Oocyte-derived forms of ruminant BMP15 and GDF9 and a theoretical model to explain their synergistic response

<p>Bone morphogenetic factor 15 (BMP15) and growth differentiation factor 9 (GDF9) are two oocyte-secreted factors with well documented effects on ovarian follicular development and ovulation-rate. The aims of these studies were to: (i) identify the molecular forms of BMP15 and GDF9 that are produced and secreted by both the ovine and bovine oocyte using highly specific monclonal antibodies; (ii) assess the biological activity of some recombinant molecular forms of BMP15 and GDF9; (iii) visualise the various molecular forms using protein modelling techniques and; (iv) provide a hypothetical model of how oocyte-secreted form(s) of BMP15, GDF9 and their cell surface receptors may interact. Using genetic modifications and transformations of HEK293 cells, recombinant forms of ovine (o) BMP15, including a BMP15 (S356C) mutant capable of forming covalent dimers, and oGDF9 were produced. The bioactivity of these proteins was established using a rat granulosa cell proliferation bioassay. The specificity of the monoclonal antibodies MN2-61A (anti-BMP15) and 37A (anti-GDF9) used in these studies, and determination of the forms they recognise, was examined by Western blotting. The recombinant forms of oBMP15 were further interrogated by purification using both immobilised metal affinity chromatography (IMAC) and reverse phase HPLC. The BMP15 and GDF9 proteins produced and/or secreted by ovine and bovine oocytes, before and after in vitro incubation, were identified and compared with the molecular forms(s) of recombinant oBMP15 or oGDF9 using Western blotting under non-reducing, reducing and cross-linking conditions. The molecular forms of recombinant oBMP15 and oGDF9 comprise mainly mature monomers with a lesser amount of the uncleaved pro-mature form. Mature domains, in the dimeric mature form, were detected for oGDF9 and oBMP15 (S356C), but not oBMP15. These mature domains were almost entirely located within high molecular weight multimeric complexes, which likely also contain the pro-region. In contrast, BMP15 and GDF9 secreted from ruminant oocytes under in vitro conditions were found mainly in an unprocessed promature form, along with some fully processed mature domains that did not interact to form detectable mature homodimers or heterodimers. Throughout ovarian follicular development, BMP15 and GDF9 are co-expressed and it has been established that these two factors have synergistic effects on granulosa cell proliferation both in vitro and in vivo and also on follicular maturation and ovulation-rate in vivo. Moreover, the recombinant proteins oBMP15 and oGDF9 generated for this study, when added together, also demonstrated a synergistic effect in the granulosa cell proliferation assay but this was not observed for oBMP15 (S356C) and oGDF9. Currently, no adequate model has been proposed to explain how interactions between the cell membrane and forms of oocyte-derived BMP15 and GDF9 achieve their synergistic effects. To investigate this, two homology models of the promature BMP15 and GDF9 proteins were generated using promature porcine TGFB1 and human BMP9 as templates. These models, together with the previously determined forms of GDF9 and BMP15 produced by the ruminant oocyte, were used to visualise their potential interactions, both with each other and with their receptors. This report describes a model showing the possible interactions involved in a synergistic response. In this model, the mature domain is presented to the type II receptor by the proregion and heterodimers form at the level of the receptor. Differences, following heterodimerisation in the conformation and orientation between GDF9 and its type I receptor, as well as between type I and type II receptors, relative to that in homodimers, could explain how heterodimerisation leads to increased Smad3 phosphorylation and subsequent down-stream somatic cell responses.</p>

Oocyte-derived forms of ruminant BMP15 and GDF9 and a theoretical model to explain their synergistic response

<p>Bone morphogenetic factor 15 (BMP15) and growth differentiation factor 9 (GDF9) are two oocyte-secreted factors with well documented effects on ovarian follicular development and ovulation-rate. The aims of these studies were to: (i) identify the molecular forms of BMP15 and GDF9 that are produced and secreted by both the ovine and bovine oocyte using highly specific monclonal antibodies; (ii) assess the biological activity of some recombinant molecular forms of BMP15 and GDF9; (iii) visualise the various molecular forms using protein modelling techniques and; (iv) provide a hypothetical model of how oocyte-secreted form(s) of BMP15, GDF9 and their cell surface receptors may interact. Using genetic modifications and transformations of HEK293 cells, recombinant forms of ovine (o) BMP15, including a BMP15 (S356C) mutant capable of forming covalent dimers, and oGDF9 were produced. The bioactivity of these proteins was established using a rat granulosa cell proliferation bioassay. The specificity of the monoclonal antibodies MN2-61A (anti-BMP15) and 37A (anti-GDF9) used in these studies, and determination of the forms they recognise, was examined by Western blotting. The recombinant forms of oBMP15 were further interrogated by purification using both immobilised metal affinity chromatography (IMAC) and reverse phase HPLC. The BMP15 and GDF9 proteins produced and/or secreted by ovine and bovine oocytes, before and after in vitro incubation, were identified and compared with the molecular forms(s) of recombinant oBMP15 or oGDF9 using Western blotting under non-reducing, reducing and cross-linking conditions. The molecular forms of recombinant oBMP15 and oGDF9 comprise mainly mature monomers with a lesser amount of the uncleaved pro-mature form. Mature domains, in the dimeric mature form, were detected for oGDF9 and oBMP15 (S356C), but not oBMP15. These mature domains were almost entirely located within high molecular weight multimeric complexes, which likely also contain the pro-region. In contrast, BMP15 and GDF9 secreted from ruminant oocytes under in vitro conditions were found mainly in an unprocessed promature form, along with some fully processed mature domains that did not interact to form detectable mature homodimers or heterodimers. Throughout ovarian follicular development, BMP15 and GDF9 are co-expressed and it has been established that these two factors have synergistic effects on granulosa cell proliferation both in vitro and in vivo and also on follicular maturation and ovulation-rate in vivo. Moreover, the recombinant proteins oBMP15 and oGDF9 generated for this study, when added together, also demonstrated a synergistic effect in the granulosa cell proliferation assay but this was not observed for oBMP15 (S356C) and oGDF9. Currently, no adequate model has been proposed to explain how interactions between the cell membrane and forms of oocyte-derived BMP15 and GDF9 achieve their synergistic effects. To investigate this, two homology models of the promature BMP15 and GDF9 proteins were generated using promature porcine TGFB1 and human BMP9 as templates. These models, together with the previously determined forms of GDF9 and BMP15 produced by the ruminant oocyte, were used to visualise their potential interactions, both with each other and with their receptors. This report describes a model showing the possible interactions involved in a synergistic response. In this model, the mature domain is presented to the type II receptor by the proregion and heterodimers form at the level of the receptor. Differences, following heterodimerisation in the conformation and orientation between GDF9 and its type I receptor, as well as between type I and type II receptors, relative to that in homodimers, could explain how heterodimerisation leads to increased Smad3 phosphorylation and subsequent down-stream somatic cell responses.</p>

PU.1 and MYC transcriptional network defines synergistic drug responses to KIT and LSD1 inhibition in acute myeloid leukemia

Activating mutations in the KIT tyrosine receptor kinase confer an adverse prognosis for patients with acute myeloid leukemia (AML). Outside of bone marrow transplantation, treatment options are limited. Here we demonstrate combined KIT and LSD1 inhibition produces synergistic cell death against KIT mutant AML cells. This combination suppresses MYC expression to drive cell cycle exit and apoptosis. This decreased MYC expression results from a loss of PU.1 binding at downstream MYC enhancers. The drug combination also inactivates PI3K/AKT/GSK3a/b signaling to decrease MYC protein abundance. KIT-mutant AML cells rapidly adapt to KIT inhibitor monotherapy by restoring PI3K/AKT activity, but cannot when treated with combined KIT and LSD1 inhibitor. In addition, we validate MYC suppression as a mechanism of synergy in KIT-mutant AML patient samples. Collectively, this work provides rational for a clinical trial to assess the efficacy of KIT and LSD1 inhibition in patients with KIT-mutant AML.Statement of significanceEffective treatment options for AML are limited. We describe the synergistic response to combined KIT and LSD1 inhibition in KIT-mutant AML and identify key biomarkers of drug response. The specificity and efficacy of this combination in cell lines and patient samples provides rationale for investigation in early phase clinical trials.

Role of Zinc-Doped Bioactive Glass Encapsulated with Microspherical Gelatin in Localized Supplementation for Tissue Regeneration: A Contemporary Review

Gelatin, a natural polymer, provides excellent tissue compatibility for use in tissue rehabilitation. Bioactive glasses (BAG) offer superior capacity in stimulating a bioactive response but show high variability in uptake and solubility. To tackle these drawbacks, a combination of gelatin with BAG is proposed to form composites, which then offer a synergistic response. The cross-linked gelatin structure’s mechanical properties are enhanced by the incorporation of the inorganic BAG, and the rate of BAG ionic supplementation responsible for bioactivity and regenerative potential is better controlled by a protective gelatin layer. Several studies have demonstrated the cellular benefits of these composites in different forms of functional modification such as doping with zinc or incorporation of zinc such as ions directly into the BAG matrix. This review presents a comprehensive perspective on the individual characteristics of BAG and gelatin, including the synthesis and mechanism of action. Further, adaptation of the composite into various applications for bone tissue engineering is discussed and future challenges are highlighted.

Interactions of Clomazone Plus Pendimethalin Mixed with Propanil in Rice

A study was conducted at the Louisiana State University Agricultural Center’s H. Rouse Caffey Rice Research Station in 2017 and 2018 to evaluate the interaction between a pre-package mixture of clomazone plus pendimethalin applied at 0, 760, 1145, or 1540 g ai ha-1 mixed with propanil at 0, 1120, 2240, or 4485 g ai ha-1. A synergistic response occurred when barnyardgrass was treated with all rates of clomazone plus pendimethalin mixed with either rate of propanil evaluated at 56 d after treatment. Unlike barnyardgrass, an antagonistic response occurred for yellow nutsedge control when treated with 760 and 1540 g ha-1 of clomazone plus pendimethalin mixed with 1120 or 2240 g ha-1 of propanil at 28 d after treatment; however, 1145 g ha-1 of clomazone plus pendimethalin mixed with 4485 g ha-1 of propanil resulted in a neutral interaction. At 28 d after treatment, Rice flatsedge treated with for all herbicide mixtures resulted in neutral interactions. The synergism of clomazone plus pendimethalin applied at 1540 g ha—1 mixed with propanil applied at 2240 or 4485 g ha-1 for barnyardgrass control resulted in an increased rough rice yield compared with 760 or 1145 g ha-1 of clomazone plus pendimethalin mixed with propanil applied at 1120 or 2240 g ha-1. These results indicate if barnyardgrass and rice flatsedge are present in a rice field the pre-package mixture of clomazone plus pendimethalin mixed with propanil can be an option for growers. However, if yellow nutsedge infest the area other herbicides may be needed.

Combinational Therapy Using Chemotherapeutic Agents and Dietary Bioactive Compounds

Diet-derived phytochemicals find prominent use in traditional medicine and have been credited with lowering cancer risk significantly. Dietary agents demonstrate anticancer activity by modulating various molecular targets and cell signaling pathways. Several studies have focused on combinations of dietary bioactive compounds and conventional chemotherapeutic agents to augment their therapeutic response and mitigate the side effects of conventional chemotherapy. The observed synergistic response heralds promise for successful future chemopreventive and chemotherapeutic strategies in cancer management. Animal models and pre-clinical trials of the effective combinations must be undertaken to clearly understand the mechanism of action. This chapter catalogues recent studies that have used dietary bioactive compounds (sulforaphane, EGCG, curcumin, genistein, resveratrol, eugenol) in combination with conventional chemopreventive agents and with other phytochemicals.

Global health security and universal health coverage: Understanding convergences and divergences for a synergistic response

Background Global health security (GHS) and universal health coverage (UHC) are key global health agendas which aspire for a healthier and safer world. However, there are tensions between GHS and UHC strategy and implementation. The objective of this study was to assess the relationship between GHS and UHC using two recent quantitative indices. Methods We conducted a macro-analysis to determine the presence of relationship between GHS index (GHSI) and UHC index (UHCI). We calculated Pearson’s correlation coefficient and the coefficient of determination. Analyses were performed using IBM SPSS Statistics Version 25 with a 95% level of confidence. Findings There is a moderate and significant relationship between GHSI and UHCI (r = 0.662, p<0.001) and individual indices of UHCI (maternal and child health and infectious diseases: r = 0.623 (p<0.001) and 0.594 (p<0.001), respectively). However, there is no relationship between GHSI and the non-communicable diseases (NCDs) index (r = 0.063, p>0.05). The risk of GHS threats a significant and negative correlation with the capacity for GHS (r = -0.604, p<0.001) and the capacity for UHC (r = -0.792, p<0.001). Conclusion The aspiration for GHS will not be realized without UHC; hence, the tension between these two global health agendas should be transformed into a synergistic solution. We argue that strengthening the health systems, in tandem with the principles of primary health care, and implementing a “One Health” approach will progressively enable countries to achieve both UHC and GHS towards a healthier and safer world that everyone aspires to live in.