable blueprint and opportunity for improved outcomes for early career professionals. Receipt of mentoring, particularly early in one’s career, can promote successful transition to professional independence . It faciMentoring is a critical ingredient for conducting innovative, impactful translational research, including implementation science (IS). The National Institutes of Health (NIH) and National Science Foundation (NSF) recognize the importance of mentorship in facilitating professional success . The NatTraditionally, mentoring is a unidirectional, hierarchical relationship between an established, senior mentor providing mentoring to a less established, junior mentee, most commonly co-located in the same institution . In thisPeer mentoring, or bidirectional mentoring between those at a similar career stage, is a well-established professional development practice, especially during career stages that are characterized by development and transition ,15. An ePeer mentoring is especially important for implementation scientists who face both typical early career challenges and challenges specific to conducting IS research. Despite rapid growth, IS remains a relatively new field, resulting in limited availability of collaborators and senior mentors . Peer meWe present a case study of a peer mentoring group featuring early career implementation scientists to highlight the value of peer mentoring for those in positions that require the production of rigorous scholarship and the acquisition of extramural funding. The case study is based on our experience in an ongoing peer mentoring group. Each of us hold K awards, which support early-stage investigators in establishing their scientific independence through mentored training and research. Though hierarchical mentoring is a requirement of these awards, peer mentoring is not, and we formed this peer group to supplement mentorship from senior colleagues. To develop this case study, we engaged in a group discussion and consensus processes to identify core components that fostered success of the group. Our case study is informed by the IMeRGE peer mentoring model  and litehttp://chipsfellows.com/) and Implementation Research Institute (http://iristl.org/about/) [This IS peer mentorship group emerged organically after two members, M.B and N.S., started biweekly one-on-one conversations as postdoctoral fellows to address challenges and progress related to applying for NIH K awards in 2015. The desire to share common professional and scientific struggles among peers was further revealed upon meeting other fellows of NIH-funded training networks, including the Child Intervention and Prevention Services Fellowship (/about/) . NetworkConsistency, routine, and having established expectations have been foundational to our group’s curriculum and structure  and a shared focus on IS, which was essential in fostering the support and impact of this group. We also previously participated in group-based NIH-supported training fellowships, which enabled foundational knowledge of NIH funding mechanisms and team science. However, we are located at different institutions with varying job expectations  and are funded by different NIH institutes. This enables representations of a variety of perspectives, while maintaining a shared focus on issues facing K awardees. Having members outside of one’s institution and/or outside the context of the hierarchy within one’s academic or research institution also enables discussion of key topics or issues without the potential for negatively impacting one’s professional development or evaluation. Additionally, our research programs share commonalities, with notable distinctions. Each is evaluating interventions to address a behavioral health problem, with a focus on understanding how the intervention can be delivered at scale leveraging IS methodology. This similarity permits meaningful and informed contributions to each other’s research challenges. At the same time, we target different health conditions  and/or use varying intervention delivery modalities , which encourages collaboration without inciting competition.current NIH practices and policies for awardees relative to prior policies or practices that might be more familiar to senior mentors. Moreover, when seemingly inconsequential, mundane, or “obvious” questions arise , it can be more appealing to ask these questions among peers than senior mentors or program officers. Examples include sharing information about rules related to awards, progress report completion, clinical trials reporting, and aspects of new grant applications. Sharing this knowledge allows us to strategically prepare for conversations with our program officials and mentors. Similarly, because our K awards and other funding streams are through different NIH Institutes, we disseminate learnings from discussions with our different institutes and expand our research funding networks. This group facilitates the responsible conduct of research, a focus within NIH K awards, by sharing resources and knowledge as well as feedback surrounding ethical issues that arise and often particularly unique in community-engaged IS research  [The second key to success is the instrumental and psychosocial support afforded by and unique to the peer mentorship structure. Because of our shared factors described above, we take advantage of the hands-on experiences we are simultaneously obtaining with our K awards and offer instrumental support in carrying out our projects and conducting responsible research. Because we are navigating this process concurrently, we often provide advice that reflects artners) .The peer mentorship model allows for opportunities to reflect on how to be a successful mentee to senior mentors, and mentor to junior trainees, unique skillsets that are necessary for early career researchers . RelyingSimilar to how we benefit from learning from each other’s mentors, we have benefitted each other’s mentees by sharing materials  and enhancing networking opportunities. Overall, peer mentoring provides a form of unique psychosocial support as compared to traditional mentoring relationships. There is a sense of psychological safety afforded by peer mentoring, which allows us to ask questions that might be difficult, inappropriate, or detrimental to discuss elsewhere.Based on our collective experiences, we offer examples of how peer mentoring activities have benefitted the authors, their institutions, and funders. We consulted and collaborated with one another around the development and submission of multiple published manuscripts and grant proposals that have been submitted or funded  and associated Matchmaker tool could be used to find investigators conducting work relevant to one’s interests. Social media platforms such as Twitter or ResearchGate could also be used to catalyze peer mentorship networks. Universities and research institutions could support peer mentorship through formal structures embedded within Clinical and Translational Science Institutes, cross-institution centers and interest groups such as the Washington University Network for Dissemination and Implementation Research  or University of California San Diego Altman Clinical and Translational Research Institute Dissemination and Implementation Science Center. (https://disc.ucsd.edu), and interdisciplinary fellowships such as the University of North Carolina at Chapel Hill Thorp Faculty Engaged Scholars Program (https://ccps.unc.edu/fes/). Although some perceived benefits of our model may not hold if translated to other peer mentoring groups, such as early career investigators at the same institution with similar institutional funding but differing research fields, alternative shared commonalities may yield other benefits to its members. Research funders could encourage peer mentorship by connecting researchers within their funding portfolios that are working in similar or adjacent areas, and affording opportunities for interaction through grantee meetings, research conferences, or other sponsored gatherings. Finally, professional societies may be uniquely positioned to connect early career researchers who have similar interests and goals. For example, the Society for Implementation Research Collaboration (https://societyforimplementationresearchcollaboration.org) has a mentorship program that links emerging scholars with more senior implementation scientists; recently, the student group has chosen to focus on peer mentorship to expand opportunities for mentorship to all students, developing students’ mentoring skills, and reducing burden on more senior researchers. However, we contend there is a need to be mindful of necessary adjustments to the process or membership of these groups to maintain the key components of peer mentoring, including engendering an equal power distribution and psychosocial safety or support.There are a variety of potential avenues for increasing the use of peer mentorship to support early career researchers. At the individual level, we encourage researchers to proactively seek and initiate peer mentorship relationships. This could take place locally within one’s institution, but it is increasingly feasible to identify and engage peers regionally, nationally, and internationally through widely available networking tools. For example, the NIH’s RePORTER , and di


MTSS1, whose downregulation is known to enhance aggressiveness of solid tumors. Here we show that low MTSS1 expression at diagnosis was associated with a poor prognosis in AML. MTSS1 expression was regulated by promoter methylation, and reduced by cytosine arabinoside and the anthracycline daunorubicin. Experimental downregulation of MTSS1 affected the expression of numerous genes. It induced the DNA damage response kinase WEE1, and rendered human AML cell lines more resistant to cytosine arabinoside, daunorubicin, and other anti-cancer drugs. Mtss1 knockdown in murine MLL-AF9-driven AML substantially decreased disease latency, and increased leukemic burden and ex vivo chemotherapy resistance. In summary, low MTSS1 expression represents a novel factor contributing to disease aggressiveness, therapy resistance, and poor outcome in AML.Despite recent approval of targeted drugs for acute myeloid leukemia (AML) therapy, chemotherapy with cytosine arabinoside and anthracyclines remains an important pillar of treatment. Both primary and secondary resistance are frequent and associated with poor survival, yet the underlying molecular mechanisms are incompletely understood. In previous work, we identified genes deregulated between diagnosis and relapse of AML, corresponding to therapy naïve and resistant states, respectively. Among them was  SOCS2 and CALCRL, which were both upregulated at relapse, on AML aggressiveness and chemotherapy resistance has already been demonstrated . We. WeMTSS1els Fig. . Bisulfi562 Fig. . One cel562 Fig. . These dlls Fig. , which wlls Fig. . Curiousent Fig. . This unMTSS1 mRNA at this disease stage  cells formed in response to araC or DNR  or a non-targeting control shRNA (shCtrl) were introduced into each of these cell lines. Efficient knockdown of MTSS1 was confirmed by immunoblot analysis . The library comprised 106 compounds representing 13 different drug classes [MTSS1 knockout and the control clones as threshold, ten drugs from various classes were found to exhibit altered—and in fact, in all cases reduced—toxicity toward the knockout clones  positivity, and transplanted into recipient mice. Knockdown of Mtss1  cells in the myeloid leukemic (CD11b+ Venus+ GFP+) compartment  also proliferated faster than their shCtrl containing counterparts (LCMLL-AF9_shCtrl) ex vivo  than LCtrl Fig. .Fig. 7KnMtss1 in an MLL-AF9-driven mouse model of AML strongly increased disease aggressiveness and the resistance of LCs to chemotherapeutic drugs.In summary, knockdown of Mtss1 knockout mice, however, developed B-cell lymphomas, pointing toward an anti-oncogenic role of this gene also in the hematopoietic system. MTSS1 was downregulated in CML, and its experimental over-expression in CML model systems impaired clonogenic growth and tumorigenesis [MTSS1 was reported to constitute part of a prognostically relevant, TET2-related 4-gene methylation signature [MTSS1 mRNA levels were high in AML with prognostically favorable CBF-rearrangements, and low in AML carrying FLT3-ITD mutations or a PML-RARA fusion, both of which are associated with a poor prognosis under araC/anthracycline based therapy [MTSS1 expression was associated with an increased risk of relapse in pediatric AML [The multifunctional protein MTSS1 acts as a tumor and/or metastasis suppressor whose downregulation was associated with a poor prognosis in several solid tumor entities –22. Mtssigenesis . In AML,ignature . MTSS1 m therapy , 45. Furtric AML , and wittric AML .MTSS1 in AML: MTSS1 was downregulated at relapse of AML, and in genetically defined intermediate and poor risk subgroups as compared to the corresponding good risk groups. Furthermore, low MTSS1 mRNA levels were associated with shorter overall survival in three independent patient cohorts. However, the latter finding could not be confirmed in the validation cohorts of GSE12417 and GSE6891. Remarkably, the majority of studies on the prognostic significance of MTSS1 in solid tumors were performed using immunohistochemistry. This raises the possibility that associations between MTSS1 expression and outcome do not always manifest on the mRNA level, but may be more reliably reflected on the protein level. In fact, MTSS1 expression appears to be regulated on several levels. Our data show that, like in prostate cancer and CML [MTSS1 in primary AML samples and AML cell lines. However, HL-60 and HNT-34 cells displayed comparable, intermediate MTSS1 methylation levels despite divergent expression levels, indicating that transcriptional regulation of MTSS1 in AML includes, but is not restricted to, promoter methylation. Accordingly, MTSS1 is a confirmed target for several transcription factors in myeloid cells, such as KAISO and REST [MTSS1 in cell lines with promoter methylation, but had the opposite effect in the MTSS1 unmethylated, MTSS1high cell lines TF-1 and KG-1. This could be due to the existence of a methylation-sensitive antisense transcript, and indeed, the 3′-end of MTSS1 overlaps with the 3′-end of one of the transcript variants of the NDUFB9 gene, whose promoter also harbors a CpG island . Alternatively, methylation-sensitive microRNAs and/or transcription factors could mediate the downregulation of MTSS1 in response to 5-aza-2′-deoxycytidine treatment. Underscoring the complexity of the regulation of Mtss1, the levels of its mRNA were reduced upon expression of bcr-abl in murine CML models, but only partially restored by tyrosine kinase inhibitor treatment [MTSS1 mRNA levels, the downregulation on the protein level was much stronger and more significant. This indicates that chemotherapy is unlikely to directly cause downregulation of the MTSS1 mRNA at relapse of AML, and points toward a post-transcriptional layer of MTSS1 regulation. In line with this, MTSS1 was shown to be a target of several miRNAs [Here, we present additional evidence for the prognostic relevance of  and CML , 28, proand REST . Peculiareatment . Furtherl miRNAs .MTSS1 in three different human AML-derived cell lines and in primary murine AML cells increased their resistance to the standard chemotherapeutics used in AML treatment. This effect was independent of TP53 status, because it was observed both in cells with wild-type  and wit MLL-AF9 ; Supplem of Rac1 , which iactivity , 50, sugs a role –22, 44.MTSS1 affected the proliferation of AML cells. Most impressively, its knockdown greatly accelerated leukemia development in an MLL-AF9-driven mouse model of AML. MLL-AF9 can transform both hematopoietic stem and progenitor cells, giving rise to very aggressive or a somewhat more slowly progressing AML-like disease, respectively, upon transplantation into congenic recipient mice [MLL-AF9-transduced common myeloid progenitors in order to facilitate detection of a disease-accelerating effect of the Mtss1 knockdown in vivo. Consistent with the in vivo phenotype, LCMLL-AF9_shMtss1 also proliferated faster than LCMLL-AF9_shCtrl ex vivo, yet their basal rates of apoptosis were comparable, similar to our observations in the human MTSS1 knockdown or knockout AML cell lines. Other than with the mouse cells, however, the knockdown also did not alter the proliferation of the cell lines. CRISPR/Cas9-mediated elimination of MTSS1 in TF-1 cells even retarded their proliferation, possibly indicating a requirement for minimal levels of the multifunctional MTSS1 protein to maintain optimal cell division rates.In addition to its effects on chemotherapy responsiveness, ent mice , 31, 41.MTSS1 is associated with relapse and shortened survival in AML. It promotes leukemogenesis in an AML mouse model in vivo, and augments resistance to drugs used in standard AML therapy, as well as to a variety of other anti-cancer therapeutics. These results suggest reduced MTSS1 expression as a novel factor contributing to disease aggressiveness, therapy refractoriness, and poor outcome in AML.In summary, our data show that downregulation of Supplementary Methods and MaterialSupplementary Table S1Supplementary Table S2ASupplementary Table S2BSupplementary Table S2CSupplementary Table S2DSupplementary Table S2E


In the translational piglet model of hypoxic encephalopathy, we have previously shown that hypoxia overactivates Ca2+/Calmodulin (CaM) signaling via Sarcoma (Src) kinase in cortical neurons, resulting in overexpression of proapoptotic genes. However, identifying the exact relationship between alterations in neuronal Ca2+-influx, molecular determinants of cell death, and the degree of hypoxia in a dynamic system represents a significant challenge.Neonatal hypoxic brain injury is a major cause of intellectual and developmental disability. Hypoxia causes neuronal dysfunction and death in the developing cerebral cortex due to excitotoxic Ca2+-influx, and Ca2+/CaM-dependent protein kinase kinase 2 (CaMKK2) expression. We then used SimBiology to build a computational model of the Ca2+/CaM-Src-kinase signaling cascade, simulating Nx, Hx, and Hx+PP2 conditions. To evaluate our model, we used Sobol variance decomposition, multiparametric global sensitivity analysis, and parameter scanning.We used experimental and computational methods to identify molecular events critical to the onset of excitotoxicity-induced apoptosis in the cerebral cortex of newborn piglets. We used 2–3-day-old piglets  for biochemical analysis of ATP production, Ca2+ level alterations following Src kinase inhibition may not be a linear predictor of changes in Bax expression. Importantly, our model indicates that while pharmacological pre-treatment significantly reduces the onset of abnormal Ca2+-influx, there exists a window of intervention after hypoxia during which targeted modulation of Src-NMDAR interaction kinetics in combination with PP2 administration can reduce Ca2+-influx and Bax expression to similar levels as pre-treatment.Our model captures important molecular trends caused by hypoxia in the piglet brain. Incorporating the action of Src kinase inhibitor PP2 further validated our model and enabled predictive analysis of the effect of hypoxia on CaMKK2. We determined the impact of a feedback loop related to Src phosphorylation of NMDA receptors and activation kinetics of CaMKII. We also identified distinct modes of signaling wherein Ca2+/CaM-Src signaling pathway leading to neuronal injury and provides a feasible framework for drug efficacy studies in translational models of neonatal brain injury for the prevention of intellectual and developmental disabilities.Our model identifies new dynamics of critical components in the CaThe online version contains supplementary material available at 10.1186/s11689-022-09431-3. Cerebral hypoxia in neonates is a potent driver of excitotoxic neuronal injury that often leads to cell death . Neurona2+ influx due to uncontrolled glutamatergic drive  = 1000–10,000 ppm, and in severe hypoxia [O2] = 0–1000 ppm  overnight at 4 °C on a rocking platform. The membranes were treated with goat-anti-rabbit HRP-conjugated secondary antibodies . The target protein bands were determined using the reagents visualized provided in the ECL+ plus kit . The immunoreactive band intensities were analyzed by imaging densitometry  using ImageJ . The protein expression is presented as optical density (OD) per mmEnergy failure caused by hypoxia in cerebral cortex tissue was confirmed by determining the levels of ATP based on an assay previously described in detail , 33. Bri2+, and 1 mM ATP. The Ca2+ influx assay was carried out at 37 °C for 2 min. Samples were then filtered on a glass fiber filter and washed three times in 20 mM Tris (pH 7.2), 100 mM potassium chloride buffer. A Rackbeta scintillation counter  was used to measure radioactivity.Neuronal nuclei (150 μg) were incubated in 300 μl medium composed of 50 mM Tris buffer (pH 7.4), 1 μM 45CaP-value of less than 0.05 was considered statistically significant.Statistical analysis for experimental measurements was performed using Graphpad Prism v. 9. We used ordinary one-way ANOVA with Tukey post hoc multiple comparisons test. A n = 5) and Hx+PP2 group (n = 3) compared to Nx controls (n = 5)  = 89.02, P < 0.0001; Tukey comparison Nx vs. Hx: P < 0.0001; Tukey comparison Nx vs. Hx+PP2: P < 0.0001). We also confirmed Ca2+ influx into neuronal nuclei  = 32.94; P < 0.0001; Tukey comparison Nx vs. Hx: P < 0.0001). PP2 treatment reduced hypoxic injury-induced Ca2+ influx (Tukey comparison Hx vs. Hx+PP2: P = 0.0116); however, Ca2+ levels in the Hx+PP2 group were still significantly higher than Nx (Tukey comparison Nx vs. Hx+PP2: P < 0.0001), indicative of a partial rescue. Western blot analysis  = 19.68; P < 0.0001; Tukey comparison: Nx vs. Hx: P = 0.0004). However, in contrast to Ca2+ influx, CaMKK2 expression was restored to almost-normoxic levels . We used the changes in Ca2+ influx and alterations in CaMKK2 expression for both validation and predictive analysis for our computational model.CaMKIV critically controls activity-dependent gene transcription in neurons . We havell: F 2, 0 = 89.02ll: F 2, 0 = 89.02ll: F 2, 0 = 89.022+ , and a decrease in concentration following Src-inhibition with PP2 (dashed line at t = 240 s). We validated our model with previously published data from Delivoria-Papadopoulos et al. . Thus, MPGSA further confirms that CREB and excitotoxic glutamate pulses significantly affect Bax expression while solely excitotoxic glutamate pulses can affect Ca2+ influx.In order to identify the particular conditions where input parameters of the GSA have differential effects on CaGSA Fig. a. Two sions Fig. b. Kolmog2+/CaM signaling critically affects Ca2+ influx and activation of neuronal death pathways in neonatal hypoxic injury [Ka = 5e−4, Bax expression significantly drops during PP2 administration; however, a drop in Ca2+ influx is delayed, but reaches significant reduction above Ka = 7e−4  resulted in a significant drop in cytoplasmic Ca2+ and returned Bax expression levels almost similar to pre-treatment  [2, in line with our previous work where ATP levels are used as a readout of tissue hypoxia [2+-mediated excitotoxic mechanisms can also lead to neuronal necrosis, which is a considerably faster form of cell death. It is likely that severe hypoxic conditions can lead to both necrosis and apoptosis. Evidence also suggests that in models of neonatal brain injury, hypoxia may induce both these forms of cell death on a continuum, rather than completely discrete events without any overlap [The dominant paradigm to explain excitotoxic neuronal damage involves an abnormal influx of Caes (ROS) . Since h hypoxia . Activat overlap , 49, and overlap . Thus, a2+/CaM pathway that cascades through a series of intermediate steps, including the activation of CaMKII, CaMKK2, nuclear CaMKIV, and finally CREB. However, the GSA and the MPGSA of our computational model suggest that, although excitotoxic hyperstimulation shows significant interaction with key signaling members of this cascade, the only other critical component that significantly interacts with excitotoxic hyperstimulation is CREB. This indicates that CaMKIV, while important, may not be a limiting component of excitotoxic mechanisms that signal via Src kinase. Another important insight from the GSA and the MPGSA points to the existence of different and discrete signaling states, where Ca2+ influx and Bax expression can be dissociated during Src inhibition. This is in line with emergent evidence on metabotropic-like signaling of NMDARs triggered by excitotoxic stimuli, wherein cell death can be dissociated from Ca2+ influx per se [Excitotoxic mechanisms have been studied in the context of hypoxic brain injury for a considerable period of time. However, few if any effective pharmacological agents that attenuate neuronal damage induced by excessive glutamatergic excitation have been identified. Our previous work strongly indicates that the Src kinase inhibitor PP2 is an effective intervention to reduce Hx-induced excitotoxic neuronal damage in the piglet neocortex , 20. Ourx per se , 13. Int2+ influx but does not significantly alter a drop in Bax expression, modulating Src activation of NMDARs in addition to PP2 administration caused a significant reduction in both Ca2+ influx as well as Bax expression . The interaction between these kinases affects multiple signaling pathways. CaMKK2 in particular serves as a hub integrating and decoding signals across diverse pathways [2+/CaM signaling cascade. Future computational studies could shed light on these kinome-related mechanisms and how they affect the Src kinase pathway in the context of neonatal brain injury.The Capathways –56, whilpathways , 58. How2 during Hx may affect these dynamics and feedback onto the Src kinase pathway. In addition, we have not included a mitochondrial Ca2+ buffering mechanism, which is likely altered in conditions that lead to neuronal death [In the present study, we have not included a detailed model of mitochondrial metabolism and how changes in extracellular Oal death –61, althImportantly, our model input is a physiologically very slow rate of glutamate release and captures slower components of excitotoxic release. It would be helpful to use faster glutamate release dynamics in future studies and adjust other temporal kinetics of the model accordingly.Finally, we did not include a comprehensive sub-model of apoptosis in our approach. Future studies should also include the expression of the pro-apoptotic protein Bad as well as anti-apoptotic proteins, such as Bcl-2 and Bcl-xl, for which experimental data is available in the piglet model of neonatal brain injury .2+/CaM pathway in neuronal excitotoxicity in a large animal model of neonatal hypoxic brain injury. Our computational model was validated with experimental measurements of critical intracellular signaling components and captures key molecular trends in this pathway. Our computational model indicates that pharmacological inhibition of Src kinase leads to signaling states wherein Ca2+ influx and the onset of Bax expression are dissociable. Finally, our computational model predicts that small molecules that specifically modulate the interaction between the NMDA receptor and Src can significantly contribute to a reduction in Bax expression. Our study combines experimental and computational methods to provide a translational platform to investigate key signaling cascades triggered by neonatal hypoxic brain injury in neurons.We have used experimental and computational approaches to determine the significance of the Src-CaAdditional file 1: Supplementary Figure 1. Representative visualization of Ca2+/CaM-Src kinase intracellular signaling computational model. Cartoon depiction of the interactions captured in our SimBiology model of excitotoxic glutamate pulses and subsequent activation of NMDARs and Ca2+/CaM signaling through Src kinase and the eventual transcription of the pro-apoptotic protein Bax. Figure created with BioRender.Additional file 2.


Disturbances in Ca2+ homeostasis and dysfunction of Ca2+ signaling pathways may have serious consequences on cells and even result in cell death. Dopaminergic neurons are particularly sensitive to any changes in intracellular Ca2+ level. The best known and studied Ca2+ sensor in eukaryotic cells is calmodulin. Calmodulin binds Ca2+ with high affinity and regulates the activity of a plethora of proteins. In the brain, calmodulin and its binding proteins play a crucial role in regulation of the activity of synaptic proteins and in the maintenance of neuronal plasticity. Thus, any changes in activity of these proteins might be linked to the development and progression of neurodegenerative disorders including PD. This review aims to summarize published results regarding the role of calmodulin and its binding proteins in pathology and pathogenesis of PD.Parkinson’s disease (PD) is a neurodegenerative disorder that manifests with rest tremor, muscle rigidity and movement disturbances. At the microscopic level it is characterized by formation of specific intraneuronal inclusions, called Lewy bodies (LBs), and by a progressive loss of dopaminergic neurons in the striatum and substantia nigra. All living cells, among them neurons, rely on Ca Parkinson’s disease (PD) is an age-related neurodegenerative disorder second in prevalence to Alzheimer disease (AD). PD manifests with rest tremor, muscle rigidity sometimes coupled with intense pain and with movement disturbances such as postural disabilities and gait disturbance. Non-motor features of PD include pain and sensory phenomena, anxiety and depression, autonomic dysfunction, cognitive impairment and dementia. It is a progressive disease, with symptoms aggravating with time. A characteristic histopathological feature of the affected brain areas of patients with PD or other synucleinopathies is the presence of specific inclusions such as Lewy bodies (LBs) and Lewy neurites (LNs) which contain mainly an aggregated form of α-synuclein . The majAppearance of motor symptoms in PD is attributed to loss of dopaminergic neurons in the striatum and substantia nigra. Usually, these symptoms develop when 70–80% of neurons are destroyed . In turn2+-binding protein, is abundantly expressed in the brain. Particularly high levels of this protein are observed in postsynaptic membranes, postsynaptic densities and synaptic vesicles [2+ concentration ([Ca2+]i). For instance, it was shown that CaM expression was higher in cells treated with rotenone, a known inhibitor of mitochondrial complex-I that increases [Ca2+]i and triggers apoptosis i in substantia nigra neurons was one of the earliest observations in PD brains [2+ fluxes and that their dysfunction caused by mutations results in serious disturbances in [Ca2+]i [2+ entry into primary neurons and astrocytes [2+]i observed in neurons of PD brain may then affect Ca2+ signaling pathways and cell functioning.Dysfunction of Cacapacity . Studiesctivated . Indeed,D brains . Further [Ca2+]i . For exatrocytes . Thus, d2+ sensor in eukaryotic cells is CaM. It is a ubiquitous low molecular weight (16.7 kDa) protein, highly conserved within the plant and animal kingdom. In mammals, there are three genes encoding CaM, namely, CALM1, CALM2 and CALM3, which differ only in the non-coding regions i, to the second one, bringing about a conformational change in the channel subunit and its deactivation i observed in dopaminergic neurons treated with 6-hydroxydopamine (6-OHDA), an agent inducing parkinsonism [Regarding the involvement of other CaM-regulated Cat brains . Moreovet brains . Expressrains [+ . OverexpR stress . Other tR stress . With reR stress . Howeverfunction . IP3Rs pchondria . The exprol rats . Howeverinsonism . The RyR2+ homeostasis is the plasma membrane Ca2+-ATPase (PMCA). PMCA is an ATP-dependent ion pump that is responsible for Ca2+ efflux from the cytoplasm [2+]i increases, PMCA is activated by CaM, acidic phospholipids and protein kinases and by other means, e.g., dimerization, and its affinity for Ca2+ increases (KD ≈ 100–200 nM). CaM binds to the C-terminal autoinhibitory domain of PMCA and relieves the inhibition i observed in PD neurons. Thus, calmodulin, by providing a negative feedback regulation of Ca2+ channel activity and stimulating PMCA activity, appears as a crucial player in sustaining Ca2+ homeostasis that is so essential for neuronal survival.As evidenced above, increased expression of Ca2+, CaM binds to CaMKII and induces its autophosphorylation/activation i is low, the autoinhibitory domain masks the catalytic core and maintains CaN in an inactive state. Increase in [Ca2+]i and binding of Ca2+ to CaM and to CaNB waives the inhibition and leads to rapid activation of the phosphatase. Neurodegeneration is characterized by activation of the CaN-NFAT signaling pathway and by pro-inflammatory gene expression i apo-CaM binds to the C-terminal part of a channel subunit with one of its lobes while the other lobe binds to a site in another subunit upon increase in [Ca2+]i. Thus, CaM confers Ca2+ sensitivity to the SK channel gating mechanism and the resultant dimerization leads to channel activation i, which is commonly observed in cells in various areas of PD brain. Upon physiological increase in [Ca2+]i, a major Ca2+ sensor, CaM, interacts with many CaMBPs, which are involved in Ca2+-homeostasis, intracellular Ca2+-signaling pathways and other cellular processes. To CaMBPs belong different types of Ca2+channels, plasma membrane Ca2+-ATPase and many enzymes, of which a kinase, CaMKII, and a phosphatase, CaN, are of most importance. It might be assumed that under sustained Ca2+- signaling, the activity of CaM is markedly enhanced and may lead to excessive activation of CaMBPs with harmful consequences for different cell types, including neurons. Indeed, experimental evidence suggests that, in most cases, inhibition of CaMBPs, by various specific drugs brings about beneficial effects both in animal models of the disease and in PD patients.Parkinson’s disease (PD) is an age-related progressive neurodegenerative disorder with symptoms aggravating with time. Appearance of PD symptoms is attributed to the loss of dopaminergic neurons in the striatum and substantia nigra. Neuronal cell death may be ascribed to increased [Ca2+ channels, RyR receptors, SK channels or NOS may protect dopaminergic neurons from apoptosis and improve motor function in mouse model of PD. Moreover, it was shown that CaMKII antagonist caused a decrease in dyskinesia and extracellular dopamine efflux in an experimentally induced PD model. Moreover, drugs that inhibited CaMKII binding partners, to which belong tryptophan hydroxylase, adenosine A2A receptor or Cdk5 kinase had a positive effect on dopaminergic cell survival. Similarly, inhibition of CaM-activated phosphatase, CaN, protected brain cells from neurotoxicity, neuroinflammation and improved cognitive function in experimental models of PD and other neurodegenerative diseases. All CaM binding proteins described in this work and their function in norm and PD pathology are summarized in As it was described in this review, published data show that inhibition of CaM-regulated proteins such as L-type Ca


The highest between-subject variability (BSV) of 69.30% was observed for tvV. Co-variates such as body weight, age, and biochemical parameters did not improve the tested model and did not contribute to explanation of the BSV. The population pharmacokinetic model of the orally administered doxycycline in rabbits should be further developed by addition of data from more animals treated with higher doses. An oral dose of 5 mg/kg could ensure percentage of the time from the dosing interval during which the concentration is above minimum inhibitory concentration (MIC) %fT > MIC of 35% if MIC of 0.18 μg·mL−1 and a dosing interval of 12 h is assumed which does not cover criteria for rational use of antibiotics.Doxycycline is a well-tolerated tetracycline antibiotic, registered for use in rabbits and administered for treatment of bacterial infections in this animal species. Nevertheless, the available pharmacokinetic data are limited and this study aimed to investigate the pharmacokinetics of orally administered doxycycline in mature and immature rabbits by application of the population approach. The rabbits were treated orally with doxycycline hyclate (5 mg/kg bw) in the form of a solid gelatin capsules. Free plasma concentrations were determined with HPLC analysis with Photodiode array detection. The estimated typical value of volume of distribution (tvV), total body clearance, and absorption rate constant were 4.429 L/kg, 1.473 L/kg/h, and 0.257 h Pasteurella multocida, Staphylococcus aureus, Bordetella bronchiseptica, Salmonella spp., Escherichia coli, Clostridium spp., and Pseudomonas spp. [E.coli and Clostridium spiroforme [The importance of rabbits in veterinary medicine is increasing due to the high nutritional value of rabbit meat and their breeding as pets ,2. This nas spp. ,4. Thesenas spp. ,7,8,9,10nas spp.  as they iroforme .1/2) of intravenously administered doxycycline to New Zealand rabbits at a dose rate of 5 mg/kg was long and the values of total body clearance (Cl) were relatively low at 8.83 ± 2.10 h and 0.46 ± 0.10 L/h/kg, respectively [max between 1.10 ± 0.00 μg·mL−1 and 2.06 ± 2.96 μg·mL−1 were reported for the drug in studies conducted with various dosage forms and doses. These concentrations were achieved at Tmax between 0.55 ± 0.33 and 0.70 ± 0.48 h for suppositories, 3.00 h for the solution and 12.00 h for chitosan microcapsulated suspension [Tetracycline antibiotics, especially from newer generations, can be applied in rabbits without fatal disruption of the balance of gastrointestinal microbiota . Doxycycectively . Adminisectively ,18. Diffspension ,18. Despspension . The citA problem encountered during pharmacokinetic experiments is related to ethical reasons and the difficulties of obtaining a sufficient number of samples due to the small size of rabbits grown for classical pharmacokinetic studies. The population pharmacokinetic analysis can be successfully applied in cases when only few samples can be obtained from an individual animal . Therefomax) and the time of Cmax (Tmax) in mature rabbits read from the observed data, were 0.58 ± 0.15 μg·mL−1 and 3.40 ± 0.96 h, respectively. These values for immature animals were 0.60 ± 0.38 μg·mL−1 and 3.63 ± 1.33 h. Plasma concentrations were below the limit of quantification (LOQ) 12 h after the treatment in mature rabbits  of the primary structural parameters of the population model, the secondary pharmacokinetic parameters, their standard errors, and the SD of the residuals are shown in The random effects, the between-subject variability (BSV) and shrinkage are presented in Rabbits belong to the group of “minor species”, animal species which have economic significance and for which there are not enough antibacterial drugs approved for treatment of bacterial infections. Emergence of resistant bacteria and increasing problems with the efficacy of the available antibiotics require knowledge of pharmacokinetics with consideration of inter-individual variability as a first step of establishment of pharmacokinetic/pharmacodynamic (PK/PD) cutoff value . Thus, t−1 and 0.25 µg·mL−1 by 5–6% and 24.6%, respectively [The applied HPLC method with Photodiode array (PDA) detection showed similar sensitivity to the methods used in the published pharmacokinetic studies in rabbits ,18. The ectively . Therefomax values were obtained four hours after the doxycycline administration in both groups of rabbits. The relatively low value of ka, calculated with the current model, confirmed a slow absorption rate after oral administration of doxycycline in rabbits, administered as capsules. The pharmacokinetic studies of doxycycline in rabbits are scarce which makes the comparison of the data from the current investigation with other studies difficult. The available literature concerns pharmacokinetics of doxycycline hyclate in the form of a solution, a chitosan microcapsulated suspension, and suppositories [−1 was similar to tvka of 0.257 h−1. Faster absorption, with value of ka 1.26 ± 0.71 h−1, was observed after oral administration of a solution of doxycycline hyclate in rabbits at a dose rate of 20 mg·kg bw [max of 1.54–2.06 µg·mL−1 achieved at Tmax of 0.55–0.7 h was reported after administration of the antibiotic as suppositories at a dose of 10 mg/kg bw [−1 and higher Cmax value of 3.20 ± 0.65 µg·mL−1 after treatment with a solution of doxycycline hyclate at an oral dose of 10 mg/kg [Doxycycline was absorbed after oral administration in immature and mature rabbits and measurable concentrations were found 0.5 h after the treatment. The Csitories ,18. The mg/kg bw . The fasmg/kg bw . Wistar 10 mg/kg .−1 obtained for other drug dosage forms orally administered in rabbits and with classical pharmacokinetic modeling [10 due to administration of different doses, drug formulations, and treatment routes in the published studies with rabbits. The published values of t1/2el of doxycycline hyclate in rabbits were between 2.12 h and 9 h, depending on the listed factors [−1 during the whole dosing interval. Despite the specific behavior of rabbits, coprophagy [0-∞ of 11.47 ± 2.91 µg/mL·h [0-∞ from 11.27 ± 3.37 to 11.79 ± 1.03 µg/mL·h were obtained with administration of suppositories, containing 10 mg/kg doxycycline and prepared with polyethylene glycol or cocoa butter, respectively [0–∞ of 11.83 ± 0.19 µg/mL·h [The primary population pharmacokinetic parameter tvV was in agreement with the results for V/F of 3.11–3.82 L·kgmodeling . The othmodeling  after ormodeling ,18. It i factors ,18. The prophagy , and exc µg/mL·h . Similarectively . Oral adThe tested model shows that high inter-individual variations can be expected in the volume of distribution. The model could not be improved although it tested the effect of several co-variables, hence they could not be used for explanation of the variability in doxycycline disposition between individual rabbits. The population analysis indicated that a larger number of rabbits should be included in further development of the model in order to obtain a more complete data set and to attempt to explain between-subject variability. This step would help to estimate more precisely the value of individual eta, even with a limited number of samples from an animal ,25, as w−1. Data on the susceptibility of pathogenic bacteria, that is relevant for rabbits, are scarce, therefore %fT > MIC was calculated by assuming a probable MIC value of 0.18 µg·mL−1 for Pasteurella spp. [fT > MIC, was 35% from a τ of 12 h. Based on the power of population modelling, the current pharmacokinetic model could be further developed by adding new data for rabbits treated with a higher dose of doxycycline. Apart from improvement of the population pharmacokinetic model, further studies should supply information about real MIC values for pathogens relevant for rabbits, identify target PK/PD values, and establish a PK/PD cut off value, based on the steps outlined by EuVetCAST procedure [The results of our study, in which an oral dose of 5 mg/kg was administered to immature and mature rabbits, revealed that the often-recommended oral dose ,14  was used for treatment of the animals. The drug was administered at the dose rate of 5 mg·kg®, HPLC grade, ≥99.9% purity, methanol HiPerSolv CHROMANORM for HPLC isocratic grade, oxalic acid 98% purity , ethylenediaminetetraacetic acid disodium salt dihydrate 99.0–101.0% (Na2H2EDTA × 2H2O) and trifluoroacetic acid ReagentPlus®, 99% purity. Analytical standards doxycycline hyclate with purity ≥ 98% and oxytetracycline hydrochloride ≥ 95% crystalline were used for preparation of standard curves during HPLC analysis.All reagents and solvents were HPLC grade and were provided by Sigma-Aldrich : acetonitrile CHROMASOLVn = 18) were enrolled in the experiments. The animals were allocated in two groups according to the age. One of the groups included six mature 5-month-old rabbits with mean body weight 3.55 ± 0.30 kg. The other group consisted of 12 immature 70-day-old rabbits with mean body weight 2.03 ± 0.21 kg. The animals were obtained from the Production experimental base to the Agricultural Institute-Stara Zagora, Bulgaria. The animals were housed in the biobase unit at the Faculty of Veterinary Medicine, Trakia University. Mature rabbits were placed in individual cages and the immature rabbits were divided in groups of four and allocated to three cages, according to the species requirements. Granulated feed  and water were supplied ad libitum. The animals were clinically healthy. The experiments started after a 10-day acclimatization period. After the end of the experiment, the animals were reared at the biobase unit.Crossbred New Zealand x Californian broiler rabbits  for determination of biochemical parameters total protein, albumin, alanine transaminase (ALT), aspartate transaminase (AST) and lactate dehydrogenase (LDH) on the same day. Biochemical analysis was performed on the Biochemistry analyzer BS-120 Mindray (P.R. China) at the Clinical Laboratory, Laboratory and Diagnostic Center, Trakia University.Doxycycline hyclate was administered orally at a dose rate of 5 mg/kg bw in the form of hard gelatin capsules. The rabbits were treated with a single dose with the help of 2 mL syringes with cut apex between 7:00 and 8:00 a.m. Two rabbits did not swallow the capsule and expelled it with damaged integrity and shell. They were excluded from the experiment because we were not sure how much of the drug had reached gastrointestinal tract. The animals had free access to feed and water at the day of the experiment.g for 10 min. Blood samples from untreated rabbits were obtained for preparation of standard solutions with vacutainers . Plasma was transferred to a new tube and stored at −25 °C until analysis.Blood samples from the mature rabbits were obtained from v. auricularis before the treatment and at the following intervals after the treatment: 0.5, 1, 2, 3, 4, 6, 8, 10, 12, and 24 h. The immature rabbits were divided in two groups of five animals each. Blood samples from group 1 (1st to 5th immature rabbits) were taken at 0.5, 2, 4, 8, and 12 h after treatment. Blood samples from the second group (6th to 10th immature rabbits) were obtained at 1, 3, 6, 10, and 24 h after treatment. The samples were immediately placed in Eppendorf tubes containing heparin-sodium and were centrifuged at 1500× g at 22 °C for 10 min. The clear upper layer was placed in HPLC vials and 20 µL was injected into the HPLC system . The drug separation was achieved by Hypersil Gold column . The analytical equipment consisted of a PDA detector  set at a wavelength of 345 nm, a Surveyor LC Pump Plus, and a Surveyor Autosampler Plus. The mobile phase consisted of acetonitrile, methanol, 0.02 M oxalic acid, and 0.02 M Na2H2EDTA × 2H2O . The flow rate was 1.0 mL.min−1. The retention times were 2.7 min for oxytetracycline and 5.66 min for doxycycline. ChromQuest Chromatography Data System  was used for peak area integrations.HPLC analysis with PDA detection was used for determination of doxycycline concentrations in plasma. The applied method is described in detail by Mileva . Briefly−1). IS was added during preparation of the samples for calibration curves. The standard curve was linear within the range of the used concentrations (R2 = 0.9981) and goodness-of-fit was proved by the lack of fit test (p > 0.08). The values of limits of detection (LOD) and quantification (LOQ) were calculated according to equations described by Shabir [−1, respectively. The accuracy was between 90.55 and 105.22% for the concentration from 1 µg·mL−1 to 0.25 µg·mL−1, respectively. The mean intra- and inter-day precision (RSD%) values were 4.88 and 10.48. The extraction recovery of doxycycline was >83.77%.The method was validated in compliance with the requirements of the international guidelines ,29,30. Ty Shabir  and were−1), medium (0.5 μg·mL−1), and high (1.0 μg·mL−1) concentrations of the antibiotic were subjected to ultrafiltration by using Ultrafree®−MC Centrifugal Filters with polytetrafluoroethylene (PTFE) membrane with 0.45 μm pore size . Subsequently, the samples were centrifuged for 4 min at 10,800× g at 22 °C and 20 μL ultra-filtrate was injected into the HPLC system. The binding percentage after this purification step was calculated according to the manufacturer’s instructions and according to the following equation:An additional purification step of plasma samples spiked with three doxycycline concentrations was used to determine the percentage of plasma protein binding after the protein precipitation step. The extraction of the antibiotic was performed according to the procedure described above. Plasma samples with low . Population pharmacokinetic parameters were computed by application of nonlinear mixed effects (NLME) approach. The one-compartment model was chosen after visual inspection of plots for all rabbits on the basis of the Log Likelyhood (-2LL) and the Akaike information criterion (AIC) to identify the model that fitted best to the observed data. Additionally, we tried to identify the sources of inter-individual variability and co-variates were incorporated in the model. The effects of different co-variates and their combinations on pharmacokinetic parameters were tested. Finally, a model without covariates was chosen based on the results of -2LL and likelihood ratio test (LRT), and the AIC. The model comparer tool of the software was also used. The LRT test was applied for statistical evaluation of goodness-of-fit of the models in order to compare more complex models to the basic model and the critical value of the χ2 distribution considered for a given nominal risk of 0.05. Finally the one-compartmental model without covariates was selected with primary estimated parameters absorption rate constant (ka), volume of distribution (V), and clearance (Cl). The terminal slope k10 was calculated according to the Equation (2):The elimination rate constant kThe elimination half-life was computed according to the Equation (3):The area under the curve was computed according to the Equation (4):ith animal) were defined as follows:i is the volume of distribution for the ith animal, tvV  is the population V, and ηi (eta) is the deviation associated with the individual animal ith from the computed population value of tvV. The other two parameters were calculated with the same algorithm. Normal distribution of etas with a mean of 0 and a variance (ω2) was assumed. Consequently, individual parameters and their etas could be correlated. Equation (6) was used for calculation of coefficient of variation (CV%):The between-subject variability (BSV) was modeled using an exponential model and the pharmacokinetic parameters  for every subject (j) is the empirical standard deviation of the jth (observation) η over all Nsub subjects, and ωj,j is the estimate of the population variance of the jth random effect, j = 1, 2, …, Neta.The shrinkage of random effects towards the means was defined according to the following equation: 2.Multiplicative  residual error was used:The first order conditional estimation extended least squares (FOCE-ELS) methodology was used for analyses. It was based on minimizing an extended least squares objective function representing the FOCE approximation to the negative log of the marginal likelihood as a function of . Censored data were not included in the modeling. A simple approach was used for estimation of typical values (tv) and their associated SE and coefficient of variation as an indication of the precision of the estimate. The goodness-of-fit of the model was evaluated by using different diagnostic plots such as visual predictive check, population predicted value based on population parameter estimates (PRED), and individual predictive value based on individual’s etas (IPRED) versus the dependent variable (DV), conditional weighted residuals (CWRES), and the fitting of the individual curves. max is maximum plasma concentration, MIC is a minimum inhibitory concentration, k10 is elimination rate constant, and τ is the dosing interval.The following formula ,32 for eThe developed population pharmacokinetic approach showed relatively low inter-individual variability in the absorption of doxycycline and higher variability in the volume of distribution. The tested co-variates could not improve the model and could not explain between-subject variability. The population pharmacokinetic model can be further developed by addition of data from rabbits. This preliminary study indicates that higher doses have to be tested in future experiments.


Pyrus pyrifolia Nakai.) is a very popular and profitable pear cultivar in China. However, its high susceptibility to cork spot disorder has limited its expansion of cultivated area. The mechanisms of cork spot disorder have been discussed extensively, focusing on Ca2+ deficiency, yet no consensus has been made. In this study, we applied nitrendipine (NI) as a Ca2+ uptake inhibitor to explore the role of calcium in cork spot disorder occurrence. Results showed that NI treatment on the fruit remarkably increased the incidence of cork spot disorder; alteration of mineral contents happened at the early developmental stage of the fruit, especially on the outer flesh and the peel of the fruit; and this gap was filled gradually along with the expansion of the fruit. Significant differences in the expression levels of Ca2+ transport-related genes were found in the inner flesh, outer flesh and peel during the fruit growth period. The observation of free Ca2+ localization indicated the intracellular imbalance of Ca2+ in the NI-treated fruit. In conclusion, NI treatment reduced the calcium content in the fruit at an early developmental stage, altered the related expression of genes and influenced the cellular Ca2+ balance in the fruit, which prompted the occurrence of cork spot disorder. Measures for the prevention and control of cork spot disorder should be taken at the early stage of the fruit development in the field.‘Akituki’ ( Pyrus pyrifolia), bred in Japan in 1988, is a very popular cultivar in China for its large fruit size, pretty fruit shape and taste. However, ‘Akituki’ is very susceptible to cork spot disorder [Cork spot is a physiological disorder of fruit which occurs most in pears and apples. The typical characteristics of this disorder in pears are brown desiccated flesh or grayish corky lesions beneath the fruit skin , and a b2+ solution three to five times during the growing period could effectively reduce the incidence of cork spot disorder in ‘Anjiu’ pears [2+-deficiency-related physiological disorder. However, a recent study found that spraying a calcium solution, a boron solution or a mixture solution of calcium and boron during the growing period could not reduce the incidence of cork spot-like physiological disorder in apples, and there was no correlation between the incidence of cork spot-like physiological disorder and Ca/B content in fruit or leaves [Similar to bitter pits  of appleu’ pears ,11,12. Tr leaves . Interes2+ on cork spot disorder, a supplement of exogenous calcium was applied in most experimental designs [2+ transport channel, is mainly used to treat heart disease [2+ through the L-type Ca2+ channel in lilies [2+ into carrot callus cells [2+ into peanut pod cells, and this inhibition was reversible when NI treatment was removed [2+ deficit on cork spot development by inhibiting the absorption of Ca2+ in pear fruit.When investigating the effect of Ca designs , but a d disease . In plann lilies  and blocus cells . It also removed ,17. Ther2+ deficit on the cork spot disorder occurrence. NI was applied in the study to inhibit the Ca2+ absorption in pear fruit, followed by the measurement of mineral content and the expression levels of Ca2+ transport-related genes, and the localization of free Ca2+. The relationship between Ca2+ deficiency and cork spot incidence was also analyzed. Results of the study are expected to provide valuable clues for the exploration of the cork spot disorder’s mechanism and to be useful for the control of cork spot disorder in orchards.The objective of the present study was to investigate the impact of CaFor a two-year consecutive investigation, the cork spot incidence of NI-treated fruit at harvest time was much higher (16%) than that of the control fruit (4%) . The corAccording to the cork spot distribution pattern , the fru2+ transport and allocation in the fruit, Ca2+ transport-related genes such as Ca2+ sensors (PpCMLs), Ca2+/H+ exchangers (PpCAX) and Ca2-ATPase (PpACA) were selected for the expression analysis. For the inner flesh, PpCML11 had higher expression levels in control fruit than in NI-treated fruit at 67 and 87 DAFB; however, its expression increased remarkably in NI-treated fruit at 107 and 127 DAFB , ACA (auto-inhibited Ca2+-ATPase) and CAX (Ca2+ exchanger) [CMLs in different positions of the fruit during the fruit growth period . PpCML25 and PpCML47 were up-regulated in the outer flesh of NI-treated fruit at 107 and 87 DAFB, respectively, which were the critical timepoints for the onset of cork spot disorder. Higher expression levels of CMLs were also shown to associate with the occurrence of other Ca2+ deficiency-related disorders in pear fruit, such as superficial scald [ACA and CAX increased as the severity of bitter pits increased [MdCAX11 and MdCAX5 were also identified to bind to the tonoplast and transport Ca2+ from the cytoplasm to the vacuole, which increased the incidence of bitter pit [PpACA4 and PpCAX4 in the inner flesh and the peel. However, no significant alterations of PpACA4 and PpCAX4 expression levels were found in the outer flesh (2+ transport-related genes and further promoted the incidence of cork spot in ‘Akituki’ pear.As well as the long-distance transport of Cachanger) . In our h period . In Arabal scald . In applncreased ,28. MdCAtter pit . Intereser flesh , which iPyrus calleryana, located at 36°53′ N and 118°42′ E in Weifang, Shandong Province, China, were used for the trial. Trees, spaced at 2 m within rows and 5 m between rows, were trained with a trellis cultivation system and were managed with full nutrition and water. NI  was dissolved in lanolin at the concentration of 0.2 mg/mL and homogeneously smeared on the fruit pedicels at 47 days after full bloom (DAFB), and solvent lanolin was used as control to smear on the fruit pedicel. Twenty fruits were selected randomly on each tree for NI treatment and control, respectively, and five trees for each trial were used for the whole trial. After NI treatment on the fruit pedicels at 47 DAFB, fruit samples were taken every 20 days until harvest for different purposes of analysis as described below.Ten-year-old ‘Akituki’ trees grafted on the rootstock of Cork spot incidence of the fruits selected for the trial were investigated at harvest time (127 DAFB) from 2019 to 2020. The fruit without any cork spots on both equatorial and longitudinal planes inside the fruit were considered as healthy fruit; otherwise any cork spot observed inside the fruit was considered as cork spot disorder.The fruit quality was evaluated at harvest time by measuring the single fruit weight, vertical and horizontal length, total soluble solids, titratable acidity and fruit firmness. All the operations and calculation referred to Cui et al. .Fruit of 67, 87, 107 and 127 DAFB were sampled for the Ca, K and Mg content analysis. For each sample, the tissues of fruit peel, outer flesh and inner flesh were separately measured for the Ca, K and Mg contents. Briefly, fruit tissues were dried in an oven at 105 °C for 30 min, and then at 75 °C until a constant weight was achieved; about 0.5 g of dried tissues were mixed with 2 mL perchloric acids and 10 mL nitric acids. After digestion at room temperature for 10 min, the levels of Ca, K, and Mg were analyzed using an IPC-OES  according to Falchi et al. . The con® 480 instrument . Gene-specific primers were designed  according to the manufacturer’s instructions. RNA quality was checked by both optical density (OD) value (1.9 to 2.1) at 260/280 nm  and electrophoresis in a 1.5% agarose gel with sharp, clear 28s and 18s rRNA bands stained by ethidium bromide according to . cDNA wadesigned  and an at method .2+ was analyzed by fluorescence imaging as previously described by Qiu et al. [The localization of free Cau et al.  with somp < 0.05 or p < 0.01 level.The statistical analysis was performed using GraphPad Prism 8 software . Significant difference was determined by Duncan’s multiple range test, and significance was tested at 2+ transport-related genes; the Ca contents in the outer flesh and peel, especially at early stage (67 DAFB); and finally, the intracellular distribution of Ca2+ in certain tissues, as summarized in 2+ uptake of the root; therefore, appropriate irrigation should be fulfilled in the areas with hot summer and less rainfall. Measures such as foliar spraying of a Ca2+ solution to prevent and control the occurrence of cork spot should be conducted as early as possible. Enough Ca up-take at an early stage of the fruit development is essential to secure a normal Ca concentration and distribution and reduce the cork spot incidence. In a future study on cork spot disorder, advanced technologies such as multi-omics analysis should be applied to explore the mechanism of cork spot occurrence at the molecular level and help propose more effective control strategies.NI treatment promoted the development of cork spot in ‘Akituki’ pear fruit by altering the expression levels of Ca


Androgen-inducible genes (AIGs), which can be regulated by androgen level, constitute a group of genes characterized by the presence of the AIG/FAR-17a domain in its protein sequence. Previous studies on AIGs demonstrated that one member of the gene family, AIG1, is involved in many biological processes in cancer cell lines and that ADTRP is associated with cardiovascular diseases. It has been shown that the numbers of AIG paralogs in humans, mice, and zebrafish are 2, 2, and 3, respectively, indicating possible gene duplication events during vertebrate evolution. Therefore, classifying subgroups of AIGs and identifying the homologs of each AIG member are important to characterize this novel gene family further. In this study, vertebrate AIGs were phylogenetically grouped into three major clades, ADTRP, AIG1, and AIG-L, with AIG-L also evident in an outgroup consisting of invertebrsate species. In this case, AIG-L, as the ancestral AIG, gave rise to ADTRP and AIG1 after two rounds of whole-genome duplications during vertebrate evolution. Then, the AIG family, which was exposed to purifying forces during evolution, lost or gained some of its members in some species. For example, in eutherians, Neognathae, and Percomorphaceae, AIG-L was lost; in contrast, Salmonidae and Cyprinidae acquired additional AIG copies. In conclusion, this study provides a comprehensive molecular phylogenetic analysis of vertebrate AIGs, which can be employed for future functional characterization of AIGs. Androgen-inducible gene (AIG) or FAR-17a is a newly identified gene first characterized in the Syrian golden hamster as an androgen-responsive gene [androgen-inducible gene 1 (hAIG1), which was found to be a homolog of hamster FAR-17a, was isolated from cultured human dermal papilla cells [hAIG1 encodes a predicted integral membrane protein with expression observed across many tissues. Additionally, the expression level of hAIG1 was upregulated by dihydrotestosterone . Then, another androgen-regulated gene, C6ORF105 (ADTRP), which encodes a transmembrane protein and shows sequence similarity with hAIG1, was identified with the characteristic FAR-17a/AIG domain in the predicted amino acid sequence. Thus, hAIG1 and hADTRP are likely to be paralogs and form an AIG family [la cells . hAIG1 eG family .hAIG1 expression was correlated with HCC patient survival rates and thus can be used as a novel biomarker for the progression of HCC [AIG1 is involved in cancer-related processes; e.g., AIG1 can form complexes with either nuclear factor 1/B in salivary adenoid cystic carcinoma or Golgi SNAR complex member 1 in T cell lymphoma [AIG1 was required for resistance to these environmental toxicants [AIG1, as an endoplasmic reticulum (ER) integral membrane protein with a sex-specific expression pattern, participates in the regulation of ER Ca2+ levels and cell death [Functionally, an initial study first revealed that the AIG1 protein can interact with the p53-induced RING-H2 protein (Pirh2) and activate the NFAT signaling pathway. In human hepatocellular carcinoma (HCC), n of HCC . Other slymphoma . Zhu et oxicants . Anotherll death .ADTRP was provoked by its potential roles in cardiovascular diseases. Tissue factor pathway inhibitor (TFPI) is a vital anticoagulation factor that inhibits factor Xa and factor VIIa to prevent prothrombotic diseases, including coronary artery disease (CAD) [C6ORF105, was positively correlated with TFPI [C6ORF105 colocalized with TFPI in endothelial cells and that androgen upregulated both TFPI and C6ORF105. Thus, the novel gene that can be regulated by androgen was named androgen-dependent TFPI-regulating protein (ADTRP) [ADTRP and CAD, which is a leading cause of death worldwide induced by both genetic and environmental factors [ADTRP locus was significantly associated with CAD, where a minor risk A allele further reduced ADTRP expression and elevated CAD risk [ADTRP levels [AIG1, androgen modulates the expression level of ADTRP by directly binding the half androgen-response element within +324 bp of the ADTRP transcription start site (TSS) [TFPI and elevates its level [ADTRP and LDLR/CD36/LOX-1 in endothelial cells was revealed, and the NF-κB and AKT pathways are possibly involved [In contrast to that of its paralog, the validation of se (CAD) ,10. Therith TFPI . A subse (ADTRP) . Later s factors ,12,13. ACAD risk . The mecP levels . MoreoveP levels ,17, and te (TSS) . Upon bits level . TFPI suts level . Positivinvolved . Additioinvolved .ADTRP and/or AIG1 is crucial. The first experiment was thus carried out in zebrafish, in which there are two hADTRP homologs, adtrp1 and adtrp2 [adtrp1 reduced the expression of tfpi, and adtrp1 was shown to be involved in the specification of primitive myelopoiesis and definitive hematopoiesis, while adtrp2 knockdown by different morpholinos did not affect the aforementioned ADTRP- and/or AIG1-related processes [adtrp1, but not adtrp2, in zebrafish development. Furthermore, another study showed that morpholino-mediated knockdown of zebrafish adtrp1 resulted in vessel development defects in zebrafish embryos [ADTRP in mammals, a global Adtrp-knockout mouse model was established by removing the sequence between exons 2 and 5. These biallelic knockout mice showed embryonic lethality to some degree and displayed defective vasculature [ADTRP is a crucial factor during vascular development in both zebrafish and mammals. Interestingly, no abnormality was observed in mice with global knockout of Aig1, Adtrp, or both using the CRISPR/Cas9 technique. The discrepancy was likely due to the residual activity of mutant AIG genes with a deletion of only 13 bp compared to the wild-type allele. In these mice, only a higher concentration of fatty acid esters of hydroxy fatty acids (FAHFAs), which are signaling lipids with anti-inflammatory and antidiabetic activities [AIG1 and ADTRP might be novel FAHFA hydrolases [In addition to in vitro and association studies, the discovery of the in vivo roles of d adtrp2 . Knockdorocesses , indicat embryos . To furtculature . These rtivities , was detdrolases ,27.AIG1 and ADTRP provided some basic information regarding the novel protein family. However, the evolutionary relationship between the members of the AIG family has yet to be determined. In the current study, vertebrate AIG-related proteins were phylogenetically investigated, and the results showed that the AIG family constitutes three major clades, AIG1, ADTRP, and AIG-L. The orthology and paralogy of vertebrate AIGs were clarified, which would facilitate the future selection of appropriate genes for modeling human AIGs function. Some evolutionarily conserved sites were identified, and some are the same as those indicated through experimental approaches. In summary, this study revealed some new information regarding the function of the AIG family from an evolutionary perspective, which may be very useful for further physiological studies of AIGs.Genetic and molecular characterization of ADTRP , AIG1 , mouse Adtrp , Aig1 , and zebrafish aigs  were used as queries to BLAST against the non-redundant protein database consisting of chordate, hemichordate, and echinoderm species through several iterations of the Phi-BLAST algorithm [AIG gene was predicted by searching these databases, the genome sequences based on synteny were downloaded and subjected to GenScan [AIG, it was concluded that AIG was lost in the species. After collection and prediction, a total of 921 entries were available (Sequences of human lgorithm . To mainvailable .Homo sapiens and Macaca mulatta), rodents (Mus musculus and Rattus norvegicus), Artiodactyla (Bos taurus and Sus scrofa), carnivores (Canis lupus familiaris and Felis catus), Lagomorpha (Oryctolagus cuniculus), Chiroptera (Myotis lucifugus), Marsupial (Sarcophilus harrisii), Proboscidea (Loxodonta africana), Monotreme (Ornithorhynchus anatinus); birds , reptiles , amphibians , lampreys (Petromyzon marinus), lobe-finned fish , 2R ray-finned fish (Lepisosteus oculatus), 3R ray-finned fishes (Danio rerio and Takifugu rubripes), 4R ray-finned fishes (Oncorhynchus mykiss and Cyprinus carpio), Chondrichthyes , Hemichordata , Echinodermata , Cephalochordata (Branchiostoma belcheri and Branchiostoma floridae), and tunicates . Invertebrates such as Echinodermata, Hemichordata, Urochordata, and Cephalochordata were used as outgroups. The protein and gene sequences from these species were collected for subsequent analyses ) were used to generate an all-protein tree using the IQ-tree with substitution matrix JTT+G4, 1000× ultrafast bootstrap [Multiple sequence alignment (MSA) was performed using Clustal Omega with default parameters . The rawootstrap  combinedootstrap . Then, aootstrap ,45.ADTRP/AIG1/AIG-L from the NCBI and Ensembl databases. The analyzed species included eutherian species, marsupial (Ornithorhynchus anatinus), monotreme (Sarcophilus harrisii); fishes , reptiles , amphibians , birds , lampreys (Petromyzon marinus), and invertebrates . For species lacking genetic information for ADTRP/AIG1/AIG-L, such as Rhincodon typus, in which only contigs include annotation of ADTRP/AIG1/AIG-L, the syntenic organization was inferred according to other closely related species.Microsynteny analysis was performed according to our previously published method . In brieAIG family, we calculated the nonsynonymous substitution rate to synonymous substitution rate ratio  using the maximum likelihood approach. The CodeML program in PAML 4.9j [The trimmed MSA results of representative proteins were converted to codon alignments by PAL2NAL . To evalAML 4.9j  was emplAML 4.9j ,49,50. TAML 4.9j . The parFirstly, site model analysis was implemented with model=0 parameter in CodeML. The parameters concerning M0, M1a, M2a, M7, M8, and M8a were evaluated, accordingly . After t2 distribution with a degree of freedom equivalent to the difference between the parameter numbers of the two models.Then, branch-site model tests were performed using either mammalian ADTRP or AIG1 clade as foreground. The null and alternative models  evaluation were performed through likelihood ratio tests, which compare twice the log-likelihood difference of selection and neutral model (2ΔlnL) to values obtained from a χIf the likelihood ratio tests of the site model or branch-site model were significant, the Bayes Empirical Bayes (BEB) approach  was perfPositively selected site identification was also performed in MrBayes to verify conclusions drawn from the results obtained with the CodeML program . The SYMAIG paralogs, while zebrafish had three. zadtrp1 seemed to be an outgroup of AIG1s and ADTRPs, and zadtrp2 was an outgroup of all AIG domain-containing genes, contrary to previous reports indicating that zadtrp1 is an ortholog of human ADTRP [AIG-related genes, we narrowed our scope to focus only on vertebrates, as the function of AIG domain-containing genes is not yet clear.An initial preliminary phylogenetic study was performed using AIG domain-containing sequences from zebrafish, bovines, chickens, humans, mice, and rats. In this study, all analyzed tetrapods contained two an ADTRP ,24. ThisADTRP, AIG1, and AIG-L. To extrapolate the phylogenetic results, all the retrieved AIG protein sequences with a sequence in Caenorhabditis elegans (NP_510364.2) were utilized to build an ML tree using IQ-TREE [Caenorhabditis elegans) to mammals, indicating that AIG-L might be the ancestral AIG. Thus, to draw a reliable conclusion regarding the AIG classification in vertebrates, another phylogenetic tree was generated with all available vertebrate AIG protein sequences. The unrooted radial tree showing aLRT [Representative species from each class of vertebrates were selected, and AIG domain-containing proteins and genes were retrieved from the databases. Maximum likelihood (ML) and Bayesian approaches were employed to construct phylogenetic trees, with sequences from Urochordata, Cephalochordata, Hemichordata, and Echinodermata forming the outgroup. All the trees represent three major clades , which w IQ-TREE . The resing aLRT  and UF-bing aLRT  values (Petromyzon marinus), nematode (Caenorhabditis elegans: NP_510364.2 and NP_001024448.1), and arthropod . As shown in Petromyzon marinus and zebrafish AIG1s were grouped together, separating from AIG-Ls. Moreover, the vertebrate AIG-Ls were placed at the base of AIG-L clade, while no one-to-one orthologous relationship is noted between different phyla/subphyla. This result indicates that the diversification of AIGs might be phyla/subphyla specific, and the classification of AIG1, ADTRP, and AIG-L of vertebrates could not be applied to other phyla. Future work concerning more invertebrates will be needed to decipher the evolution of AIGs in other species.To figure out whether invertebrate species embody the same classification of AIGs, a preliminary analysis was carried out using AIG sequences from available invertebrate chordates, hemichordates, echinoderms, zebrafish, lampreys  that took place 450 million years ago (Mya) gave rise to the current vertebrate genomes [AIG copies in synteny, similar to that in Petromyzon marinus, where AIGs were adjacent to either EDN-like genes or HIVEP-like genes , indicahostomes ,64. In tSarcophilus harrisii has a copy of the AIG-L gene. To determine whether AIG-L was preserved in mammalian genomes, another phylogenetic analysis was performed using all the available mammalian AIG protein sequences. As shown in AIG paralogs of AIG1 and ADTRP, except for two species of metatherians, Sarcophilus harrisii and Phascolarctos cinereus, which preserve AIG-L. As shown by the synteny analysis, AIG-L in all tetrapod species resides in a relatively conserved synteny group  does not have AIG-L; Oncorhynchus mykiss  has two AIG1s, two AIG-Ls, and one ADTRP; and Cyprinus carpio has three AIG1s, 1 ADTRP, and 1 AIG-L. These results indicate that the AIG family underwent several different duplications, losses, and rearrangements in these teleosts, possibly due to 3R or 4R WGD events [AIG copies in zebrafish, originally named adtrp1, adtrp2, and aig1, are phylogenetically clustered with AIG-L, ADTRP, and AIG1 clades, respectively. Therefore, they were renamed aig-l, adtrp, and aig1. Hence, the previous adtrp1 is actually paralogous to hADTRP. According to functional studies of zebrafish and mice, the paralogs zaig-l and mAdtrp play similar roles in vascular development and can regulate TFPI expression. However, the mammalian ADTRP ortholog zadtrp does not exert a similar function [zadtrp and zaig-l. Gene duplication is the driving force for speciation and adaptation [AIG, vascular development-related function was preserved in tetrapod ADTRPs and ray-finned fish AIG-Ls.An analysis of ray-finned fish genomes led to 277 hits of AIG sequences, most of which belonged to Percomorphaceae, Salmonidae, and Cyprinidae . As showD events ,73,74,75function ,24, indiaptation . Most duaptation ,79. ThusAIG evolution in fish, another phylogenetic tree was established using Urochordate, Chondrichthyes, and Osteichthyes AIGs  have four copies of AIGs with three potential protein-coding genes and one pseudogene (Lepisosteus oculatus), has three AIGs, similar to those in Chondrichthyes and tetrapods. A clustering analysis revealed that Acipenser ruthenus has one AIG1 and two AIG-Ls, and the other two basal Actinopterygian species have one ADTRP, one AIG1, and one AIG-L. The differences between Acipenser ruthenus and the two other basal fish might be explained by Acipenser ruthenus-specific genome duplication [To obtain a more comprehensive view of yes AIGs . This treudogene , and anolication .AIGs [AIG evolution in other teleost orders and families. Of the Percomorphaceae, which includes Anabantiformes, Synbranchiformes, Batrachoidiformes, Carangiformes, Pleuronectiformes, Centrarchiformes, Labriformes, Perciformes, Spariformes, Tetraodontiformes, Gobiiformes, Kurtiformes, Beloniformes, Cyprinodontiformes, Blenniiformes, Cichliformes, and Syngnathiformes in our study, only AIG1 and ADTRP were maintained without the presence of AIG-L. Other exceptions were found in Salmonidae and Cyprinidae, in which 4R WGD occurred independently [AIG copies with one ADTRP, two AIG1s, and two AIG-Ls, resulting from a common auto-tetraploidization event ~100 Mya [ADTRP  and one AIG-L and 2–3 copies of AIG1. Even the closely related Carassius auratus (goldfish) and Cyprinus carpio (common carp), whose common ancestor underwent WGD ~8 to 12 Mya, have different AIG1 copies [Approximately 320 Mya, another teleost-specific WGD, occurred after the divergence of the Holostei and Teleostei, and rediploidization resulted in most orders of teleosts maintaining three copies of AIGs ,73,81, sendently ,83,84,85~100 Mya ,83,86. O1 copies ,87.AIG genes may experience purifying selection or adaptive evolution, which make them functionally conserved or diversified, respectively. To test which evolutionary selection predominated, multiple sequence alignments of AIG protein sequences were converted to codon alignments using PAL2NAL [AIG family were evaluated using the Nei-Gojobori method in MEGA X [AIG family . It is common, and possibly always true, that many amino acid residues in a given protein sequence undergo strong purifying selection, making the average dN/dS ratio less than 1 [After gene duplications, the duplicated copies of the  PAL2NAL . Then, on MEGA X ,89 and tn MEGA X ,91. Boths than 1 ,93. Thuss than 1 . Next, MAIG1 and ADTRP were classified as atypical integral membrane hydrolases of FAHFAs, where genetic ablation or pharmacological inhibition of either protein could elicit an increase in endogenous FAHFA without any cardiovascular defects [AIG family show a conserved evolutionary pattern and might have a conserved function.In two recent functional studies,  defects ,27. Muta defects . In our  defects . TherefoAIG family from an evolutionary perspective. Through phylogenetic and syntenic analyses, vertebrate AIGs were categorized into three major groups, ADTRP, AIG1, and AIG-L. We also proved that vertebrate AIGs might evolve from invertebrate AIG-L genes with the expansion that occurred in the common ancestor of agnathans and gnathostomes. During evolution, some taxonomic units might have lost or gained AIGs; however, extant AIGs may have a conserved function because of purifying selection. Importantly, our study clearly presented human, mouse, and zebrafish ADTRP/AIG orthologs, which lays the foundation for future reliable molecular characterization of AIGs.Our study, for the first time, characterized the vertebrate


Phlpp1 floxed mice with mice harboring LysM-Cre. Micro-computed tomography of the distal femur of 12-week-old mice revealed a 30% increase in bone volume per total volume of Phlpp1 female conditional knockouts, but we did not observe significant changes within male Phlpp1 cKOLysM mice. Bone histomorphmetry of the proximal tibia further revealed that Phlpp1 cKOLysM females exhibited elevated osteoclast numbers, but conversely had reduced levels of serum markers of bone resorption as compared to littermate controls. Osteoblast number and serum markers of bone formation were unchanged. In vitro assays confirmed that Phlpp1 ablation enhanced osteoclast number and area, but limited bone resorption. Additionally, reconstitution with exogenous Phlpp1 suppressed osteoclast numbers. Dose response assays demonstrated that −/−Phlpp1 cells are more responsive to M-CSF, but reconstitution with Phlpp1 abrogated this effect. Furthermore, small molecule-mediated Phlpp inhibition enhanced osteoclast numbers and size. Enhanced phosphorylation of Phlpp substrates—including Akt, ERK1/2, and PKCζ—accompanied these observations. In contrast, actin cytoskeleton disruption occurred within Phlpp inhibitor treated osteoclasts. Moreover, Phlpp inhibition reduced resorption of cells cultured on bovine bone slices in vitro. Our results demonstrate that Phlpp1 deficiency within myeloid lineage cells enhances bone mass by limiting bone resorption while leaving osteoclast numbers intact; moreover, we show that Phlpp1 represses osteoclastogenesis and controls responses to M-CSF. Prior work demonstrated that Phlpp1 deficiency alters trabecular bone mass and enhances M-CSF responsiveness, but the cell types and requirement of Phlpp1 for this effect were unclear. To understand the function of Phlpp1 within myeloid lineage cells, we crossed  Enhanced bone resorption is associated with conditions such as osteoporosis, cancer-associated bone loss, rheumatoid arthritis, and periodontitis. Bone resorption is accomplished by osteoclasts, the large, multinucleated cells that line bone surfaces. Osteoclasts arise via iterative fusion of myeloid progenitor cells and form within bone. Because disruptions of osteoclast differentiation and/or activity impact bone resorption, a better understanding of osteoclast biology will help design strategies to limit bone loss.PH Domain and Leucine Rich Repeat Protein Phosphatase 1  is a serine/threonine protein phosphatase. Phlpp1 and its related isoform, Phlpp2, are metal-dependent type 2C protein phosphatases .PHLPP1 dephosphorylates and inactivates several kinases that regulate osteoclastogenesis and bone resorption including Akt, a kinase that is well established in promoting osteoclast differentiation, survival, and activity . PHLPP1 Phlpp1 germline knockout mice  are viable, but show enhanced growth factor responsiveness and cellular proliferation [Phlpp1 germline deficiency decreased body size, long bone length and bone mass [Phlpp1 within Ctsk-Cre expressing cells enhanced bone mass [Phlpp1 deletion within myeloid lineage cells on bone mass and osteoclastogenesis. feration . Our groone mass . In contone mass , but theone mass ,15,16,17Phlpp1 limited acquisition of peak bone mass [Phlpp1 within myeloid progenitor cells affected bone mass, we crossed Phlpp1 floxed [Phlpp1 cKOLysM and control littermates were aged to 12-weeks and femora and tibiae were collected. Micro-CT analyses revealed a 30 percent increase in trabecular BV/TV of Phlpp1 cKOLysM females within the distal femur  [Phlpp1 ablation may likewise enhance osteoclast progenitor proliferation and/or migration leading to increased osteoclast numbers. Phlpp1, because it reduces anabolic signaling, suppresses cellular proliferation and survival of a many different cell types. Increased osteoclastogenesis occurring with myeloid lineage rophages . Since tenitors) , Phlpp1 + mononuclear cells. Fusion and fission of osteoclast lineage cells could likewise be altered; as we see disruptions to the actin cytoskeleton of mature osteoclasts, this is a possibility worth exploring in the future. Phlpp1 also dampens pro-survival signaling; thus, diminished osteoclast survival could also account for lowered osteoclast numbers observed with AdPhlpp1 transduction. Diminished osteoclastogenesis observed with forced Phlpp1 expression  could reflect several possibilities. Decreased osteoclast numbers could be a result of a permanent lineage change. As our models system uses unfractionated bone marrow macrophages, which are a heterogeneous population of cells, differential effects within this population is possible. Phlpp1 also targets phosphorylation of Histone 3, leading to altered acetylation and chromatin compaction . AlteredWe found that small molecule-mediated inhibition of Phlpp isoforms likewise enhanced osteoclastogenesis, but limited bone resorption ex vivo. NSC 117079 inhibits the activity of PHLPP1 and PHLPP2, but inhibition of PHLPP2 activity is greater . PHLPP1 To mitigate bone loss, therapies target one or more aspects of bone remodeling. While anti-resorptive therapies  effectively limit bone resorption, they also decrease osteoclast number leading to disruptions in the reversal phase ,31,32,33Phlpp1 allele were crossed with mice expressing Cre recombinase driven by the Lyz2 promoter (LysM-Cre). We crossed Phlpp1fl/fl: Cre− animals with Phlpp1fl/+: Cre+ animals to generate Phlpp1 conditional knockout mice (referred to as Phlpp1 cKOLysM) and were genotyped as previously described [−/−Phlpp1 animals was performed as previously described [Mice harboring a floxed escribed ,18. Geneescribed . All aniPhlpp1 cKOLysM mice (n = 6 per sex) and their control littermates  were isolated and fixed in 10% neutral buffered formalin for 48 h, then stored in 70% ethanol. Scanning was performed using a Scanco Viva40 micro-CT at 70 kV, 221 ms with a 10.5-μm voxel size. For trabecular measurements, a region of interest was defined at 10% of total femur length starting immediately proximal to the growth plate; samples were analyzed using a 220-threshold air filling correction.Femora from 12-week-old male and female Phlpp1 cKOLysM mice (n = 6) and their control littermates  were isolated and fixed in 10% neutral buffered formalin for 48 h, decalcified in 15% EDTA for 14 days, then stored in 70% ethanol. Tissues were paraffin embedded and 7-μm sections were collected and TRAP/Fast Green stained  or Masson’s trichrome stained as previously described [Tibiae from 12-week-old male and female escribed . Standarescribed . Phlpp1 cKOLysM (n = 6) female mice and their control littermates  and stored at −80 °C. Enzyme-linked immunosorbent assays (ELISA) for bone resorption (CTX-1) were performed in duplicate using 20 μL of serum from each mouse according to the manufacturer’s specifications (Ratlaps (CTX-1) #AC-06F1, Immunodiagnostics Systems). Bone formation was assessed using the ELISA for serum P1NP performed in duplicate using serum from each mouse as described by the manufacturer (Ratlaps (P1NP), #AC-33F1, Immunodiagnostics Systems, East Boldon, UK).Serum was collected from 12-week-old Phlpp1 cKOLysM mice or −/−Phlpp1 mice and their control littermates as previously described [6 cells/cm2 [−/−Phlpp1 cells, cells were exposed to increasing M-CSF concentrations and infected with adenoviral (Ad) GFP or AdPhlpp1 at an MOI of 10 as previously described [+ cells with 3 or more nuclei was determined as previously described [Bone marrow macrophages were collected from escribed . Cells wells/cm2  and infeescribed ,42. On described . All expn = 3) were also cultured in the presence of the small molecule Phlpp inhibitor NSC 117079  starting on day 0 of osteoclastogenesis assays. Cells were fed on day 3 with osteoclastogenic medium containing NSC 117079 or control. On day 4, cells were TRAP stained. Bone marrow macrophages were also seeded onto bovine bone disks. Cells were cultured on bone slices for 10 days and feed with osteoclastogenic medium containing NSC 117079 or control every 3–4 days. On day 10, cells were lysed in 5% domestic bleach and bone slices were stained with toluidine blue as previously described [Bone marrow macrophages from C57Bl/6 females , 20 μg of total proteins per sample was resolved by SDS-PAGE and transferred to PVDF membranes. Western blotting was performed with antibodies (1:2000 dilution) for PHLPP1 , phospho-Ser 660 PKC , phospho-Ser 410 PKCζ , PKCζ , phospho-Akt2 , Akt , phospho-Ser2448 mTOR , mTOR , phospho-Ser56 4E-BP1 , phospho-Ser371 p70 S6K , phospho-Thr389 p70 S6K , phospho-Ser10 Histone 3 , acetyl-K27 Histone 3 , Histone 3 , Tubulin , and corresponding secondary antibodies conjugated to horseradish peroxidase (HRP) . Protein detection was accomplished using the Supersignal West Femto Chemiluminescent Substrate . Experiments were repeated three times with biological replicates with representative data shown.For Western blotting, cell lysates were collected in SDS sample buffer on ice. After determining total protein concentrations using the Bio-Rad Dt-test or AVOVA as appropriate. Comparisons where p < 0.05 were considered statistically significant.GraphPad Prism 8 software  was utilized for statistical analyses. Comparisons were made using a Student’s