Here, we hypothesize that oligodendrocyte progenitor cells (OPCs) cultured with BMSCs containing their native Treg population show greater cell viability, less pro-inflammatory cytokine secretion and greater myelin production after exposure to oxygen-glucose deprivation and reoxygenation (OGD/R) than OPCs cultured without Tregs. OPCs were cultured and then exposed to OGD/R. BMSCs with or without Tregs were added to the co-culture immediately after ischemia. The Tregs were depleted by running the BMSCs through a column containing a magnetic substrate. Fibroblast growth factor beta (FGF-β) and interleukin 6 (IL-6) ELISAs determined BMSC activity levels. Immunohistochemistry assessed OPC differentiation. OPCs cultured with BMSCs containing their endogenous Tregs showed increased myelin production compared to the BMSCs with depleted Tregs. IL-6 and FGF-β were increased in the group cultured with Tregs. Collectively, these results suggest that BMSCs containing Tregs are more therapeutically active, and that Tregs have beneficial effects on OPCs subjected to ischemia. Tregs play an important role in stem cell therapy and can potentially treat white matter injury post-stroke.Stroke-induced hypoxia causes oligodendrocyte death due to inflammation, lack of oxygen and exacerbation of cell death. Bone marrow-derived stem cells (BMSCs) possess an endogenous population of T-regulatory cells (T Although stroke rates are declining, cerebrovascular accidents present a major obstacle for public health in the United States. Stroke is responsible for approximately 1 in 20 deaths in Americans , and theregs), a subtype of T-cell, have recently discovered immunosuppressive properties. Tregs express the biomarkers FoxP3, CD25 and CD4 and have been found to exist as a small subpopulation of BMSCs [reg population, at present about 0.4% of BMSCs, was shown to confer increased cell viability via anti-inflammation in a set of neurons subjected to oxygen-glucose deprivation and reoxygenation (OGD/R). After OGD/R, pro-inflammatory cytokine IL-6 decreased with increasing concentrations of Tregs in co-culture [regs varied inversely with basic fibroblast growth factor beta (FGF-β), suggesting that increasing the number of Tregs beyond that of the native population within BMSCs may have deleterious effects on the survival and differentiation of the BMSCs [regs can mitigate inflammation after ischemia by influencing macrophage polarization toward the more regenerative M2 phenotype [regs is seen after ischemic stroke, which could play a crucial role in exacerbating an inflammatory state and causing further cell death [Clinical safety and efficacy of stem cell transplantation is still being determined . Bone maof BMSCs . The nat-culture . There w-culture . Intereshe BMSCs . M2 micrhe BMSCs . Tregs chenotype ,18,19. Ill death ,21,22.Gray matter has been a focus as a potential therapeutic intervention site for ischemic stroke, but the role of white matter damage following ischemia has been neglected. Stroke in areas of the brain containing white matter tracts represents a large minority of patients . Myelin-regs contained within BMSCs in ischemic stroke and white matter recovery. We hypothesized that depletion of Tregs before BMSC co-culture with OPCs subjected to OGD/R would result in decreased myelination  and not co-cultured with BMSCs (control). The second group contained OPCs exposed to OGD but not co-cultured with BMSCs (OGD). The third group contained OPCs that were exposed to OGD and co-cultured with BMSCs containing Tregs (OPCs with BMSCs T+reg). The fourth group contained OPCs that were exposed to OGD and co-cultured with BMSCs that had been depleted of their Tregs (OPCs with BMSCs T−reg). Lastly, the fifth group contained OPCs that were exposed to OGD and co-cultured with BMSCs that were run through the depletion column, but not depleted of their Tregs (OPCs with BMSCs no magnet). We used 5 groups to assess the effects of BMSCs and Tregs, at a concentration of 40,000 cells per well for three more days . Magnetically labeled cells were isolated by passing the cell suspension through a column containing magnetic metal substrate. CD25+ positive cells were depleted by passing the BMSCs through the column. The resulting eluent was a population of BMSCs that did not contain native Tregs. To mimic the conditions of the OGD with BMSCs T−reg group, the OGD with BMSCs no magnet group was similarly run through the depletion column, but the cells were not magnetically labeled and the magnet was inert. We compared this group to the other BMSC groups in order to determine whether the mechanical stress from the depletion column affected the secretion of cytokines or the expression of myelin basic protein (MBP) or O4. BMSCs were thawed and run through the depletion column approximately one hour before the co-culture was created. Anti-CD4 and CD25 antibodies were used to label Tg for 15 min and supernatant was collected and frozen at −80 °C. The supernatant was later processed and analyzed using ELISA kits specific to IL-6  and FGF-β  with absorbance measured at 450 nm on a Synergy HT plate reader .After OGD/R, culture medium was centrifuged at 3000× 2, n = 32/group) per treatment condition to quantify cell staining for MBP and/or O4 . Stained cells were digitally captured under microscope (20×). In our 8 well plates, one photo was captured from each quadrant in each well (four photos per well). The cells with positively stained nuclei only for MBP and O4 were not counted as MBP+ and O4+ respectively when calculating the percentage of positive cells, as this does not indicate typical morphology. O4+ and MBP+ cells were counted as positive if cells showed processes extending beyond the cell nucleus, as this is consistent with the shape generally assumed by OPCs and oligodendrocytes. Clusters of cells were not considered O4+ or MBP+ if obvious processes extending beyond their nuclei could not be identified.After cell culture was completed, cells were fixed for 30 min in 4% paraformaldehyde, permeabilized with a 0.1% tween solution, and incubated with fluorescent antibodies with affinity for MBP and oligodendrocyte marker O4 at the manufacturer’s recommended dilutions. Comparison of cell viability between the control and OGD groups was performed using a DAPI nuclear stain . Images of approximately 50 cells/picture were captured in 32 randomly selected areas . The sample size (eight replicates) was based on our previous study allowing rigorous testing of cultured cells for recognizing a 25% statistically significant difference between treatments and controls [For categorical independent variables with more than one dependent continuous variable, a one-way ANOVA test was used to determine significance. A controls . Pairwisp < 0.05). This survival rate was similar to what was obtained in our previous study [Cells were counted using ImageJ software after they were imaged with a confocal microscope. Exposure to OGD/R caused a reduction in number of surviving OPCs to 56.03% of the control (us study . The OPCp < 0.001)  . There wp < 0.001), again implying that BMSCs secrete growth factors necessary for oligodendrocyte differentiation (+reg and BMSC no magnet groups showed increased MBP positive cells compared to the BMSC T−reg group (both ps < 0.01). Since the BMSC no magnet group was not depleted of its Tregs, this suggests that the presence of the endogenous Treg population within BMSCs influences the outgrowth and production of myelin processes. Qualitatively, the BMSC T+reg and BMSC no magnet group had more delineated and robust myelin processes than the BMSC T−reg group. No significant differences in staining for either O4 or MBP were seen between the OGD with BMSCs T+reg group and the OGD with BMSCs no magnet group, and this therefore suggests that running the cells through the column did not alter O4 or MBP expression. Groups co-cultured with BMSCs showed increased myelination compared to groups not co-cultured with BMSCs . Interestingly, the BMSC T+reg showed increased IL-6 production compared to both other groups (both ps < 0.001) (−reg group (p < 0.05). The BMSC T+reg group displayed more secretion of FGF-β than the BMSC T−reg group, suggesting that the BMSCs were more active in this group (p < 0.05). There was no significant difference between the FGF-β secretion in the BMSC T+reg group and the BMSC no magnet group (p = 0.72). We assayed both the control and OGD supernatants in both ELISAs, but no results were obtainable because both ELISA kits were customized to detect human cytokines only. Extracellular secretion of cytokines FGF-β and IL-6 was different among the BMSC groups (< 0.001) . There wreg cells within a BMSC population on stroke-induced white matter injury in OPCs. Our main findings are as follows: (1) OGD reduced cell viability of OPCs; (2) OPCs treated with BMSCs containing their endogenous Treg population displayed increased myelin production compared to BMSCs with depleted Tregs; (3) co-culture with BMSCs with and without Tregs induced differentiation of immature OPCs into myelin-producing oligodendrocytes compared to control; and (4) extracellular cytokines were increased in the T+reg groups. In addition, treatment with BMSCs in general (with or without Tregs) was associated with increased OPC differentiation into myelinating oligodendrocytes. This implies that stem cell transplantation into injured white matter may aid in healing and quicker remyelination of damaged tissue after ischemic stroke.The present study tested the effects of Tregs are upregulated compared to other BDNF positive CD4+ T-cells in the human brain following ischemic stroke, suggesting that Tregs may influence oligodendrocyte differentiation via the BDNF pathway [regs in directing OPC-mediated myelination [regs act as a key mediator of myelination, perhaps through the BDNF pathway.Our results are supported by a plethora of studies showing the beneficial effects of BMSC transplantation on white matter after ischemic stroke. Mice and rats subjected to middle cerebral artery occlusion (MCAO) displayed an increased expression of MBP or O4 following BMSC transplantation compared to the MCAO group devoid of BMSC treatment ,33. Simi pathway . Moreove pathway ,41. A-kilination . While tregs are powerful negative regulators of pro-inflammatory cytokine secretion, specifically IL-6, the results of the current study reveal a unique neuroprotective property of IL-6 in OPCs [regs. This may be due to increased BMSC activity in this group, which is supported by our results that there was increased FGF-β secretion in both the BMSC T+reg and BMSC no magnet group. Although many studies have labeled IL-6 as a pro-inflammatory cytokine, there is equally compelling evidence suggesting that it also has neuroprotective properties and that it is involved in crosstalk with the BDNF pathway [Although our previous study showed that T in OPCs . We saw  pathway ,44. Inde pathway . IL-6 ha pathway . Thereforegs displayed increased MBP production compared to the T−reg depleted BMSCs. To date, limited studies demonstrate a potential interaction between Tregs and BDNF/Shh signaling in the brain. Of note, Shh signaling was necessary for expansion of Tregs by dendritic cells, but this was not in the context of the CNS [regs result in increased MBP expression. Additionally, treatment of cells belonging to the oligodendrocyte lineage showed an increase in myelination after exposure to IL-6 fused with its receptor in a dose-dependent manner [regs [regs use the cytokine transforming growth factor beta to modulate levels of IL-6 secreted by mast cells, controlling the inflammatory milieu and increasing neutrophil clearance with increasing levels of IL-6 secretion [regs may be direct modulators of its secretion. Effective treatments targeting white matter injury will ensure survival, differentiation and protection of OPCs from inflammation, as this is critical for the remyelination process [regs and BMSCs should be investigated to further elucidate the unique roles that each plays in influencing the oligodendrocyte lineage.As stated above, the group treated with BMSCs containing T the CNS . This int manner . This fuer [regs . More specretion . Therefo process . More spregs or stem cells has shown to result in increased cell viability in neurons and oligodendrocytes after ischemic conditions [regs on white matter injury due to ischemic stroke to segue into the clinic. Recognizing that BMSCs are unlikely to transdifferentiate into myelin-producing oligodendrocytes before six days after induction, we did not include stem cell markers in our immunocytochemistry assays in order to distinguish between the original OPCs and stem cells . Future nditions ,29,30. Wreg exposure resulted in more robust expression of MBP. These results confirmed prior research highlighting the beneficial effects of BMSCs after ischemic stroke. Our novel finding consisted of the increased expression of MBP due to exposure to Tregs, but the exact mechanism by which this occurs is unclear at this stage. Limitations including the restricted clinical application and the lack of visualization of stem cell markers should be taken into consideration in future research. However, the finding that Tregs resulted in overall healthier oligodendrocytes emphasizes their potential therapeutic benefit to white matter recovery after ischemic stroke.In conclusion, we found that BMSC co-culture was associated with increased differentiation of OPCs after ischemic injury and that T


Deletion of long arm of chromosome 20 [del(20q)] is the second most frequent recurrent chromosomal abnormality in hematological malignancies. It is detected in 10% of myeloproliferative neoplasms, 4–5% of myelodysplastic syndromes, and 1–2% of acute myeloid leukaemia. Recurrent, non-random occurrence of del(20q) indicates that it is a pathogenic driver in myeloid malignancies. Genetic mapping of patient samples has identified two regions of interest on 20q – the “Common Deleted Region” (CDR) and “Common Retained Region” (CRR), which was often amplified. We proposed that the CDR contained tumor suppressor gene(s) (TSG) and the CRR harbored oncogene(s); loss of a TSG together with over-expression of an oncogene favored development of myeloid malignancies. Protein Tyrosine Phosphatase Receptor T (PTPRT) and Hemopoietic cell kinase (HCK) were identified to be the likely candidate TSG and oncogene respectively. Retroviral transduction of HCK into PTPRT-null murine LKS+ stem and progenitor cells resulted in hyperproliferation in colony forming assays and hyperphosphorylation of intracellular STAT3. Furthermore, over half of the murine recipients of these transduced cells developed erythroid hyperplasia, polycythemia and splenomegaly at 12 months, although no leukemic phenotype was observed. The findings suggested that HCK amplification coupled with PTPRT loss in del(20q) leads to development of a myeloproliferative phenotype. It is associated with myeloid malignancies, detected in 10% of myeloproliferative neoplasms (MPN)2, 4–5% of myelodysplastic syndromes (MDS)3, and 1–2% of acute myeloid leukemia (AML)4. Del(20q) can result from a simple deletion or an unbalanced translocation5. Del(20q) can occur as the sole cytogenetic abnormality or as the first cytogenetic abnormality in a more complex karyotype6.Deletion of the long arm of chromosome 20 [del(20q)] is the second most frequent recurrent chromosomal abnormality in hematological malignancies7. Indeed, mapping analyses of patient samples detected a frequently deleted region on chromosome 20q, named the “Common Deleted Region” (CDR). The CDR approximates 2.6 Mb in MPN samples and 2.7 Mb in MDS and AML samples, with an overlapping CDR region of 1.7 Mb2. Aziz and colleagues found that loss of L3MBTL1 and SGK2, two imprinted genes located in the CDR, resulted in hyperplasia of early erythroid progenitors but did not exclude a contribution from other genes located in the CDR to the del(20q) phenotype8.The role of del(20q) as a primary recurrent cytogenetic abnormality in myeloid malignancies led to the hypothesis that one or more “myeloid” tumour suppressor genes (TSG) exist on chromosome 20q12. The intracellular segment of PTPRT contains its catalytic region13, with the juxtamembrane domain followed by two phosphatase domains. The phosphatase D1 is responsible for the phosphatase activity, while the “pseudophosphatase” D2 is thought to modulate D117. Recent studies demonstrated PTPRT’s role as a TSG in a wide range of human malignancies, including colorectal, lung and gastric cancers19. PTPRT is the most highly mutated tyrosine phosphatase in human colon carcinomas. Wang and colleagues19 examined 175 colorectal cancer samples for somatic mutations, and discovered that PTPRT mutations were the most common amongst 87 tyrosine phosphatase genes analysed. Moreover, PTPRT overexpression in these colorectal cancer cells inhibited their growth. PTPRT knockout mice have also been found to be highly susceptible to azoxymethane induced colon cancer20. Analyses of PTPRT mutations in lung, gastric and breast cancers21 suggested they were loss-of-function mutations resulting in diminished phosphatase activity. PTPRT mutations were shown to be common in head and neck squamous cell carcinomas by Lui and colleagues22. PTPRT mutation was also found in AML. Ley and colleagues used massively parallel sequencing technology to sequence a patient with AML. Eight heterozygous, non-synonymous somatic single nucleotide variants were detected, including PTPRT23.Protein Tyrosine Phosphatase Receptor T (PTPRT) is a recognized TSG residing in the CDR. It is a member of the type IIb protein tyrosine phosphatase family. The extracellular segment of PTPRT contains a meprin-A5 antigen-PTP (MAM) domain, an immunoglobulin-like (Ig-like) domain, and four fibronectin type III (FNIII) repeats. The MAM, Ig domains and FNIII repeats are involved in cell adhesion5. In addition to the CDR, these samples were demonstrated to have a “Common Retained Region” (CRR) at 20q11.224. Further definition of the CRR using Fluorescence In Situ Hybridisation and array Comparative Genomic Hybridisation discovered tandem sequence amplification in five cases5. A 250 kb region represented the region of shortest DNA sequence with greatest amplification, up to 9 copies in one patient5. The CRR contained four complete genes of which the most likely candidate oncogene was the Src family protein tyrosine kinase Hemopoietic cell kinase (HCK). HCK has been implicated in AML, chronic myeloid leukemia (CML) and acute lymphoblastic leukemia25. HCK was found to be overactivated in Philadelphia positive murine myeloid cell line27 and there is evidence that HCK is required for BCR-ABL1 induced oncogenesis28. Pene-Dumitrescu and colleagues showed that HCK overexpression was sufficient to induce imatinib resistance in CML cells, apparently by direct phosphorylation of BCR-ABL1 by HCK29. HCK could also transmit antiapoptotic signals in CML, via BCR-ABL1 and STAT5. These data supported a role for HCK in BCR-ABL1-induced proliferation and survival30. Increased HCK expression and activation was also found in accelerated phase and blastic phase CML32. Furthermore, HCK has been shown to contribute to AML proliferation and survival. Dos Santos and colleagues reported that AML samples often co-expressed multiple activated Src family kinases, with most expressing both LYN and HCK. They also demonstrated that siRNA knockdown of HCK inhibited AML progenitor survival and proliferation. Lastly, HCK has also been shown to be highly differentially expressed in human primary AML stem cells compared to normal hemopoietic stem cells33, linking its presence in AML progenitors to recurrence of the disease.We have reported on our analysis of patients with del(20q) MDS and AML using multicolour banding34 by dephosphorylation, while HCK phosphorylated and activated STAT335.Many oncoproteins have been identified to be the endogenous substrates of PTPRT and HCK. STAT3  is an oncoprotein involved in many hematological malignancies. HCK and PTPRT demonstrate opposing effect on STAT3 activity: PTPRT has been characterised as an important inhibitor of STAT3Therefore, the abnormal chromosome 20 in del(20q) contained two regions of interest – the CDR and the CRR, that harbored the candidate TSG PTPRT, and the candidate oncogene HCK respectively. We hypothesized that PTPRT loss and HCK amplification cooperated to initiate myeloid malignancies, through the STAT3 pathway.PTPRT−/− and wild type (WT) LKS+ haematopoietic stem and progenitor cells (LKS + HSPC) were retrovirally transduced with either HCK or control vector MIGR1 and cultured in methylcellulose media. Colony numbers and size (numbers of cells per colony) were enumerated. Over-expression of HCK only in PRTPRT−/− HSPCs demonstrated significantly higher colony numbers Fig.  and coloWe predicted that over-expression of HCK would lead to hyperphosphorylation of the second messenger STAT3 and this effect would be maximal in PTPRT−/− HSPCs as phosphoSTAT3 is a physiological substrate of PTPRT. The intracellular phosphoSTAT3 was quantified by flow cytometry. Figure in vivo by reconstituting sub-lethally irradiated CD45.1+ murine recipients with LKS+ HSPC from PTPRT−/− and WT mice retrovirally transduced with either HCK or MIGR1 control virus. All recipients were monitored up for 12 months and then culled to assess for features of abnormal hematopoiesis or leukemia. While PTPRT/HCK HSPC murine recipients did not develop overt myeloid leukemia, five out of nine recipients developed splenomegaly with the remaining four recipients demonstrating normal splenic weights . Figure Bone marrow myeloid maturation was resolved using Gr1 and CD11b expression into immature (Gr1 dim) and mature (Gr1 bright) granulocyte maturation. There was no difference between PTPRT−/− HCK-transduced HSPC recipients and other groups Fig. .Morphology assessment of the bone marrow trephine paraffin sections after haematoxylin-eosin stain and TER119 immunohistochemistry showed that, despite the aberrant erythroid maturation in PTPRT/HCK HSPC recipients, there was no alteration in the overall bone marrow cellularity and normal trilineage haematopoiesis observed without evidence of leukemia, again indicating that the aberration is with maturation, not with the quantity Fig. .Mice were analysed at 12 months. Lineage negative cells were resolved as LKS+ and LKS- populations. There were no differences in the proportion of LKS+ cells between the PTPRT−/− HSPCs transduced with HCK and other recipients Fig. . LKS- prSince the primary murine recipients of PTPRT−/− HCK-transduced HSPCs demonstrated aberrant bone marrow erythropoiesis, splenomegaly and polycythemia without overt leukemia, BM HSPCs from the four cohorts were used to reconstitute lethally-irradiated secondary recipients. Two primary recipients of PTPRT−/− HSPCs transduced with either HCK or MIGR1 control at 7 months of age were used to harvest whole bone marrow cells for transplantation. The secondary recipients (three for each cohort) were monitored and then culled after 8 months. The analyses were identical to those of the primary recipients.Similar to the primary recipients, the examination of blood film, full blood counts, bone marrows and spleens did not demonstrate evidence of acute leukemia in the secondary recipients (data not shown). One out of three recipients of PTPRT−/− HSCs transduced with either HCK or MIGR1 control developed splenomegaly Fig. . Althougin vitro evidence of signalling cooperation to result in proliferative advantage. In addition, we characterized the capacity of HCK amplification to lead to leukemia or MPN by reconstitution of mice with HSPCs overexpressing HCK.Amplification of HCK and deletion of PTPRT are recurrent abnormalities in AML with 20q deletion. One possible mechanism of cooperation in oncogenic signalling is the hyperphosphorylation of STAT3 by HCK particularly in the absence of PTPRT, which serves as an endogenous brake by its capacity to dephosphorylate phospho-STAT3. We utilized retroviral transduction of HCK in PTPRT−/− HSPCs to provide There have been other published data that support our findings regarding a role for HCK and PTPRT in oncogenesis. HCK has been implicated in myeloid malignancies. In chronic myeloid leukemia, BCR-ABL1 activates several pro-survival substrates, including HCK, supporting a role for HCK in BCR-ABL1-induced proliferation and survival. Evidence suggests that BCR-ABL1 induced oncogenesis might require HCK, and HCK overexpression has been shown to be sufficient to induce imatinib resistance in CML. Increased expression and activation of HCK was also reported in accelerated phase or blastic phase CML. HCK has also been shown to contribute to AML proliferation and survival, as siRNA knockdown of HCK inhibited AML progenitor survival and proliferation. HCK has also been found to be highly differentially expressed in human primary leukemic stem cells compared with normal hematopoietic stem cells. The obvious importance of this finding is the contribution of AML progenitors to recurrence of the disease. Similarly, PTPRT mutation was also detected in AML using massively parallel sequencing technology to sequence the genomic DNA of both tumor and normal skin cells from a patient with AML. Many oncoproteins have been shown to be endogenous substrates of PTPRT, including STAT3. PTPRT has been characterised as an important direct inhibitor of STAT3, and aberrant STAT3 activation has been detected in a variety of hematopoietic malignancies such as AML.In this study, overexpression of HCK was associated with increased STAT3 phosphorylation in HSPCs containing deletion of PTPRT. It was interesting to note that deletion of PTPRT alone was not adequate to cause hyperphosphorylation of STAT3 by endogenous murine HCK. These data correlated with alterations in colony numbers and size with significant increase noted only with over-expression of HCK in PTPRT−/− HSPCs and without proliferative advantage with over-expression of HCK in WT HSPCs that contain functional PTPRT. This raises the possibility that a double hit of HCK amplification and PTPRT loss is essential for providing proliferative advantage.Our evidence demonstrates that PTPRT−/− HSPCs over-expressing HCK were hyperproliferative when cultured in methylcellulose media compared with colony numbers derived from both PTPRT−/− HSC with MIGR1 vector and WT HSCs over-expressing HCK. This correlated with higher intracellular phosphoSTAT3 in PTPRT−/− HSCs with HCK overexpression and is noteworthy because it represented hyperactivation of a well-recognised leukemogenic pathway. Therefore, an aberrantly activated STAT3 as a result of HCK amplification and PTPRT loss could be postulated as the driver mechanism for the myeloproliferative/leukemic phenotype noted in some del(20q) myeloid malignancies.in vivo reconstitution experiment however did not recreate the phenotype of leukemia as seen in those patients with del(20q) who were initially studied to identify evidence of HCK amplification. Leukemia was not noted even in mice reconstituted with PTPRT−/− HSPCs overexpressing HCK even after 12 month post primary transplantation and after secondary transplantation. However, five out of nine mice did exhibit an interesting phenotype of abnormally high bone marrow immature nucleated erythroid cells, splenomegaly and polycythemia. The three characteristics appeared to be strongly correlated and supports the hypothesis that HCK amplification on the background of PTPRT depletion had directly resulted in aberrant bone marrow erythroid development, which subsequently led to the establishment of polycythemia and splenomegaly. These findings resembled signs associated with MPN, specifically polycythemia vera (PV), a myeloid malignancy that is most frequently associated with del(20q). Interestingly, two out of the five del(20q) patients that were examined to map out the CRR by MacKinnon and colleagues had erythroleukemia(5), however, this was not observed in the mice.Our There are some caveats to our findings. Firstly, in the affected PTPRT/HCK HSPC recipients, the myeloproliferative phenotype was incomplete – the bone marrow cellularity and hematopoiesis were normal, and the bone marrow erythroid population was not increased. Secondly, the phenotype was not fully penetrant, with five out of nine PTPRT/HCK HSPC recipients developing the aberrant features. Finally, the decreasing percentage of peripheral GFP positive cells suggested that PTPRT−/− HCK-overexpression did not confer a survival advantage to HSPCs. This posed an interesting question as to the mechanisms behind these myeloproliferative changes. Although outside the scope of our study, several mechanisms have been proposed: firstly, autocrine or paracrine cytokine production by the PTPRT/HCK HSC could have contributed to these changes. These cytokines could have an effect on the neighbouring non-GFP positive cells by promoting an immature erythroid development. Secondly, the bone marrow microenvironment played an important role in the maturation of hematopoietic cells. Whether some of the PTPRT/HCK HSC transformed into support cells in the microenvironment or influenced the non-hematopoietic bone marrow microenvironment cells to alter their support potential and thereby had an influence on the differentiation of the erythroid population would also be an informative question to answer, but is beyond the scope of this study. Finally, hematological malignancies such as acute myeloid leukemia with 20q loss usually have other relevant copy number abnormalities, hence other mutations are likely needed in addition to our model, explaining the partial penetrance in the mice.There is considerable heterogeneity in human disease with del(20q) as a sole abnormality being recognised as a favorable cytogenetic prognostic marker in MDS. Transformation to leukemia in some del(20q) patients implies the presence of additional events and one of these could be variability in HCK amplification in terms of copy numbers. All mice reconstituted with PTPRT−/− HCK-over-expressing HSPCs revealed hyperphosphorylation of STAT3 but the precise copy numbers of HCK were not ascertained and it is possible that there is a gene dosage effect that is necessary to establish a MPN/leukemia phenotype. Furthermore, we cannot exclude a contribution from other candidate TSG located in the CDR, such as L3MBTL1 and SGK2, to the del(20q) phenotype, as described in other contiguous gene deletion syndromes like the 5q- syndrome.There is variability of phenotypes in JAK2 mutation-containing myeloproliferative neoplasms. PV is the myeloid malignancy that is most frequently associated with del(20q), and is characterized by erythrocytosis. Almost all patients with polycythemia vera have a gain-of-function mutation in the Janus kinase 2 gene (JAK2) with the resultant constitutively activated JAK/STAT signaling pathway causing the MPN phenotype. Allele burden of the JAK2 mutation also has an influence on disease phenotypes seen in polycythemia vera versus essential thrombocythemia, another related MPN in which about 50% of patients would harbor the JAK2 mutation.Possible reasons why the combination of PTPRT depletion and HCK overexpression has an effect only on an erythroid lineage include: Firstly, the level of hyperphosphorylation, and consequent variations in aberrant activation of the STAT3 pathway, might have a crucial role in the phenotype of the murine recipients. Therefore, a higher level of STAT3 hyperphosphorylation and activation might lead to phenotype penetrance on other hematopoietic linages. The second reason could be that the combination of genetic changes and effect of signalling on erythropoiesis via the EPO pathway, may have skewed towards an erythroid phenotype, though this was unable to be delineated in our work.in vitro, and aberrant bone marrow erythroid maturation, polycythemia and splenomegaly in vivo. We surmised that the dysregulation of these two genes in some del(20q) malignancies at least have a contribution in the development of a myeloproliferative neoplasm, such as PV.To summarize, the combination of HCK overexpression and PTPRT loss was associated with some myeloproliferative characteristics such as hyperproliferation and STAT3 upregulation All animal experiments were approved and conducted in accordance with the guidelines of the St. Vincent’s Health Animal Ethics Committee.4 LKS+ HSPC were added to each well. The HSC-Retronectin suspension plate was centrifuged at 400 g for 1.5 hours and then incubated at 37 °C. Cells with either MIG or HCK transduced were sorted by FACS (GFP positive).Ficoll-Paque was used to collect mononuclear cells from C57BL6 wild type (WT) and PTPRT-null (PTPRT) mice and LKS+ hematopoietic stem and progenitor cells (HSPC) were collected by FACS sorting. Retroviral transduction of either MIG control or HCK was performed using Retronectin spinofection. 500 μl of Retronectin (40 μg/ml) was added to four wells  on a 24-well suspension culture plate and incubated overnight at 4 °C. Excess Retronectin was removed, and the Retronectin-coated wells were blocked using 300 μl of PBS + 2% BSA per well. This was incubated at room temperature for 30 minutes. The blocking solution was removed, and the wells were washed twice with PBS. Then either MIG or HCK retroviral supernatant was added to the wells (multiplicity of infection of 10x). The suspension plate was incubated at 37 °C for one hour, then centrifuged 1100 g at room temperature for two hours. The retroviral supernatant from the suspension plate was removed and 2 × 104/ml). 2.5 ml of methylcellulose media was aliquoted into four tubes, one for each cell type, to which 25 μl of cells was added. The media contained recombinant murine SCF and IL-3, as well as recombinant human IL-6. The tubes were mixed and then 1 ml of mixture was plated per 35 mm petri dish. Duplicate plates and an extra 35 mm dish with sterile PBS (maintain moisture) were also placed in the 100 mm petri dish and incubated at 37 °C. The colonies (>50 cells) were counted at day 7 and 12. The total number of cells per dish were also enumerated by dissolving the methylcellulose media using PBS and counting the cells.WT/MIG HSC, WT/HCK HSC, PTPRT/MIG HSC, and PTPRT/HCK HSC were resuspended in PBS to 50 cells/μL . The cell suspensions were added to the cytocentrifuge, and spun at 600 rpm for 10 minutes. The cells were then transferred to the slides in the chambers. The cytospin slides were removed from the cytocentrifuge and allowed to dry. They were stained with hemotoxylin and eosin and analyzed by light microscopy with regards to their morphology.Dataset 1, 2


Saccharomyces cerevisiae genome to a commercial wine yeast can generate novel phenotypes ranging from wine flavor and aroma diversity to improvements in targeted fermentation traits. In the current study we utilized a novel approach to screen isolates from an evolving population for increased fitness in a S. cerevisiae × S. uvarum interspecific hybrid previously generated to incorporate the targeted phenotype of lower volatile acidity production. Sequential grape-juice fermentations provided a selective environment from which to screen isolates. Chromosomal markers were used in a novel approach to identify isolates with potential increased fitness. A strain with increased fitness relative to its parents was isolated from an early timepoint in the evolving population, thereby minimizing the risk of introducing collateral mutations and potentially undesirable phenotypes. The evolved strain retained the desirable fermentation trait of reduced volatile acidity production, along with other winemaking traits of importance while exhibiting improved fermentation kinetics.Wine yeast breeding programs utilizing interspecific hybridization deliver cost-effective tools to winemakers looking to differentiate their wines through the development of new wine styles. The addition of a non- An increasingly competitive global market requires winemakers to minimize production costs and target market niches by differentiating their wines through, for example, development of novel wine styles. One way of achieving these ends is to generate new yeast strains with improved fermentation traits and/or novel phenotypes that shape wine flavor and aroma. Such yeasts provide winemakers with tools that are readily and easily introduced into the winery without incurring additional costs or requiring processing interventions.Saccharomyces cerevisiae. However, more recently the potential to bring a higher level of phenotypic diversity into wine yeast by hybridization with non-S. cerevisiae species of the Saccharomyces clade has been realized. This has led to the generation of interspecific hybrids for use in a range of beverage industries, particularly brewing  with a commercial S. cerevisiae wine yeast strain (Saccharomyces species (Saccharomyces mikatae) as a genetic contributor generated hybrid strains capable of producing novel, not previously recognized, flavor-active metabolites in wines (S. cerevisiae × S. bayanus (var. S. uvarum) interspecific hybrids  showed exceptional promise in terms of what it could bring to wine quality  and spores from Saccharomyces uvarum strain AWRI 1176 as described in MATa, BY4743  and 53-7 .Selection was performed in filter-sterilized Chardonnay juice fermentations : total sugars (glucose and fructose) 225 g/L, yeast assimilable nitrogen 226 mg/L, pH 3.39. This juice was supplemented with the 300 mg/L di-ammonium phosphate. Yeast strain AWRI 1572 was pre-cultured in YEPD medium  for 2 days with an incubation temperature of 22°C and agitation rate of 150 rpm. The cells were then acclimatized to the higher sugar concentration of grape juice by 2 days of growth in ½ X Chardonnay juice (diluted with sterile water) before being inoculated from a cell density of 2 × 10eviously . At comp® micromanipulator was used to pick 20 individual cells from each replicate fermentation culture onto YEPD plates , which were then incubated at 22°C until clonal colonies had formed. PCR-RFLP analysis on genomic DNA prepared from clonal colonies was performed using species-specific markers targeting each arm of the 16 Saccharomyces spp. chromosomes as previously described (Table 1) using S. cerevisiae S288C and S. uvarum (formerly bayanus) MCYC 623 sequences. Primer locations for each set of primers were assessed against work reported on S. cerevisiae and S. uvarum genomes  instrument equipped with a 150 milliwatt DPSS laser emitting at 488 nm. Cells were detected at 583/26 nm using a Yellow B PMT filter with a flow rate of 7 μl/min and fluorescently plotted to a linear scale. Five thousand cells per sample were analyzed to obtain cell DNA intensities. Analysis was undertaken on six biological replicates of control ploidy strains and ancestral hybrid AWRI 1572 and 60 isolates from each fermentation series. Duplicate fluorescent readings were taken of all samples.Colonies from YEPD plates were inoculated into liquid YEPD medium. Cells were harvested after 5 days of growth at 22°C and prepared in triplicate using a propidium iodide staining protocol for FACs analysis as described in TM weighing meter) as a measure of CO2 egress from fermentation vessels.Fermentations were conducted in two Chardonnay juices: the original Chardonnay juice containing 225 g/L reducing sugars, and the same juice supplemented with an addition of glucose and fructose, increasing the total reducing sugars concentration to 350 g/L. Fermentations were carried out in triplicate under conditions previously described  with a s8 cells per ml in liquid YEPD for 2 days at 22°C and 5 μl of 10-fold serial dilutions spotted to plates and then further incubated at 22°C until sufficient colony growth could be observed.Ethanol tolerance of yeast strains was determined by plating onto YEPD agar medium containing a range of ethanol concentrations . Plates were prepared when YEPD (plus 2% agar) medium was cooled to 50°C by the addition of a requisite volume of absolute ethanol. Strains were pre-cultured to a cell density of 2 × 10Wines were analyzed using HPLC to determine concentrations of organic acids , residual sugars, glycerol, and ethanol as described previously .t-test (p < 0.5) was used to determine differences between wines produced by different yeast strains.A Student’s 6 cells/ml . At the end of each fermentation single cells were harvested. Chromosomal compositions determined by PCR-RFLP revealed that loss of S. uvarum Chromosome 14 was the overriding chromosomal alteration occurring during the fermentation series .In order to generate an evolved hybrid strain with increased fitness in a fermentation context, consecutive grape juice batch fermentations were undertaken. Triplicate fermentations of Chardonnay grape juice inoculated with AWRI 1572 were serially passaged four times with cells harvested from each end-of-fermentation seeded into the next ferment at a rate of 2 × 10th isolate  . Additional markers for Chromosome 14; 14ML , 14LC , and 14RC  (Table 1) were designed and used to confirm loss along the entire S. uvarum chromosome. The frequency of loss continued to increase in the Stage 3 ferments with 12 isolates of 20 from Replicate A, all 20 from Replicate B, and 18 of 20 from Replicate C having lost the complete S. uvarum Chromosome 14 , culminating at Stage 5 with complete S. uvarum Chromosome 14 loss in 57 isolates while the remaining three isolates (two from Replicate A and one from Replicate C) showed partial loss, retaining the right arm .Analysis of cells harvested from Stage 2 fermentation series revealed a level of Fluorescence flow cytometry analysis was used to confirm that fitness improvements of hybrid isolates were not due to a major change in ploidy. While this analysis is not sufficiently sensitive to distinguish differences in DNA content arising from loss of a single chromosome in a triploid background, it can be used to determine larger scale changes.0 peaks showed diploid and tetraploid strain fluorescent levels approximately double or quadruple that of the control haploid strain respectively, while hybrid strain AWRI 1572 gave a G0 fluorescent peak level midway between diploid and tetraploid intensities. Fluorescence cell flow cytometry showed no discernible difference in ploidy status between the triploid ancestral hybrid strain AWRI 1572 and isolates  from the Stage 1 ferment  and while isolates from Stage 3 and Stage 5 ferments showed greater diversity of DNA intensities, no isolates showed a gain or loss of complete ploidy level.Fluorescence peak intensities for non-dividing GS. uvarum Chromosome 14 in the evolved hybrid strain had not compromised fermentation proficiency. One isolate, AWRI 2530, was chosen from Stage 3 that showed only loss of S. uvarum Chromosome 14 with an apparent 3n ploidy equivalent to the original hybrid strain.Fermentations in Chardonnay juice were undertaken to establish that the loss of S. cerevisiae AWRI 838, S. uvarum AWRI 1176) and hybrid strains  were evaluated in two juices: the original Chardonnay juice containing 225 g/L reducing sugars, and a high-sugar juice fermentation with an addition of glucose and fructose to the Chardonnay juice increasing the total reducing sugars concentration to 350 g/L. The evolved hybrid strain (AWRI 2530) fermented at much faster rate than the original hybrid strain (AWRI 1572) in both fermentations with only a slightly reduced rate relative to the S. cerevisiae wine yeast parent strain, AWRI 838, in the 225 g/L sugar juice  while matching the fermentation kinetics of the S. cerevisiae parent strain in the high-sugar ferment . Isolates with only S. uvarum Chromosome 14 loss from each independently-evolving adaptive evolution replicate have been tested for fermentation efficiency. All of these isolates performed similarly  showed the slowest fermentation rate in both fermentations and chemical analysis of the final wines established that this yeast was unable to complete fermentation in the 225 g/L sugar Chardonnay juice with a residual fructose concentration of 30g/L fermentation. Similar residual sugar levels were observed for the S. cerevisiae wine yeast parent strain and both hybrid strains in the high-sugar (350 g/L) ferments, but again, the S. uvarum parent strain was unable to utilize sugars to the same degree. Importantly, chemical analysis of the wines confirmed that the evolved strain AWRI 2530 had retained the desirable low-acetic acid production phenotype for which the ancestral hybrid strain was generated .To verify that the important fermentation trait of high ethanol tolerance was retained in the evolved hybrid strain, growth on high ethanol medium was performed. Assay plating confirmed that the evolved strain AWRI 2530 retained the high ethanol tolerance trait of ancestral hybrid strain AWRI 1572, with tolerance slightly higher than the original A competitive growth assay was carried out in Chardonnay juice to validate the improved fitness status of the evolved isolate relative to the ancestral hybrid strain in a fermentation context.6 cells/ml) of AWRI 1572 and AWRI 2530 were then co-inoculated into triplicate, full strength Chardonnay juice. At fermentation completion, cells were then passaged into a second stage ferment at 2 × 106 cells/ml.A co-fermentation of AWRI 1572 and AWRI 2530 was conducted in triplicate using Chardonnay juice. Both strains were subcultured individually in YEPD for 2 days and then acclimatized to Chardonnay grape juice by growth in 1/2X juice for 2 days. Equal numbers of cells . One hundred cells from each fermentation medium were picked to YPD plates using a Singer manipulator.Figure 7) with replicate ferments having AWRI 2530 populations ranging from 86 to 94%. However, only a slight growth advantage was observed during first ferment as AWRI 2530 cell numbers increased to a final proportion of only 55% from the initial inoculum of 50%.Colonies were identified through PCR-RFLP species specific marker (14L) using primers targeting the left arm of Chromosome 14 which was missing in the evolved hybrid (AWRI 2530). Analysis of cells harvested from the final stage of the competition fermentation revealed that AWRI 2530 had out-competed the ancestral strain and cell numbers had risen to 90% of the population  during the experiment, separate ferments where undertaken in parallel in which this strain was the sole yeast inoculated into the grape must. PCR-RFLP analysis of 100 clones from each triplicate AWRI 1572-solo ferment confirmed that the ancestral strain AWRI 1572 was stable with respect to S. uvarum Chromosome 14 in the context of these two sequential Chardonnay juice fermentations  round ferment revealed that chromosomal mutations in S. uvarum Chromosome 14 occurred in all isolates analyzed: 95% of isolates lost the entire chromosome while 5% still retained the right arm.Hybrid isolates recovered from the final  and the frequency of this karyotype increased to 100% over subsequent passaging steps. A possible reason for this was that loss of S. uvarum Chromosome 14 leads to increased fitness in this interspecific hybrid.While minor Chromosome 14 instability was identified in isolates from the first stage fermentation, by the completion of the second fermentation stage it was evident that S. uvarum chromosomes containing translocations can be attributed to marker location  a number of unmatched losses were detected in marker 16R from the Stage 3 ferment. Interestingly, no loss of marker 16R was identified in cells harvested from the final stage of adaptive evolution and perhaps loss of the right arm of S. uvarum Chromosome 16 resulted in decreased fitness relative to the rest of the population. Given that the frequency of S. uvarum Chromosome 14 loss was 100% by the end of the adaptive evolution series (95% showing loss of the entire chromosome), and that an evolved strain with only loss of S. uvarum Chromosome 14 identified displayed increased fitness relative to the ancestral hybrid strain, it is unlikely that these other changes, at least in the genetic background of AWRI 1572, and in Chardonnay juice led to increased fitness.Loss of other chromosomal markers during later stages of the fermentation series was also identified  but none of these showed a frequency increase in all replicates over the course of the experiment. While some of the single arm marker loss identified in S. uvarum Chromosome 14 but no other detectable changes in karyotype was chosen for further characterization. A competitive growth assay was carried out in Chardonnay juice to validate the improved fitness status of the evolved isolate relative to the ancestral hybrid strain in a fermentation context. As the first evidence of S. uvarum chromosome 14 genomic instability in the adaptive evolution fermentation experiment occurred in isolates harvested from the end-of-fermentation Stage 2 culture, sampling of the 2nd stage competition ferment was undertaken very early in the fermentation (at day 3: it took 14 days for the fermentation to reach completion). Separate ferments with ancestral parent hybrid AWRI 1572 alone were used to establish that Chromosome 14 instability was not an issue for the duration of the competition experiment. Serial growth competition in Chardonnay juice between the original hybrid (AWRI 1572) and the above isolate demonstrated that loss of S. uvarum Chromosome 14 is likely to contribute to the observed increase in fitness, but we cannot exclude the possibility that other mutations contributed to this phenotype.An isolate (AWRI 2530) with loss of S. cerevisiae × S. kudriavzevii hybrid showed a loss of DNA content, stabilizing at a level similar to triploid when undergoing fermentation stresses (S. cerevisae × S. bayanus hybrids increased their DNA content by 60% (approximating triploid ploidy levels) after 50–80 vegetative generations . Interestingly, other studies describing the stabilization of synthetic polyploid interspecific hybrids have reported ploidy stabilization to triploid levels; a tetraploid stresses , while ierations .S. uvarum Chromosome 14 loss from each independently-evolving adaptive evolution replicate have been tested for fermentation efficiency. All of these isolates performed similarly  and genomic rearrangements  brings even greater novelty to an organism, enhancing genetic and biochemical flexibility relative to the parents. On the other hand, plant studies have shown that interaction between different genomes with inherent incompatibilities can lead to genomic instability with alterations such as chromosomal losses, translocations, gene repetitions, and silencing . Studies regions , while c regions . In addingements .Brassica interspecific hybrids have shown that rapid and extensive genomic changes can occur within five generations of hybridization and that a relationship exists between frequency of change and divergence of parental genomes , has been proposed to explain the roles of changing clonal population during yeast fermentation . This moS. cerevisiae × S. uvarum diploid hybrids exposed to nitrogen limiting conditions has been reported earlier by MEP2 (a high-affinity ammonium permease) occurred, resulted in chimeric chromosomes each carrying a MEP2 fusion gene. This chromosomal rearrangement also increased hybrid fitness, allowing evolved hybrid strains to grow faster under nitrogen-limitation than ancestral hybrids. An evolutionary study involving a different alloploid interspecific hybrid strain, Saccharomyces pastorianus, also showed instability in Chromosome 14 with loss of S. cerevisiae right arm copy number  had retained the S. uvarum Chromosome 14, two S. cerevisiae × S. kudriavzevii natural wine yeast hybrids (Enoferm Assmunshansen and Maurivin EP2) lost the non- S. cerevisiae Chromosome 14 .Recently, genomic sequencing of a small number of natural wine yeast hybrids was reported  and, whiS. uvarum Chromosome 14 was identified by the chromosomal marker system used, with only six isolates from a total of 240 revealing partial alteration. This could mean that partial loss of Chromosome 14 is rare, fundamentally unstable, or leads to decreased fitness.In the current study, little evidence of partial loss of S. uvarum Chromosome 14 may, for example, have impacted on acclimatization to fresh medium leading to a decreased lag phase, faster growth in exponential phase, increased cell population at stationary phase, the ability to tolerate stresses such high sugar and high ethanol concentrations or the ability to uptake and metabolize sugars at a faster rate. While there is high DNA sequence divergence between S. cerevisiae and S. uvarum  S. cerereported  and therWe report the successful generation of an evolved interspecific wine yeast hybrid with increased fitness in a fermentation context relative to the ancestral hybrid strain. This was achieved using a novel screening approach that utilized chromosomal mutations as markers for the trait of interest. The evolved hybrid strain retained the targeted fermentation trait of reduced volatile acidity production while exhibiting improved fermentation kinetics. The chromosomal marker system employed allowed the pattern of genomic plasticity that arose during the evolution of the interspecific hybrid to be exposed and future work on individual isolates from the evolution study may reveal information about genomic alterations that lead to interspecific yeast hybrid stabilization.JB designed and undertook all the experimental work, interpreted the data, and wrote the manuscript. CF, AB, and PC assisted in experimental design, supervised experimental work, helped to evaluate, and edit the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Heterotrimeric G protein, composed of Gα, Gβ, and Gγ subunits, modulates plant adaptations to environmental stresses such as high salinity, drought, extreme temperatures and high light intensity. Most of these evidence were however derived solely from conventional genetics methods with which stress-associated phenotypes were compared between wild type and various G protein mutant plants. Recent advances in systematic approaches, mainly transcriptome and proteome, have contributed to in-depth understanding of molecular linkages between G proteins and environmental changes. Here, we update our knowledge on the roles of G proteins in abiotic stress responses. Furthermore, we highlight the current whole genome studies and integrated omics approach to better understand the fundamental G protein functions involved in abiotic stress responses. It is our purpose here to bridge the gap between molecular mechanisms in G protein science and stress biology and pave the way toward crop improvement researches in the future. Plants experience frequent changes in their growth environments which impede or alter their normal development. Environmental conditions include biotic stresses such as pathogen infection and abiotic stresses such as drought, high salinity, heat, cold, excessive light, high ultraviolet B (UVB) radiation, nutrient deficiency and accumulation of toxic metals in the soil. Due to the increased frequency of extreme weather and climate change in recent years, the adverse effects from those abiotic stresses have been accelerated in plants Zhu, . As a seGCR1)  and forms an inactive complex with an obligate Gβγ dimer. Upon nucleotide exchange on Gα for a guanosine triphosphate (GTP), GTP-bound Gα dissociates from Gβγ then modulates the activity of downstream signaling proteins , three XLG , one Gβ (AGB1) and three Gγ  genes. Genetic ablation of some G protein genes confers various anomalous morphologies including leaf and flower shape, hypocotyl elongation and root mass and architecture. G protein mutations also alter the sensitivity to growth hormones including auxin, gibberellic acid (GA) and brassinosteroid (BR). In general, Gα mutants are hyposensitive to auxin, GA and BR, while Gβ mutants are hypersensitive to auxin but hyposensitive to GA and BR  mutants exhibit a higher photosynthetic rate, root to shoot ratio and greater stomatal conductance under drought stress -induced inhibition of stomatal opening and promotion of stomatal closure, suggesting that GCR1 and GPA1 have an opposite effect in ABA signaling in guard cells  suppresses plant growth and reduces net photosynthesis, which is considered to cause the reduction of crop yield by 5–15% annually  generation that causes stomatal closure  mutants and nia1-2/nia2-5 (defect in NO production) mutants, Gα is further confirmed to act as an upstream positive regulator of H2O2-dependent NO production in in UVB induced stomata closure  gene expression. The expression of APX2 gene was 3–5 fold higher in gpa1 and agb1 mutants as compared to WT when grown under high light  condition and consequently absorb more light energy than required for photosynthesis, therefore the excess excitation energy has to be dissipated Baker, . In ArabImmunoglobulin-Binding Protein 3 (BiP3), Protein Disulfide Isomerase (PDB) and several other components comprising a protein-folding machinery in ER. Wang et al. proposed that Arabidopsis agb1-2 mutation ameliorates leaf senescence under tunicamycin (Tm) treatment, possibly due to reduced expression levels of BiP3 and PDB transcripts   protein COLD1 interacts with RGA1 and slightly accelerates its GTPase activity to activate Cas Figure .Studies on single gene or protein are insufficient to reveal the correlation or the crosstalk between different stress signaling pathways as stress response is a dynamic rather than a static cellular process within a single organism and across different organisms. Moreover, abiotic stress responses in plants involve multiple signaling processes coordinately. Omics studies have the highest potential to uncover the spatiotemporal signaling events for plant stress responses. Various systems biology approaches, for example, transcriptomics, metabolomics, proteomics and interactome have been employed to understand the G protein signaling in abiotic stress responses. These systematic methodologies allow us to not only discover new gene functions in a complex cellular environment, but also characterize genome-scale relationships between genotypes and phenotypes deeply. In this chapter, we discuss established and cutting-edge techniques of OMICs approaches which are applicable to G protein studies.gpa1 and gcr1 mutations dramatically alter the expression pattern of abiotic stress-related genes, mainly genes related to transcription factors, secondary metabolism and hormone responses -based quantitative proteomics approach -MS based interactome was established to detect the time- and glucose- dependent RGS1 interacting networks  based interactome studies identified hundreds of G protein-interacting partners (Klopffleisch et al., The above-mentioned systematic approaches greatly improved our knowledge in plant abiotic stress responses and the involvement of G protein pathways, however the application of these advanced approaches is still limited to specific cases such as signals evoked by ABA or some stress treatments. With the importance and complexity of G proteins-by-environment relationship, more profound and broader studies are required. This section describes novel approaches that have yet to be applied to plant G protein signaling.Given the rapid development of OMICs tools, an increasing number of researches are utilizing these techniques to construct comprehensive visualization of stress responses in plants. These techniques enable detection and quantification of dynamic intracellular changes from gene expressions to post-translational modifications of proteins during the course of stress response and development. For example, Geng et al captured spatiotemporal transcriptional changes in different cell types during Arabidopsis root development under salt treatment (Geng et al., ChIP-sequencing identifies binding targets of multiple TFs and their directly regulatory genes. Through integrated-TF networks of ABA-induced drought response, Song et al identified 21 ABA-related TFs and novel genome-wide binding sites in Arabidopsis (Song et al., In addition, ChIP-sequencing is capable of detecting chromatin dynamics and variations. Environmental factors induce epigenetic changes such as chromatin modification, which are tightly correlated with transcriptional response. For example, drought, flooding, temperature fluctuation and high salinity affect the methylation or acetylation of DNA and histones (Pandey et al., snrk2 mutants and WT, Umezawa et al identified new direct substrates of ABA-activated SNRK2 (Umezawa et al., 2+ signal-related proteins and HSPs (Rampitsch and Bykova, 2+ signaling pathways. Future studies should investigate the whole genome PTM status in G protein mutants in comparison to WT under various stresses.PTM, particularly phosphorylation, participates in the signal transduction in abiotic stress conditions. Several quantitative phosphoproteomes of drought stressed plants (Umezawa et al., E. coli. This model is trained based on multi-omics and interaction data and then predicts multi-omics expression under untested novel conditions. As a result, it could precisely predict and integrate different layers of OMICs data and could be broadly applicable for biological discovery (Kim et al., As discussed, OMICs data are one of the higher order dimensional data with complex multi-level structures, and therefore they have become promising input data for machine learning and deep learning approaches for analysis and interpretation of biological data. Indeed, machine learning has been implemented in abiotic stress research recently (Ma et al., Evidence to date leave no doubt that G proteins are involved in abiotic stress response in many plants. However, to access the in-depth knowledge on how G proteins are involved in stress response, more genome-wide analyses are still needed. As discussed, the methodologies and experimental designs in systems biology help us answer the complex questions in abiotic stress responses Figure . We coulStress response is by no means a steady-state response and could be well-explained by the changes captured in just a few time points under stress treatment. However, stress responses in plants are extremely dynamic along the time. Consequently, the time resolution of exact kinetic changes in G proteins and their regulated genes after stress signal perception would be an interesting topic in the future.Previous studies suggested that G proteins acquired different specificities in different cell types in response to ABA. Plants also show different responses to various abiotic stresses in different cell types. Hence, it would be interesting to elucidate G protein regulatory network in distinct cell types in different developmental stages under stress condition. Besides, there are several public databases regarding gene expression patterns in different root or seed cell types along the course of development. By comparing gene expressions changed by abiotic stresses and G proteins using the publicly-available data, we might gain insight into the common and distinct regulatory modules associated with G protein signaling under stress conditions.By analyzing the expression patterns of G protein genes from public data, it is clear that the expression levels of G protein genes are relatively consistent, only exhibiting some slight changes under certain conditions Figure . While mIt has been a long-standing question how plants sense different stress conditions and activate different stress response genes. Abiotic stress responses are mediated by several signaling pathways: some are shared by different stresses while others are unique to a specific stress type. G proteins are involved in many stress tolerance, but little is known about their selectivity. A possible strategy adopted by plants would be switching interacting partners based on the type of stresses. More specifically, G proteins may form a protein complex with different regulatory partners upon different types of stresses. Interactome or proteome experiments could be established to determine the protein components associated with G protein complex under different stress conditions.To date the evidence show that part of the G protein response to stress is similar in Arabidopsis and several crops including rice and maize (Urano et al., In conclusion, with the development of OMICs tools and integration of massive amounts of quantitative dataset, we are able to understand the molecular and biochemical aspects of G protein regulation in various environmental stresses. The long-term goal is to integrate the outcomes from previous conventional studies and future genome-wide studies to find new stress tolerance gene candidates and mechanisms in plants. The new knowledge will help us to genetically design crops which are able to flourish in changing environment.T-YW and DU contributed to the conception, design and writing of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Here we have compared comprehensively the bone health of clotting factor VIII, factor IX, and Von Willebrand Factor knockout  mice both in the absence of joint hemorrhage and following induced joint injury. We found FVIII−/− and FIX−/− mice, but not VWF−/− mice, developmentally have an osteoporotic phenotype. Unilateral induced hemarthrosis causes further bone damage in both FVIII−/− and FIX−/− mice, but has little effect on VWF−/− bone health, indicating that the FVIII.VWF complex is not required for normal bone remodeling in vivo. To further investigate the bone healing following hemarthrosis in hemophilia we examined a two week time course using microCT, serum chemistry, and histological analysis. Elevated ratio of osteoprotegerin (OPG)/receptor activator of nuclear factor-kappa B ligand (RANKL), increased osterix+ osteoblastic cells, and decreased smoothness of the cortical bone surface were evident within several days of injury, indicative of acute heterotopic mineralization along the cortical surface. This was closely followed by increased interleukin-6 (IL-6) levels, increased osteoclast numbers, and significant trabecular bone loss. Uncoupled and disorganized bone formation and resorption continued for the duration of the study resulting in significant deterioration of the joint. Further elucidation of the shared mechanisms underlying abnormal bone homeostasis in the absence of FVIII or FIX is needed to guide evidence-based approaches to the screening and treatment of the prevalent bone defects in hemophilia A and B.While joint damage is the primary co-morbidity of hemophilia, osteoporosis and osteopenia are also observed. Coagulation factor VIII deficient (FVIII Although the primary crippling, painful symptoms are experienced at the joint articulation, osteopenia and osteoporosis in individuals with hemophilia also are widely documented8. Recent studies of hemophilia A mice suggest that increased bone resorption and low bone mineral density may be associated with factor VIII deficiency even in the absence of gross hemarthrosis10.Hemophilia A and hemophilia B are X-linked recessive inherited bleeding disorders caused by deficient activity of blood coagulation factors VIII (FVIII) and IX (FIX), respectively, resulting in severely deficient thrombin generation. Severe hemophilia is associated with chronic degenerative joint and bone disease12.Tight control of bone structure and phosphocalcic balance is essential to maintain skeletal integrity and depends upon the proper coordination of bone osteoclast and osteoblast activity. Bone remodeling, particularly following injury, can be rapidly modulated by a number of factors including pro-inflammatory mediators present due to injury and the final molecular effectors receptor activator of nuclear factor-kappa B (RANK), RANK ligand (RANKL), and osteoprotegerin (OPG), the natural decoy receptor for RANKL14 and RANKL, and inhibits osteoclastogenesis via interaction with these mediators15. The FVIII.VWF complex may act as an anti-resorption factor both in normal physiology and following hemarthrosis. An increased incidence of low bone mineral density, osteoporosis, or abnormal structural integrity of bone has not been described in humans with VWD.Von Willebrand factor (VWF) is a multimeric protein that is required for platelet adhesion and whose lack results in von Willebrand Disease (VWD), the most common coagulation disorder in humans. VWF also serves an important function as a carrier protein for FVIII in circulation, decreasing the clearance of FVIII from plasma. Recent work has demonstrated that the FVIII.VWF complex associates physically with OPG−/− FIX−/− VWF−/−, respectively, and their WT littermate controls), we compared congenital bone structure as well as remodeling phenotypes following joint hemorrhage. The scope of our studies sought to model the observed clinical association of severe deficient factor VIII and bone abnormalities in males with hemophilia A. Using dual energy X-ray absorptiometry (DXA) and microcomputed tomography (microCT) measurement we found that in the absence of any observed injury or bleeding FVIII−/− mice display significantly low bone mineral density (BMD) and other deficits of trabecular bone integrity. FVIII is a cofactor for the protease factor IXa in the complex that activates factor X, leading to the ultimate amplification of thrombin generation. Therefore we also hoped to dissect whether the absence of the FVIII protein itself, or absence of an intact complex of VWF protein with FVIII protein, were necessary to promote the abnormal bone phenotype previously described in male hemophilia mice and in males clinically. We examined in parallel the phenotype that develops in age-matched male mice that express FVIII but have isolated complete absence of FIX or have isolated complete absence of VWF. Our findings demonstrate that mice congenitally deficient for either factor VIII or factor IX demonstrate similar deficits of BMD and trabecular bone integrity. Further, despite the close physiologic association of VWF with FVIII, we report that male VWF−/− mice do not display this congenital abnormal bone phenotype.Utilizing mouse FVIII, FIX, and VWF gene knockout disease models . The difference in BMD at 16 weeks is 4.54% lower in FIX−/− mice than WT littermates (p = 0.0057). When examined at 22 weeks of age, mean BMD and BMC were lower in FVIII and FIX deficient mice, however the differences from WT littermates were statistically different only for BMC in the FIX−/− mice. Unlike FVIII−/− and FIX−/− mice, VWF−/− mice showed no difference in whole body BMD compared to their WT littermates at any time point  had a 27.8% lower measured vBMD at baseline than WT littermates (p = 0.0057). FIX−/− mice had 15.5% lower vBMD at baseline than WT littermates, in the absence of injury or observed hemorrhage (p = 0.0447). VWF−/− mice did not differ in vBMD from WT littermates, as measured by microCT. MicroCT examination of additional measures of trabecular integrity also demonstrated that significant parallel deficits exist in FVIII−/− and FIX−/− mice. FVIII−/− mice displayed a 17.3% increase in the trabecular space, associated with a 33.4% decrease in trabecular bone connectivity density and a 14.8% decrease in trabecular number. Uninjured FIX−/− mice had similar differences of 18.6%, 26.6%, and 12.7% in these respective parameters, when compared to uninjured WT littermates. VWF−/− mice at skeletal maturity did not differ in measures of trabecular bone health from WT littermates  , it was not significantly elevated compared to normal littermate controls of the WT VWF deficient mice that were either injured or non-injured animals and still falls in the 1 to 0 range considered normal.The histopathological features of hemophilic synovitis following knee joint bleeding challenge were quantified by the Valentino scaleper Fig. . WT hemoely Fig. . In contes) Fig. . Injured−/− transgenic mice appeared minimal. Surface smoothness analysis supported the visual findings, as the smoothness ratio was significantly reduced in the FVIII−/− and FIX−/− groups , but not in the VWF−/− group. The injured limbs from the FVIII−/− and FIX−/− mice each exhibited further deterioration in vBMD versus the contralateral control limbs , while the WT and VWF−/− groups showed no differences. Though there were no phenotypic differences found in trabecular bone thickness, both FVIII−/− and FIX−/− cohorts showed significant differences in the injured limb versus the contralateral uninjured control limb    Fig. . Similar04) Fig. . The hig19, it’s most potent direct action is on osteoclasts as a pro-resorption factor20. At two weeks post-injury we found significant IL-6 elevations in both the FVIII−/− and FIX−/− injury groups in comparison to injured WT littermates, 6.2 and 8.3 fold higher respectively   Fig. . Here we−/− mice using microCT, immunohistochemical staining, calcein incorporation, and serum cytokine assays   Fig. , and ser01) Fig. . Additio22. MicroCT measurements of vBMD and trabecular thickness decline rapidly indicating significant bone loss at day 7 and further decline at day 14 post-injury resulting in a 27% reduction in vBMD and 11% reduction in trabecular thickness compared to non-injured limb. Like the acute heterotopic bone formation, bone resorption persisted throughout the duration of the study.Bone resorption occurred as a second, somewhat overlapping process. Osteoclast numbers, determined by TRAP staining, increase 3–7 days post injury (p < 0.01) Fig. . At 7 da+ osteoblastic cells and bone resorption by TRAP+ osteoclasts associated with a dysregulation between OPG and RANKL levels. The OPG/RANKL ratio therefore signifies the sum of bone remodeling influences at a given time, where increases in the OPG/RANKL ratio indicate a deregulated bone remodeling. Indeed, RANKL plays a pivot role in the bone resorption process by coupling RANKL producing cells  and RANK+ osteoclastic precursors20. RANKL is produced locally and its functional impact is tightly regulated by OPG, a decoy receptor that blocks the binding of RANKL to RANK, disrupts RANK/RANKL signalling and the osteoclastic differentiation/activation20. RANKL is considered as mandatory factor for osteoclastogenesis. Even if there is no clear evidence that the values of circulating OPG and RANK reflect the local production of both factors in bone, previous reports showed a correlation of OPG/RANKL ratio with the severity of bone loss24. The increase of OPG/RANKL ratio may be considered as a homeostatic response to prevent bone loss and consequently to maintain bone mass even if this system may be insufficient. Any influence that uncouples this process can result in an overall change in bone density. IL-6, a pro-inflammatory cytokine released by local inflammatory cells in response to injury, negatively regulates osteoblast differentiation25 and bone resorption27 through osteoblastic production of downstream effectors such as RANKL that activate osteoclasts20.Bone density is determined by a continuous process of coordinated bone formation by osterix30. The independent clinical risk of hemophilia B is more difficult to determine, as most population studies have either not included hemophilia B or have not analysed hemophilia B separately from hemophilia A28. Studies performed by the same group of investigators and using identical methods provide an exception, separately analysing severe hemophilia A and severe hemophilia B populations, as well as a population with combined factor V and VIII deficiency, and demonstrate similar trends in bone outcomes32. A clinical association of low BMD with VWD has never been shown.Low bone mineral density is an increasingly recognized complication in the severe hemophilia population. Multiple epidemiologic studies  document this risk in hemophilia A adult and pediatric populationsin vivo investigation of FVIII−/− mice by two different sets of investigators showed that complete factor VIII deficiency is associated with congenital low bone density phenotype in the absence of injury or observed haemorrhage10. The congenital bone deficits and the abnormal bone remodeling phenotype described by Liel et al. and Lau et al., respectively, were studied only in FVIII−/− mice and WT mice. Therefore, no inference could be made as to whether the abnormal bone homeostasis was mechanistically attributable to a specific property of the factor VIII molecule versus defective hemostasis, inflammation, or some other more global phenomenon33. Aronovitch et al., in contrast, note that thrombin exerts multiple effects on osteoblasts including induction of differentiation and inhibition of apoptosis. They hypothesized that complete absence of factor VIII leads to deficient thrombin generation, resulting in ineffective thrombin-mediated signalling through protease activated receptor 1 (PAR1) and abnormal bone remodeling by PAR1-positive endosteal cells22. In support of this hypothesis, they demonstrated bone structural abnormalities were similar in mice with a complete knockout of either FVIII or of PAR1.Separate 34. VWF also directly binds to FVIII in circulation, protecting it from proteolytic inactivation35. Adhesion of VWF to the subendothelial matrix at sites of vascular damage not only initiates formation of a platelet plug but brings FVIII in proximity to the site of vascular damage to promote thrombin and fibrin generation36. Recently, the FVIII.VWF complex has been shown in in vitro studies to interact directly with OPG to enhance its inhibition of RANKL induced osteoclastogenesis, whereas FVIII alone had no effect on RANKL mediated osteoclastogenesis15. We examined bone homeostasis in two strains of mice with a severe bleeding tendency due to severely deficient thrombin generation (complete knockout of either zymogen factor IX or its cofactor FVIII in the complex that activates factor X). In parallel we examined mice with a severe bleeding tendency resulting from severely deficient platelet function due to knockout of VWF. VWF−/− mice have ~20% of normal circulating factor VIII  which could exacerbate bleeding, but would be expected to support a degree of thrombin generation not present in FIX−/− or FVIII−/− mice39. We hypothesized that VWF−/− mice would exhibit decreased bone density and bone resorption, particularly following injury15.VWF is essential for normal platelet-to-platelet and platelet-to-matrix binding−/− or FVIII−/− mice, as is defective bone remodeling in bones adjacent to joint hemorrhage. We found that serum OPG/RANKL ratios and low IL-6 serum levels after injury initially favour a pro-bone formation phenotype in FVIII−/− and FIX−/− animals. IL-6 has been associated with bone/joint health  in hemophilia mice and and IL-6 signalling blockage by using IL-6 receptor antagonist may be used as an adjunct to replacement hemostasis and an approach to minimize hemophilic joint degeneration40. Accordingly, we saw increases in osteoblast number at 1 day post-injury and roughening of the cortical bone surface by 3 days post-injury. This acute heterotopic mineralization was described by Lau et al., at two weeks post-injury in hemophilic mice; they demonstrated the new material was porous and rough while the underlying original cortical bone maintained its density3. Through time course analysis we report that this new material is indeed calcified and appears in the presence of osteoblasts, implying this is acute heterotopic bone formation. Heterotopic bone formation has also been reported in hemophilia following elective joint replacement41. One week post-injury IL-6 levels shifted dramatically to create a pro-bone resorption environment. Osteoclast numbers elevated and trabecular thickness and vBMD dropped demonstrating significant uncoupled bone resorption particularly within the trabecular cavity. Bone formation and resorption continued in a disorganized fashion for the duration of the study. However, in the absence of observed injury we found no significant difference in BMD  is not pivotal to the bone pathology observed in hemophilia. The findings also must be considered in the context of prior studies that demonstrate low bone mineral density and suggest primary bone pathology in children and young adults with haemophilia49. A limitation of the study that complicates interpretation of bone phenotype changes after injury is that the differential hemorrhage in joints is not quantified across the strains. Specifically, if the VWF−/− do not bleed in this injury model, then the relative contribution of VWF to bone homeostasis after injury cannot be interpreted. The results of both the joint diameter measurements at days 1, 2, 3 and 7 after injury, and also the small but significant change in synovitis score, confirm that the VWF−/− mice experience bleeding. Furthermore, the time course of the VWF−/− soft tissue response to bleeding is qualitatively more like hemophilia mice than WT mice, although quantitatively less great at all time points. This is despite our results suggesting that endpoints of cortical porosity and trabecular volume/structure become abnormal in hemophilia mice with complete deficiency of FVIII or FIX (and a severe defect in amplification of thrombin generation); the same endpoints are maintained in the VWF−/− mice with the severe defect of platelet associated hemostasis (combined with a mild-moderate FVIII deficiency). Our studies cannot rule out the possibility that there may be a threshold effect of bleeding, wherein mild/moderate amounts of joint hemorrhage do not significantly affect bone health until a threshold amount of bleeding is exceeded, after which the events proceed as detailed in our time course  developed diagnostic criteria for osteoporosisrse Fig. . AlternaIn conclusion, mice with complete FIX or FVIII deficiency (but not VWF deficiency) display similarly defective congenital bone homeostasis and bone remodeling following hemarthrosis. Our results are consistent with the hypothesis that thrombin generation and signalling are important for the maintenance of normal bone phenotype. Further study of potential mechanisms of primary bone deficits in hemophilia is needed to guide appropriate screening and treatment guidelines. In addition, the effectiveness of recommended interventions for low BMD/osteoporosis in hemophilia , if prescribed, deserve careful prospective evaluation to establish their effectiveness, in particular if implemented without prophylactic factor replacement to address hemophilia’s primary congenital deficiency.−/− and FIX−/− mice were originally supplied by Dr. H. H. Kazazian Jr50. and by Dr. D.W. Stafford respectively51. VWF+/- mice52 were purchased from Jackson laboratory (USA). Each knockout strain was bred in house and back-crossed 12 generations with C57Bl/6J mice. All the animals used in the experiment were male clotting factor deficient mice or male wild type (WT) littermates of the corresponding transgenic strain. At 22 weeks of age, the mice were subjected to knee joint hemorrhage induced by introducing a 30.5 needle with 5 microliters of saline into the joint space of the left knee as described previously18. The three strains of mice did not produced pups equally on both total numbers and numbers based on gender. The number of animals produced by colonies and used in each experiment exactly matched the real scenario at the time of experiments carried out and describe in the manuscript.All investigations were approved by the University of North Carolina-Chapel Hill Institutional Animal Care and Use Committee and were performed in accordance with relevant guidelines and regulations. FVIII53.At two weeks post-injury mice (n = 4–23 per group) were euthanized for histopathological examination. Mice were examined at time of necropsy for gross signs of hemorrhage. Knee joints were immersion-fixed in 10% neutral buffered formalin, trimmed, processed, sectioned, and stained with hematoxylin and eosin by routine methods. The synovium was evaluated microscopically for histopathological changes which were quantified using a 0–10 scale of murine synovitis, the Valentino scale, consisting of synovial proliferation, hemosiderin staining, villous formation, neoangiogenesis, the presence of blood, and loss of cartilage integrity as previously describedin vivo using DXA. Measurements were collected at 16, 22, and 24 weeks of age using a Lunar PIXImus Densitometer . Animals (n = 26–34 per group) were anesthetized using 2.5% isoflurane prior to and during the measurement at each time point.Bone mineral density (BMD) and content (BMC) were assessed Hind limbs were imaged post mortem with the knee joint intact using microCT with a voxel size of 10 microns . The scan region spanned from the third femoral trochanter through the proximal metaphysis of the tibia. In order to quantify bone surface roughness, the proximal end of the tibia (spanning 1.5 mm) was subjected to a smoothing algorithm to remove any pits and fissures present on the surface due to heterotopic mineralization. This was accomplished by first removing any disconnected elements remaining from the joint structure. The surface of the remaining object was then dilated and subsequently eroded by 10 of voxels to produce the smoothed object. This was done using the Scanco IPL software. The surface area of the smoothed object was used in tandem with the normal, unsmoothed surface area to compute a smoothness ratio to quantify the post-injury surface mineralization. The proximal tibia was further analyzed in a region beginning immediately below the epiphyseal plate extending 1 mm distally. The trabecular bone was isolated using manually-drawn contours. Micro-architectural parameters were then measured using a visually-determined threshold of 500 mg HA/ccm of the maximum grayscale value. Cortical bone measurements were similarly obtained from the mid-diaphysis of the femur, in a volume spanning 0.5 mm axially immediately distal to the third trochanter. The threshold used was visually set at 775 mg HA/ccm of the maximum value. All measurements were obtained using the Scanco analysis software.Serum GM-CSF, IFN-γ, IL-1β, IL-12p70, IL-13, IL-18, IL-2, IL-4, IL-5, IL-6, TNF-α, IL-10, IL-17A, IL-22, IL-23, IL-27, and IL-9 concentrations were assessed by Bio-Plex® system  and RANKL concentrations by using ProcartaPlex kits . OPG, DKK-2, and SOST concentrations were evaluated using Milliplex MAP kits . The procedure was performed according to the manufacturer’s protocols. Each target concentration was calculated using a 5-parameter logistic fit curve generated from the standards.54. Immunostaining of osteoblasts was performed by using a rabbit polyclonal Osterix antibody , as previously described55. All slides were counterstained with Gill2 hematoxylin. Stained sections were automatically numerized  before observation with the NDP view virtual microscope (Hamamatsu). Pictures were saved using tiff format and analysed by ImageJ software . A specific region of interest (ROI) of 0.5 mm beginning from the growth plate and containing most of the primary bone spongiosa was selected. Each staining was analysed to determine both relative length contact of osteoclasts to bone perimeter and osterix+ number (precursors of osteoblasts and mature osteoblasts) to bone perimeter (per millimeter).Tibias collected from euthanized mice were fixed in 4% neutral buffered paraformaldehyde and decalcified in 4.13% EDTA, 0.2% paraformaldehyde pH 7.4, at 50 °C in KOS microwave tissue processor . Decalcified specimens were then dehydrated and embedded in paraffin. Three μm thick sections distant each from 100 µm were carried out. Tartrate-resistant acid phosphatase (TRAP) staining was performed on the tibia sections to identify osteoclasts, as previously describedResults were analyzed using GraphPad Prism 6.0 software . For the cytokine assays, outliers were removed using the ROUTT test, the number of samples removed is indicated in the corresponding figure legend. A one-way analysis of variance with multiple planned comparisons, or a non-parametric two-way analysis of variance  was performed followed by a Sidak post-hoc test.Supplemental Table 1Supplemental Table 2Supplemental Table 3Supplemental Figure 1Supplemental Figure 2