However, long-term use of l-DOPA leads to side effects such as dyskinesias and motor fluctuation. Since purines have neurotransmitter and co-transmitter properties, the function of the purinergic system has been thoroughly studied in the nervous system. Adenosine and adenosine 5′-triphosphate (ATP) are modulators of dopaminergic neurotransmission, neuroinflammatory processes, oxidative stress, excitotoxicity and cell death via purinergic receptor subtypes. Aberrant purinergic receptor signalling can be either the cause or the result of numerous pathological conditions, including neurodegenerative disorders. Many data confirm the involvement of purinergic signalling pathways in PD. Modulation of purinergic receptor subtypes, the activity of ectonucleotidases and ATP transporters could be beneficial in the treatment of PD. We give a brief summary of the background of purinergic signalling focusing on its roles in PD. Possible targets for pharmacological treatment are highlighted.Parkinson’s disease (PD) is the second most common neurodegenerative disorder, characterized by progressive loss of dopaminergic neurons that results in characteristic motor and non-motor symptoms.  The ongoing degeneration of this peculiar pathway causes the characteristic motor symptoms such as resting tremor, rigidity, bradykinesia and postural instability [Parkinson’s disease (PD) is the second most common neurodegenerative disorder, characterized by progressive loss of dopaminergic neurons in the tability , 2. Besitability . In spittability –7. Amongtability . Dysfunctability , 10. Simtability , 12. Thitability . Therefotability , 15.l-3,4 dihydroxyphenylalanine (l-DOPA) [l-DOPA is able to cross the blood–brain barrier and converts into DA that engages specific DA receptor subtypes (D1 to D5) [l-DOPA leads to a dysbalance of striatal circuits of the motor system and leads to side effects such as l-DOPA induced dyskinesias and motor fluctuation in 50% of patients after 5 years of continuous treatment [As for the symptomatic treatment of PD, the clinical break-through came with the first clinical trials of DA replacement therapy using the high dosage of the DA precursor (l-DOPA) –19. l-DO1 to D5) . Howeverreatment , 22. Thereatment .The concept of purinergic signalling, being adenosine 5′-triphosphate (ATP) as an extracellular signalling molecule with neurotransmitter properties was proposed in the early 1970s , 25. A c1, A2A, A2B, A3). P2 receptors are subdivided into two classes: P2X(1-7) ionotropic receptors, activated by ATP and G protein-coupled metabotropic P2Y receptors, activated by ATP, adenosine diphosphate (ADP), uridine di- and triphosphate (UDP and UTP), or UDP-glucose depending on the receptor subtype [+, K+ and Ca2+. The activation of these ionotropic receptors is important for Ca2+-induced intracellular signalling pathways [2+ levels, which modulate the activity of several secondary messengers involved in physiological processes [There are two families of purinergic receptors, which are distinguished by their main agonists . P1 rece subtype –40. ATP pathways –43. Deperocesses –46. The rocesses –66. Purirocesses . Not onlrocesses , 68–70.1 and A2A receptors have higher affinity  and A2B and A3 receptors have lower affinity (activated by higher extracellular levels of adenosine) for the ribonucleoside [1 and A2A receptors are highly expressed in the brain and CNS, where they have profound influence on neuronal activity. Adenosine A1 receptor is the dominant adenosine receptor subtype in the CNS. Adenosine A1 receptors can be found in various cortical and subcortical regions of the brain, while A2A receptors are mainly expressed in the striatum [2B and A3 receptors are mainly found in peripheral tissues, even though low levels of these receptors are also expressed in some regions of the brain [Adenosine is the predominant, presynaptic modulator of neurotransmitter release in the CNS, although ATP has presynaptic modulator effect as well –73. Adencleoside –77. The striatum –81  polymorphisms  were inversely associated with genetic PD risk, moreover, there was evidence of interaction with coffee consumption [GRIN2A encodes an N-methyl-d-aspartate-2A (NMDA) glutamate receptor subunit involved in central excitatory neurotransmission, which is associated with A2A receptor activation. Carriers of GRIN2A rs4998386-T allele had a lower risk of PD, than carriers of rs4998386-CC genotype among heavy coffee drinkers [GRIN2A coffee drinkers, which demonstrates an example of a genetic factor interacting with environmental factors exemplifying the complexity of environment–gene interactions in the progression of PD [7 receptor 1513A>C polymorphism is a risk factor for sporadic PD, late-onset PD and male PD in Han Chinese population [Two sumption . CYP1A2asumption . Humans sumption . GRIN2A drinkers . There ion of PD . In addipulation .2A receptors are enriched in dopaminergic brain areas (the highest expression of these receptors are in the striatum), thus pointing to a significant role of purines in motor control [2A and DA D2 receptors are mainly expressed in the neurons of the indirect pathway of striatal circuits projecting to the globus pallidus, in contrast to A1 and DA D1 receptors, which are mainly found on the neurons of the direct pathway of motor control projecting to the internal globus pallidus and substantia nigra pars reticulata. The main adenosine signalling mechanism is via the cyclic adenosine monophosphate (cAMP)-dependent pathway. Activated A2A receptors stimulate the enzymatic function of adenyl cyclase that increases cAMP levels and depresses the signalling mediated by D2 receptors. Activation of protein Gi-coupled DA D2 receptors leads to reduction in the cAMP level. There is a reciprocal situation in the direct pathway of motor control with protein Gs-coupled D1 and protein Gi/o-coupled A1 receptors. Generally, adenosine acts as a negative modulator of D1- and D2-mediated actions in the direct and indirect pathways [A control . A2A andpathways –112.2A and DA D2 receptors may depend on the formation of receptor heterodimers (A2A-D2 heteroreceptor complexes) in the striatum thereby balancing the inhibitory and excitatory impulses in the striatal circuits [2A receptors may involve interactions with various non-dopaminergic receptors, possibly by forming heterodimeric and/or multimeric receptor complexes [2A receptors may adjust the actions of striatal adenosine A1 receptors (A1-A2A heteroreceptor complexes), metabotropic glutamate receptors (mGlu) 5 (A2A-mGlu5 heteroreceptor complexes), cannabinoid receptor type 1 (CB1) receptors (A2A-CB1 heteroreceptor complexes) and serotonin 1A (5-HT1A) receptors [2A-D2-mGlu5 and A2A-CB1-D2 receptor complexes in the striatum [The antagonistic functional interaction between adenosine Acircuits . Not onlomplexes . Thus, aeceptors –115. Morstriatum , 117. Thstriatum . In genestriatum .2A receptor antagonists, such as methylxanthines, caffeine, or selective A2A antagonists) have been shown to enhance therapeutic effect of l-DOPA in a wide range of animal models of PD [2A homoreceptor complexes are in balance with DA D2 homoreceptor complexes in intact striatum [2A receptors and decreased signalling via DA D2 receptors. These changes explain the beneficial effect of A2A receptor antagonists on increasing motor functions without worsening l-DOPA-induced dyskinesias [Adenosine receptor antagonists  [2A receptor antagonists. Most of them were shown to be safe, well tolerated and beneficial; however, the majority did not reach the regulatory threshold for efficacy to be approved as PD drugs [2A-D2 heteroreceptor complexes acting on A2A and DA D2 receptors may be a good therapeutic approach in the future. Heterobivalent drugs offers the opportunity to target the orthosteric sites of the receptors in the heterodimer with a higher affinity and a higher specificity versus corresponding homomers and reduce the dose required for therapy and, accordingly, the side effects [APD Table . Istradeh l-DOPA . The drureatment . In cont effects , 131. Prff state , 133. Vieceptors . Its devherapies , 135–137cytosis) . There hPD drugs , 140. De effects .Table 3P2A receptor antagonists may also involve direct or indirect actions at microglia and inflammatory processes. Pre-treatment of slices from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-injected mice with preladenant facilitates the ability of activated microglia to respond to tissue damage [1/A2A adenosine receptor antagonist caffeine and the selective A2A receptor antagonist (KW-6002) had anti-inflammatory potential in a rat model of lipopolysaccharide (LPS)-induced neuroinflammation [Adenosine Ae damage . The nonammation .2A receptor expression was observed as an early pathological event in PD and increased A2A receptor expression was detected after hippocampal injection of α-synuclein in mice [2A receptor-knock out mice showed resistance against α-synuclein induced insults [2A receptor antagonism restrained hyperactivation of NMDA-glutamate receptors and decreased the aggregation of α-synucleins [2A receptors seem to have role in the pathological process of synucleinopathy [Increased striatal A in mice , 144. A2 insults . A2A recnucleins . Based uinopathy .1 and P2X1-4, 6 receptor protein subtypes are expressed in dopaminergic neurons with co-expression of P2X1 with DA D1 receptors, therefore stimulation of P2 receptors by ATP induces an increased release of DA in the striatum [1 receptor activation [P2 ionotropic and metabotropic receptors are widely expressed in basal ganglia and in various cell types, such as neurons and astrocytes , 148. 6-striatum –152. In tivation .7 receptor in PD. P2X7 receptor antagonism with A-438059 or Brilliant Blue G (BBG) prevented DA deficit in the striatum and 6-OHDA-induced hemiparkinsonian behavior [7 receptor deficiency or inhibition did not promote the survival of dopaminergic neurons in rotenone and MPTP induced animal models of PD [substantia nigra, which activates cell death pathways via purinergic receptors and is able to activate further purinergic subtypes [7 receptors. In the same study, activation of P2X7 receptors lead to ATP release with the recruitment of the pore forming protein pannexin1, whilst α-synuclein decreased the activity of extracellular ecto-ATPase which is responsible for ATP degradation [7 receptor by extracellular α-synuclein increased oxidative stress, which was prevented with the use of P2X7 receptor antagonist [Many data have implicated the role of P2Xbehavior , 155. Hols of PD . It is psubtypes . Permanesubtypes . The accradation . Stimulatagonist .1 receptor is involved in the regulation of calcium signalling [1 and P2Y4 receptors [6 receptor in PD patients younger than 80 years were higher than healthy controls and multiple system atrophy (MSA) patients and P2Y6 receptor could thereby be a potential clinical biomarker of PD. P2Y6 receptor was also upregulated in LPS-treated microglial cells and involved in proinflammatory cytokine release through UDP secretion [6 receptor on neuronal SH-SY5Y cell is associated with the progression of oxidative stress and cell death induced by 1-methyl-4-phenylpyridinium (MPP+) [6 receptor with MRS2578 [6 receptor subtype seems to be involved in the process of neuroinflammation in PD and blocking UDP/P2Y6 receptor signalling could reverse these pathological processes [DA neurotransmission has been linked to calcium signalling. There is data that P2Ygnalling . Neurodeeceptors . Recentlecretion . Anotherm (MPP+) . In vivo MRS2578 . Based orocesses .2A, P2X1, P2X7 and P2Y6 receptor subtypes is a promising weapon against PD via various ways: reducing l-DOPA induced dyskinesia, influencing neuroinflammation, preventing α-synuclein aggregation, reducing microglia activation. Development of new bivalent compounds to target A2A-D2 heteroreceptor complexes, which are orally bioavailable and can cross the blood–brain barrier could be a potential therapeutic tool. In addition, multi-target compounds targeting self-amplifying circuits controlled by purinergic and non-purinergic receptors could be a viable strategy to obtain the desired disease-modifying effect [In general, many data confirm the involvement of purinergic signalling pathways in PD. Modulation of purinergic receptor subtypes, the activity of ectonucleotidases and ATP transporters could be beneficial in the treatment of PD. Antagonism of Ag effect . Additio


Furthermore, treatment with palmitic acid (PA) led to significant upregulation of SLAMF3 on human T cells primed by anti-CD3/CD28 antibodies and on Jurkat cells, a human T-cell line. RNA sequencing revealed strong activation of the PI3K/Akt signaling pathway in T cells that were primed with PA. Further mechanistic studies showed that inhibition of PI3K/Akt signaling, or its upstream mediator STAT5 can prevent PA-induced SLAMF3 upregulation on T cells. These results indicate that SLAMF3 upregulation is associated with T-cell activation and cytokine production in T2D patients, and suggest that elevated saturated fatty acids in T2D patients may induce SLAMF3 upregulation on T cells via activation of the STAT5-PI3K/Akt signaling pathway.Metabolic stress-induced low-grade chronic inflammation plays an important role in the development of insulin-resistance and type 2 diabetes (T2D). Emerging evidence highlights the importance of directly elucidating T-cell activation under the obesity-induced metabolic stress condition, as T cells primed under such conditions were found to acquire a unique phenotype and function. Herein, we found a significant upregulation of signaling lymphocytic activation molecule family member 3 (SLAMF3) expression on T cells from T2D patients compared to those of healthy controls. Importantly, SLAMF3 upregulation was associated with an increased ability to produce proinflammatory cytokines. Significantly increased SLAMF3 expression was seen in T2D patient T cells that produce IFN-γ or IL-17 upon short (4-h) stimulation, compared to non-cytokine-producing T cells. In line with this finding, SLAMF3 T2D patients have been reported to exhibit a decrease in NK, Th2, and regulatory T cells, an increase in Th1 and Th17 cells3, and a higher production of inflammatory cytokines such as IFN-γ, IL-17, and TNF-α4. Previous studies revealed that T-cell activation and infiltration into obese adipose tissues play an important role in the initiation and progression of adipose tissue inflammation, by recruiting macrophages and inducing obesity-associated insulin resistance6. In line with these findings, immunotherapy using T-cell-targeted antibody was effective in reversing high-fat diet (HFD)-induced insulin resistance7.As a metabolic disorder characterized by insulin resistance and hyperglycemia, type 2 diabetes (T2D) is also associated with low-grade chronic inflammation that may result in a series of diabetes-related complications, including cardiovascular diseases, nephropathy, retinopathy, and increased risk for bacterial infection8. It has been reported that palmitic acid (PA), a major component of the saturated NEFAs in HFD, as well as in the plasma of individuals with obesity and T2DM, is a detrimental factor that promotes inflammation and insulin resistance8. PA could induce inflammatory injury through an agonist membrane receptor, such as Toll-like receptor 4 (TLR4)9, and stimulate T cells to produce reactive oxygen species (ROS) and cytokines10. A recent report demonstrated that incubation of mouse CD4 T cells with PA induces CD4 T cells to acquire a CD44hiCCR7loCD62LloCXCR3+LFA1+ proinflammatory phenotype11. Although the exact mechanisms are not understood, these reports indicate that PA plays a critical role in inducing inflammatory profiles in T2D.An increase in nonesterified fatty acids (NEFAs) is involved in obesity-associated insulin resistance and linked to T2DM12. T cells harvested from SLAMF3−/− mice exhibit poor proliferation and little IL-2 production in response to suboptimal stimulation by anti-CD3 antibody in vitro, indicating that SLAMF3 signaling is involved in T-cell action13. Similarly, SLAMF3 has been found to be upregulated in patients with autoimmune disorders, such as systemic lupus erythematosus, supporting its role in promoting T-cell responses15. However, SLAMF3-deficient mice develop spontaneous systemic autoimmunity, suggesting that SLAMF3 may also serve as a receptor contributing to immune tolerance16. The reasons for these contradictory observations remain unclear. In this study, we found that SLAMF3 is upregulated on T cells from T2D patients. Importantly, increased SLAMF3 expression on T cells was associated with improved potential to produce proinflammatory cytokines and enhanced responses to TCR stimulation. Further mechanistic studies showed that exposure of T cells to PA could significantly upregulate SLAMF3 expression through activation of the STAT5-PI3K/Akt signaling pathway, providing direct evidence linking chronic inflammation with elevated plasma NEFAs in T2D patients.The signaling lymphocytic activation molecule family member 3 (SLAMF3) is a member of the SLAMF receptors that are widely expressed on lymphohematopoietic cells and regulate immune function through homophilic interactionsn = 76) and healthy control subjects  were recruited from the Department of Endocrinology and Metabolism and the physical examination center of the First Hospital of Jilin University. Exclusions were severe complications , chronic or common infections , cancer, long-term use of immunosuppressive agents, or merging with other autoimmune disease. Umbilical cord blood was collected from the Department of Obstetrics of the First Hospital of Jilin University with informed consent for human T-cell function measurement. Protocols involved in the use of human samples were approved by the Institutional Review Board, and all of the experiments were performed in accordance with the protocols.Subjects diagnosed with T2DM  were isolated from heparinized whole blood using density gradient centrifugation from T2D patients or HCs, and stained with various combinations of the following fluorescence conjugated antibodies: anti human-CD3, CD4, CD8, CD25, CD229 (SLAMF3), IL-17, and IFN-γ (BD Pharmigen). BD Cytofix/Cytoperm Fixation and Permeabilization Kits (BD) were used for intracellular staining assays to detect cytokine production of T cells from T2D patients that were stimulated for 4 h with Leukocyte Activation Cocktail . Flow cytometric measurement was performed with a FACS Fortessa (BD Biosciences) and the data were analyzed by FlowJo (version 10).+ cells by MACS positive selection. Briefly, the cells were incubated firstly with biotin-conjugated antihuman CD4 antibody (BD) and then streptavidin-conjugated Micro-beads (Miltenyi Biotec) after washing. CD4+ T cells were collected from the cells retained in LS column under magnetic field. The purity of sorted cells was more than 97% as confirmed by flow cytometric analysis.Mononuclear cells were prepared from umbilical cord blood by density gradient centrifugation using Histopaque , and then sorted for CD4+CD4+CD25−SLAMF3high (SLAMF3high CD4 T cells) and CD3+CD4+CD25−SLAMF3low (SLAMF3low CD4 T cells) were purified by FACS cell sorting (BD Influx) and the purity was confirmed by flow cytometric assays (>97% purity). SLAMF3high or SLAMF3low CD4 T cells were incubated in the presence of precoated antihuman CD3 and soluble antihuman CD28 antibodies at the indicated concentrations in 37 °C incubator with 5% CO2. Seven days later, the cell expansion was measured by flow cytometric analysis and the images under microscope.CD3+ cord blood T cells were stimulated with CD3/CD28 antibodies (as detailed above) for 72 h and analyzed for SLAMF3 expression by flow cytometry analysis. We also measured SLAMF3 expression on human T cell Jurkat cells (pass mycoplasma contamination test) after incubated for 72 h under similar conditions without addition of antihuman CD3/CD28 antibodies in the absence or presence of PA , PI3K inhibitor , and/or STAT5 inhibitor .CD4+ cord blood T cells were stimulated with CD3/CD28 antibodies in the absence or presence of 0.3 mM PA for 72 h in 37 °C incubator with 5% CO2. After confirming SLAMF3 expression by flow cytometry analysis, total RNA was extracted from the cells by TRIzol (Invitrogen) and RNA sequencing was performed by Novogene. Differentially expressed genes between CD4 T cells that were stimulated in the absence or presence of PA were identified using the significance of digital gene expression profiles17. Prior to differential gene expression analysis, for each sequenced library, the read counts were adjusted by edgeR program package through one scaling normalized factor. Differential expression analysis of two conditions was performed using the edgeR package18. The p values were adjusted using the Benjamini and Hochberg method. Corrected p value of 0.05 and absolute foldchange of two were set as the threshold for significantly differential expression. Kyoto encyclopedia of genes and genomes (KEGG) pathways or Disease Ontology (DO) terms were considered. The method of calculating the p value was performed traditionally19. Then, the enriched significance p value was adjusted using the Benjamini and Hochberg algorithm20. Finally, KEGG pathways or DO terms with adjusted p values < 0.05 and including at least two differentially expressed genes were considered.CD4t-test. For all analysis, statistical significance was reported as follows: *p < 0.05; **p < 0.01; ***p < 0.001; ns, no significance.All analyses were performed with GraphPad Prism version 6. Control and experimental results were compared with the nonparametric Wilcoxon/Kruskal–Wallis or the paired two-tailed Student’s 2 for T2D patients, and 49.37 years and 25.14 kg/m2 for HCs. T2D patients had an average HbA1c level of 8.81% and significantly higher fasting blood glucose  than HCs (5.23 ± 0.47 mmol/L). T2D patients also exhibited significantly increased triglyceride  and decreased high-density lipoprotein cholesterol  compared to HCs.Seventy-six T2D participants that met the inclusion criteria and 74 healthy controls (HCs) were enrolled in the study Table . The mea+ T cells, but the level of CD3+CD4+ T cells was significantly increased in T2D patients compared to HCs. Interestingly, a notable change in T2D patients was the upregulated surface expression of SLAMF3 on T cells, including both CD4+ and CD4− T-cell subsets  were found at increased levelsMF3 Fig. . Meanwhiion Fig. . These dhigh (MFI = 677) and SLAMF3low (MFI = 318) CD4+CD25− T cells . Differential gene expression analysis revealed that 989 genes were upregulated and 1462 genes were downregulated in PA-treated human T cells . Again, PA treatment significantly upregulated SLAMF3 expression on Jurkat cells, but this SLAMF3 upregulation on PA-treated cells was significantly and dose dependently inhibited by LY294002 Fig. . The eff1. There is emerging evidence for obesity-induced immune responses. Recently, it was reported that memory T cells from hosts with or without obesity-induced metabolic stress are different in both phenotype and function11, highlighting the importance of characterizing biased T-cell activation under obesity conditions. Although there are still some controversies, previous studies provided strong evidence supporting the role of SLAMF3 in promoting T-cell activation24. Here we found that SLAMF3 is significantly upregulated on T cells from T2D patients; importantly, its upregulation on T cells was associated with increased potential to produce proinflammatory cytokines. In agreement with these observations made in T2D patients, human SLAMF3high T cells were found to be more sensitive to TCR stimulation with anti-CD3/CD28 antibodies. Although our data cannot firmly demonstrate that SLAMF3 upregulation is necessary and/or sufficient for the observed chronic inflammatory phenotypes of T cells in T2D patients, these results indicate that SLAMF3 upregulation provides a marker for activated T cells that are the major source of T cell-derived proinflammatory cytokines in T2D patients.Low-grade chronic inflammation is considered a causative factor in the development of insulin resistance and T2D8. PA was reported to interact with agonist membrane receptors 9 and to induce ROS and cytokine production10. PA can also convert naïve T cells to a proinflammatory phenotype11. These studies raise the possibility that PA is an important factor driving T-cell activation and inflammation in T2D patients. In support of this possibility, we found that PA treatment significantly upregulated SLAMF3 expression on human T cells through induction of SLAMF3 gene transcripts  and triglyceride (TG). Importantly, human T cells from HFD-treated mice showed a significantly upregulated SLAMF3 expression compared to those from HD-fed mice  is an important factor that upregulates SLAMF3 expression on T cells, and this process requires activation of the STAT5-PI3K/Akt signaling pathway. These results provide insight into the immunopathogenesis of T2D and potential molecular targets for T2D therapies.Supplemental material


Phosphophoryn (PP) and dentin sialoprotein (DSP) are two of the most abundant dentin matrix non-collagenous proteins, and are derived from dentin sialoprotein-phosphophoryn (DSP-PP) mRNA. Mutations in the DSP-PP gene are linked to dentinogenesis imperfecta II and III. Previously, we reported transient DSP-PP expression in preameloblast cells first, followed by co-expression in preameloblasts and young odontoblasts, and finally sustained expression in odontoblasts. This phenomenon raised the possibility that DSP/PP proteins secreted by preameloblasts might promote dental pulp cell migration toward the dental pulp border and promote dental pulp cell differentiation. To examine the effects of DSP/PP proteins on dental pulp cell development, we investigated:(1) native PP effects on dental pulpcell migration and matrix protein expression; and (2) recombinant DSP/PP protein effects on cell proliferation and differentiation. We found that PP promoted cell migration and the expression of high levels of Col type I and PP in dental pulp cells. The addition of recombinant DSP/PP proteins affected cell proliferation and differentiation in a dental pulp cell line. These findings strongly suggest that DSP/PP may modulate cell migration, cell proliferation and differentiation, thus leading to dentin formation. DSP/PP protein may be useful clinically for pulp tissue regeneration. Dentin sialoprotein (DSP) and phosphophoryn (PP) are two major dentin non-collagenous extracellular matrix proteins, which are derived from the dentinsialoprotein-phosphophoryn  gene. Briefly, DSP-PP mRNA is transcribed from the DSP-PP gene and then translated into DSP-PP precursor protein, which further undergoes cleavage by peptidasesbone morphogenetic protein BMP1) and/or tolloid-related-1 (TLR1) to generate mature DSP and PP proteins  and/or t,3. DSP/PThe transient expression of DSP protein and DSP-PP transcripts occurs first in the preameloblasts, and next in both preameloblasts and young odontoblasts. Finally, DSP-PP transcripts exhibitsustained expression in odontoblasts ,10. SincTo better understand the potential role of transient DSP-PP mRNA or DSP/PP protein expression in preameloblasts, a rat dental pulp cell line (MRPC-1) was used to examine whether native PP could affect dental cell migration and matrix protein synthesis. We further investigated whether recombinant DSP/PP proteins could affect dental pulp M2H4 cell proliferation and cell morphology as well as DSP-PP mRNA expression.Ascorbic acid can stimulate type I procollagen mRNA and collagen synthesis in the mouse calvaria-derived cell line MC3T3-E1, which was used to study the role of collagen synthesis in osteoblast differentiation . Rat den523 is the dominant transcript, which generated rat PP with 523 amino acids [2: 95%air and 100% relative humidity. The medium was changed every other day.Native rat PP was kindly provided by Mary E. Marsh  and the preparation of native rat PP was described previously ,7. Multino acids . One per2. The precipitate was then dissolved in 0.1 M EDTA and loaded onto polyacrylamide gel for electrophoresis. The gels were stained with Stains-All to detect the presence of PP.Agarose containing 1 μg native PP was cultured in DMEM medium with 10% FBS and P/S (100 IU/mL and 100 μg/mL)and the culture medium was collected on Day 1 (24 h), and Day 2 to Day 4 to test the PP release. Because PP was soluble in 5% trichloroacetic acid (TCA), the collected cultured medium was treated with 5% TCA, which precipitated the non-PP proteins in the medium . The preTo examine the cell migration under the influence of various agarose bead concentrations  of PP, each group with three wells was observed on Day 2 under microscope. The cells located next to the agarose beads were counted in each group. The cells in the control group with no agarose were also counted randomly.Six more wells in each group at Day 2were fixed with 4% form aldehyde. Three of these wells were used to detect collagen type I (Col I) expression with anti-collagen type I (Col I) antibodies at 200X dilution in 1% blocking agent with maleic acid in no agarose cell culture, and in Group 1 (agarose-no PP), Group 3 (agarose-1 μg PP) and Group 4 (agarose-5 μg PP) cell cultures. Next, diluted secondary biotinylated antibody was added and then shed and quenched of endogenous peroxidase activity. Finally, Vectastain Elite ABC reagent was added to the cultures and four drops of DAB stock solution (Vector laboratories) were added to each well for color observation. Following a similar protocol, another three wells were used to detect PP expression with anti-PP antibodies at 100X dilution in 1% blocking agent with maleic acid in four groups .Three wells from each group at Day 4 were fixed with 4% formaldehyde and used to detect PP expression with anti-PP antibody as described above.240 cDNA in a baculovirus expression vector pVL1392 was used to generate recombinant DSP430/PP240  proteins in the insect sf9 cell culture medium [307 cDNA was derived from a naturally occurring DSP-only transcript present in tooth germs [307 protein  was also generated in a baculovirus system, and secreted into the insect culture medium (data not shown). These sf9 cell supernatants, from either anti-sense DSP-PP240 cDNA infected sf9 cell conditioned medium containing no recombinant proteins or sense-infected DSP307 cDNA or DSP-PP240 cDNAsf9 cells containing recombinant DSP307 or DSP430/PP240 protein mixture, were used directly for this study.Rat DSP-PPth germs . RecombiWhen we were ready to test recombinant DSP/PP effects on dental pulp proliferation, MRPC-1 cells did not function well in Ritchie lab. Thus, we developed a M2H4 cell line , which w5 cells/T-25 flask) were cultured in 5mL of M2H4 growth medium  containing 100 IU/mL penicillin and 100 ug/mL streptomycin (P/S) and 10% fetal bovine serum (FBS)). Once the cells reached 90% confluency, 1 mL of conditioned medium  and/or ascorbic acid (50 μg/mL) was added to the flasks separately. The flasks were changed every other day with the appropriate conditioned medium. Cells were treated with 1X trypsin-EDTA. The cell number per flask was determined by hemocytometer. Once cells were treated with 1X trypsin-EDTA at Day 2, Day 4 and Day 6, the flasks were discarded. Data are presented as the mean ±S.D. of triplicate samples.M2H4 cells (8 × 105 cells/T-25 flask) were cultured in 5mL of M2H4 growth medium. Once the cells reached 90% confluency, 1 mL each of: (1) condition medium (containing no recombinant protein); (2) condition medium with 50 μg/mL ascorbic acid; and (3) recombinant DSP/PP240 protein mixture plus 50μg/mL ascorbic acid, was added to the flask separately. The flasks were cultured for a total of eight days. The flasks were changed every other day with appropriate medium. Triplicate samples were for each group.M2H4 cells  primer/random primer and reverse transcriptase to generate cDNA pools for PCR analyses. DSP-PP upper primer (5’GAAGCATGTCCTTCTG3’) and DSP-PP lower primer (5’CTATCCTGCTGTGTCC3’) sequences were used to generate a 220bp DSP-PP DNA fragment. G3PDH upper primer (5’TGTTTGTGATGGGTGTGAACC3’) and lower primer (5’ACAGTCTTCTGAGTGGCAGT3’) sequences were used to generate a 175 bp PCR G3PDH DNA fragment was used as an internal experimental control. RT-PCR amplification was performed as follows: denaturation for 30s at 94 °C, annealing for 30s at 55 °C and extension for 1 min at 72 °C, for 30 cycles. The PCR products were analyzed by gel electrophoresis with Ethidium Bromide staining.5 cells/T-25 flask) were cultured in 5mL of M2H4 growth medium as described in the previous section. Once the cells reached 90% confluency, 1 mL of conditioned medium was added to the following groups of T-25 flasks: (1) conditioned medium containing no recombinant protein;(2) conditioned medium containing no recombinant protein with ascorbic acid (50 μg/mL); (3) conditioned medium containing recombinant DSP307 protein; and (4) conditioned medium with recombinant DSP430/PP240 protein mixture. Medium from these cells was changed every other day for eight days. Triplicates were set up for each group. At Day 8, the cells from each group were examined by light microscopy.M2H4 cells  by personnel at the Center for Statistical Consultation and Research Center of the University of Michigan.Results are presented as means ± standard deviation (S.D.). Two-sample To determine the rate of PP release from the agarose gel, agarose beads contained 1 μg of PP were cultured in DMEM with 10%FBS and P/S (100 IU/mL and 100 μg/mL). The culture medium was collected on Day 1 (24 h), and Day 2 to Day 4. We found that PP was completely released in 24 h into the culture medium. No further PP release was detected after 24 h . After tSince PP was fully released into the culture medium within 24 h, we examined three wells from each group on Day 2. In general, all three wells for each group were examined under the microscope A. The coUsing anti-Col I antibodies, immunohistochemistry showed weak Col I expression in control (no agarose) cultures and in Group 1 (agarose-no PP). Strong Col I expression in Group 3 (agarose-1 μg PP) and less Col I expression in Group 4 (agarose-5 μg PP). Overall, strong Col I expression appeared in Day 2 cells bordering Group 3 (agarose-1 μg PP) agarose beads .Using anti-PP antibodies, the PP expression was more intense  than tha240 protein mixture± ascorbic acid. 240 protein mixture, cell proliferation was significantly reduced. Similarly, in the presence of DSP-PP240 and ascorbic acid treatment, M2H4 proliferation was further reduced, as shown in To test whether DSP and PP proteins could alter M2H4 dental pulp cell developmental programs, we first sought to determine whether recombinant DSP and PP proteins could alter M2H4 cell proliferation. Cells were incubated for six days in anti-sense conditioned media ± ascorbic acid, as well as sense conditioned media containing recombinant DSP/PP240 proteins on DSP-PP mRNA expression in M2H4 cells, we compared its effect with those of sf9 condition medium in the presence or absence of ascorbic acid. We found that sf9 insect conditioned medium (without ascorbic acid) showed weak DSP-PP mRNA expression. In the presence of ascorbic acid, DSP-PP mRNA expression was slightly elevated. However, the addition of both recombinant DSP/PP240 and ascorbic acid to M2H4 cells significantly up-regulated DSP-PP mRNA expression  for eight days showed highly packed rounded cells a. M2H4 ccultures c,d .Even though non-collagenous proteins (NCPs)account for less than 20% of dentin’s organic components, their participation in dentinal mineralization is well established. PP is the most abundant NCP in dentin . PreviouBetween the control group (no agarose) and Group 1 (agarose-no PP) at Day2, we found no difference between cell morphology and cell numbers.In Day 2 cultures, MRPC-1 cells migrated slightly to the border of the agarose beads containing 0.2 μg PP. More cells migrated to the border of agarose beads containing 1 μg PP and cells completely encircled the agarose beads containing 5 μg PP . Thus, wAt higher PP concentration (5 μg), cell migration declined . On Day Col I is the major dentin matrix protein and provides the framework for the deposition of mineral crystals via interaction with non-collagenous proteins such as PP . In thisAmong various PP concentrations, the agarose-1 μg PP induced the most significant cell migration in MRPC-1 cells. The agarose-1 μg PP also exhibited a high level of Col type I expression on Day 2 and a high level of PP expression on Day 4. Taken together, agarose-1 μg beads promoted better cell migration, stronger Col I and PP expression.The expression of Col I first followed by PP expression in MRPC-1 cells mimicked the Col I and PP expression patterns in vivo . Likely These results lend support that PP plays a role during epithelial–mesenchymal interactions. PP could be used to recruit dental pulp cells to the odontoblast layer during dentin formation and dentin repair. Thus, PP could potentially be used as a bio-mimetic material for tooth repair and tissue engineering.240 conditioned medium containing no recombinant protein displayed higher cell proliferation  on Day 6   . Cells tto Day 6 . The compression .307 and DSP/PP240 sense-conditioned media, respectively (307 and DSP/PP240 protein mixture might strongly promote dental cell differentiation and matrix synthesis.Cells cultured in medium containing no recombinant protein and no ascorbic acid were rounded a, which ectively c,d. BothIn summary, our cell culture results suggest that native PP could promote dental pulp cell migration and induce cell differentiation causing Col I and DSP/PP protein expression. Recombinant DSP/PP proteins may also reduce cell proliferation. Taken together, these findings support our hypothesis that native PP and recombinant DSP/PP influence cell migration from dental pulp to the peripheral area, reduce proliferation and increase differentiation to enable pulp cells to develop into odontoblast-like cells and produce Col I and DSP/PP proteins.These studies suggest the potential use of the native PP and the recombinant DSP/PP proteins as novel clinical capping agents for caries treatment.


The M segment of the 2009 pandemic influenza A virus (IAV) has been implicated in its emergence into human populations. To elucidate the genetic contributions of the M segment to host adaptation, and the underlying mechanisms, we examined a panel of isogenic viruses that carry avian- or human-derived M segments. Avian, but not human, M segments restricted viral growth and transmission in mammalian model systems, and the restricted growth correlated with increased expression of M2 relative to M1. M2 overexpression was associated with intracellular accumulation of autophagosomes, which was alleviated by interference of the viral proton channel activity by amantadine treatment. As M1 and M2 are expressed from the M mRNA through alternative splicing, we separated synonymous and non-synonymous changes that differentiate human and avian M segments and found that dysregulation of gene expression leading to M2 overexpression diminished replication, irrespective of amino acid composition of M1 or M2. Moreover, in spite of efficient replication, virus possessing a human M segment that expressed avian M2 protein at low level did not transmit efficiently. We conclude that (i) determinants of transmission reside in the IAV M2 protein, and that (ii) control of M segment gene expression is a critical aspect of IAV host adaptation needed to prevent M2-mediated dysregulation of vesicular homeostasis. Influenza A virus (IAV) pandemics arise when a virus adapted to a non-human host overcomes species barriers to successfully infect humans and sustain human-to-human transmission. To gauge the adaptive potential and therefore pandemic risk posed by a particular IAV, it is critical to understand the mechanisms underlying viral adaptation to human hosts. Here, we focused on the role of one of IAV’s eight gene segments, the M segment, in host adaptation. Comparing the growth of IAVs with avian- and human-derived M segments in avian and mammalian systems revealed that the avian M segment restricts viral growth specifically in mammalian cells. We show that the mechanism underlying this host range phenotype is a dysregulation of viral gene expression when the avian IAV M segment is transcribed in mammalian cells. In particular, excess production of the M2 protein results in viral interference with cellular functions on which the virus relies. Our results therefore reveal that the use of cellular machinery to control viral gene expression leaves the virus vulnerable to over- or under-production of critical viral gene products in a new host species. Influenza A virus (IAV) epidemics and pandemics result in widespread and often severe disease –3, as weChanges to viral receptor specificity and polymerase function necessary to overcome host species barriers have been well documented –11, but in silico identification of positive selection acting on the M segment of the Eurasian avian-like swine lineage, following transfer of this IAV from birds to swine .To assess the contribution of the M segment to host adaptation, we generated a panel of IAVs possessing human- or avian-derived M segments in the A/Puerto Rico/8/34 (H1N1) [PR8] background . Also, under high MOI growth conditions in DF-1 and QT-6 cells, the growth phenotypes of the PR8 NL09 M and PR8 avian M viruses were similar, although the NL09 M-containing virus trended towards increased growth relative to the avian M-containing viruses in DF-1 cells , and additionally from high MOI in human 293T cells . In each case, PR8-based viruses carrying the NL09 M segment grew with more rapid kinetics and to higher titers than isogenic viruses carrying avian M segments, although the differences in growth from low MOI did not reach statistical significance. To extend our findings to an independent human lineage of IAV, we evaluated the growth from high MOI in A549 cells of PR8 Pan99 M and PR8 Beth15 M viruses, each of which have an M segment derived from the human seasonal H3N2 lineage . We found that both of these human-adapted M segments supported significantly faster kinetics and higher magnitude of growth than the dkAlb76 89G M segment . Thus, results from multiple different avian and mammalian culture systems reveal defects in the kinetics and magnitude of viral growth associated with avian IAV M segments specifically in mammalian host cells and provide evidence of the acquisition of adaptive changes in the M segment of pH1N1 and H3N2 human influenza A lineages.The relative fitness of PR8-based viruses carrying M segments from the human-adapted NL09 virus or from avian IAVs was evaluated in three avian substrates: embryonated chicken eggs (ECE), chicken origin DF-1 cells, and quail origin QT-6 cells. When grown from low multiplicities of infection (MOI) in either ECE or DF-1 cell substrates, all viruses exhibited similar kinetics and reached comparable titers . As shown in Fig 3B, fewer guinea pigs were productively infected with 10 PFU when the inoculating virus carried an avian M segment. Differences in the kinetics of viral growth were determined by assessing changes in growth over time using repeated measures ANOVA . Each virus encoding an avian host-derived M segment replicated with significantly slower kinetics than the PR8 NL09 M virus.To evaluate the impact of M segment host adaptation on viral phenotypes in an intact host, we evaluated the PR8 NL09 M virus and the four PR8-based viruses carrying avian M segments in a guinea pig model ,55. A loFig 3B). Thus, data from a mammalian animal model reveal defects in infectivity, growth and transmission associated with avian IAV M segments. Our results suggest the acquisition of adaptive changes in the M segment of pH1N1 human IAV since its introduction from the avian reservoir into mammals.The efficiency of transmission was assessed by calculating the proportion of contact animals that contracted infection, considering only cages in which donor guinea pigs were productively infected through intranasal inoculation. While transmission occurred in 7/9 instances for PR8 NL09 M virus, a significantly lower proportion of animals  were infected with a virus carrying an avian M segment transmitted from her cage mate . When M1 and M2 levels were normalized to those of vinculin and the relative levels of M1 and M2 calculated, the NL09 M2 protein comprised <10% of the total M segment-derived protein expression. Very similar results were seen for four PR8-based viruses carrying avian M segments grown in DF-1 cells. In each case, M2 comprised <20% of the total M segment-derived protein, and in the case of three avian M segment-containing viruses , the levels of M1 and M2 proteins were not significantly different from those expressed by the PR8 NL09 M virus .In the avian cell line, the PR8 NL09 M virus expressed lower levels of M2 than M1 . In contrast, avian M segments yielded markedly higher levels of the M2 protein than were observed for the same viruses grown in avian cells. Here, M2 levels were approximately equal to those of M1 . Moreover, when compared to the NL09 M segment in human cells, levels of M2 protein were significantly higher for avian M segments (P<0.0001) . Thus, compared to matched virus-host pairings in both human and avian systems, M2 was markedly overexpressed when avian-adapted M segments were introduced into human cells.In A549 cells, the PR8 NL09 M virus expressed M2 at ~20% of the total M protein, similar to the expression levels observed in infected DF-1 cells (7), which can be spliced by cellular splicing factors to yield mRNA10 or mRNA11 ATP and 10 U of T4 PNK (New England Biolabs) in 70 mM Tris-HCl (pH 7.6) containing 10 mM MgCl2 and 5 mM DTT. Radiolabeling reactions were carried out at 37 ºC for 1 h and diluted to 30 μL with RNase-free water after termination of the reaction. Radiolabeled primers were stored at –20 ºC until needed. 500 ng of total RNA was incubated with 0.45 μL unlabeled 5S rRNA primer, 0.05 μL radiolabeled 5S rRNA primer, 0.25 μL radiolabeled IAV segment 7 vRNA primer, and 0.25 μL radiolabeled IAV segment 7 mRNA primer in a final volume of 5 μL. Primers were annealed to RNA by sequential incubations at 95 ºC for 3 min and ice for 3 min. Reactions were pre-warmed at 50 ºC for 5 min. Transcription was initiated by the addition of 50 mM Tris-HCl (pH 8.3) buffer containing 75 mM KCl, 3 mM MgCL2, 10 mM DTT, 2mM dNTP, and 50 units of SuperScript III Reverse Transcriptase (Thermo Fisher Scientific). Reactions were incubated at 50 ºC for 1 h and terminated by the addition of Gel Loading Buffer II (Thermo Fisher Scientific) and incubation at 95 ºC for 10 min. Reactions were resolved on denaturing sequencing polyacrylamide gels . Gels were subsequently dried and exposed to a phosphor storage screen overnight. The intensity of bands was analyzed using a Typhoon Trio Imager  and quantified with ImageQuant software  after normalization to 5S rRNA. At least three biological replicate viral infections were carried out, with two technical replicates of the RT primer extension assay for each infection. Data are shown with error bars representing SD and statistical analysis was carried out using one-way ANOVA with Tukey’s multiple comparisons in GraphPad Prism software.Radiolabeling of primers: 1 μmol of oligo DNA primer was incubated with 10 μCuries of [γ-To allow tracking of LC3B II and viral protein, cells grown on collagen-coated coverslips were transduced with lentivirus expressing LC3B-GFP , as follows.18h post-transduction, 293T or A549 cells were infected with the indicated IAVs at an MOI of 5 PFU/cell and incubated at 37°C. At 8h or 12 h post-IAV infection, cells were washed once with PBS and fixed with 4% paraformaldehyde. Following permeabilization with 0.1% Triton X-100, cells were incubated overnight at 4°C with anti-M2 monoclonal antibody . Three washes were performed prior to addition of goat anti-mouse Alexa Fluor-647 secondary antibody  and incubation for 2 h at room temperature. Antibody dilutions and washes were performed with PBS plus 1% Tween-20. Cells were treated with DAPI, and coverslips were mounted on slides using Vectashield (Vector Labs) mounting medium. Images were obtained with on a Nikon FV1000 confocal microscope at the Emory Integrated Cellular Imaging core facility.Where indicated, chloroquine (Invitrogen) was added to cell culture medium at a final concentration of 60 or 120 μM. Amantadine HCl  was added to cell culture medium at a final concentration of 200 μM. Both drugs were added to infected cells 1 h after IAV inoculation.Statistical analyses were performed in GraphPad Prism.Multi-cycle and single-cycle growth in cell culture was assessed in three independent experiments, with three technical sample replicates per experiment. Overall, statistical significance was determined using repeated measures, two-way, multiple comparisons ANOVA on log-transformed mean values, with Bonferroni correction applied to account for comparison of a limited number of means, as well as by assessing pairwise comparisons of viruses at each time point, except for single-cycle growth in cell culture in the presence of amantadine, which was assessed in four independent experiments, with three technical sample replicates per experiment. Overall statistical significance was determined using repeated measures, two-way, multiple comparisons ANOVA on log-transformed values, with Sidak’s correction applied, as well as by assessing pairwise comparisons of viruses at each time point.Growth of each virus in the guinea pig model was assessed in three independent experiments with four guinea pigs per experiment. For transmission analysis, each guinea pig was treated as an independent biological sample. Statistical significance in the magnitude of replication was determined by comparing AUC of growth curves using two-tailed Student’s t-test. Statistical significance in transmission efficiency was determined using unpaired two-tailed Student’s t-test.Significance of differences in the kinetics of replication was determined by assessing the interaction of time and virus using repeated measures, two-way ANOVA on mean log-transformed values, with Bonferroni correction applied to account for comparison of a limited number of means.For experiments presented throughout the manuscript, unless specific values are provided, P values are represented as follows: *< 0.05; **<0.01; ***< 0.001; ****< 0.0001.S1 Fig(A) The M segment of influenza virus is template for synthesis of mRNA7 (encoding M1), mRNA M10 (encoding M2), and mRNA11 (which encodes a putative but unconfirmed 10 amino acid peptide from a short open reading frame). (B) Pandemic H1N1 influenza virus M1 and M2 proteins differ from the avian consensus sequences by 9 residues in M1 and 7 residues in M2. The M segments differ by 8.3% at the nucleotide level. (C) Seasonal H3N2 influenza virus strain A/Panama/2007/99 M1 and M2 proteins differ from the avian consensus sequences by 11 residues in M1 and 14 residues in M2. The M segments differ by 9% at the nucleotide level. (D) Seasonal H3N2 influenza virus strain A/Bethesda/55/15 M1 and M2 proteins differ from the avian consensus sequences by 11 residues in M1 and 16 residues in M2. The M segments differ by 9.5% at the nucleotide level.(PDF)Click here for additional data file.S2 Fig(A), or A549 cells (B). Cells were incubated at 37°C for up to 24 h. Virus released into supernatant was collected at the indicated time points, and virus growth was measured by plaque titration. Data obtained from viruses possessing human M segments are represented with blue lines: A/NL/602/09 M , A/Panama/2007/99 M (B), and A/Bethesda/15 M (B), while data from viruses encoding avian M segments are represented with red lines. In each cell type, the human M segments conferred more rapid kinetics and higher peak titers of growth than any avian-origin M segment. Single-cycle growth was assessed in three independent experiments, with three technical sample replicates per experiment. Graphs show the means with SD for the three experiments. Statistical significance was determined using repeated measures, two-way, multiple ANOVA on log-transformed data, with Bonferroni correction applied as there were a limited no of means to compare.PR8-based viruses were inoculated at a MOI of 5 PFU/cell onto human-derived 293T cells (PDF)Click here for additional data file.S3 Fig(A-E) Virus replication in nasal wash of inoculated animals was measured by plaque titration at days 2, 4, 6, and 8 post-infection and the titers at each time point were plotted (dotted lines). The differences between PR8 NL09 M and each avian M-encoding virus were considered significant. Statistical significance in kinetics of growth was determined by assessing the interaction of time and virus using repeated measures, two-way, multiple comparisons ANOVA on mean values, with Bonferroni correction applied to account for comparison of a limited no of means.Groups of four guinea pigs were inoculated with 10 PFU of each avian M-encoding virus, or NL09 M-encoding virus, as indicated. Graphs show individual titers obtained from animals used in three independent experiments. (PDF)Click here for additional data file.S4 Fig(A) and MDCK cells (G). Vinculin expression was measured to allow normalization of viral protein levels. NP expression was measured to assess viral replication. Levels of M1 and M2 protein expression were assessed using an antibody (Mab E10) to a common epitope at the amino terminus of M1 and M2 proteins, allowing relative expression to be assessed. Levels of LC3B I and II were assessed using an antibody that detects both the precursor and activated forms of LC3B protein.  M1 protein and  M2 protein were normalized to vinculin, quantitated and displayed as a percentage of total protein expressed from the M gene.  The ratio of M1:M2 protein expression.  LC3B I protein and  LC3BII protein were normalized, quantitated and displayed as a percentage of total LC3B protein. Graphs in B-F, and H-K show the means with SD from three independent experiments. For each experiment, two replicate Western immunoblots were performed and quantitated. Statistical significance was assessed using ordinary one-way ANOVA.293T and MDCK cells were inoculated at a MOI of 5 PFU/cell with PR8 viruses encoding avian or human-derived M segments and incubated at 37°C for 8 h, then cells were lysed. Western immunoblot analysis of virus-infected 293T cells (PDF)Click here for additional data file.S5 FigA549 cells were inoculated at a MOI of 5 PFU/cell with PR8 viruses encoding avian or human-derived M segments and incubated at 37°C for 8 h, then cells were lysed. Human M segments were derived from the following viruses: A/NL/602/09 (H1N1) (NL09); A/Panama/2007/99 (H3N2) (Pan99); and A/Bethesda/15 (H3N2) (Beth15). Vinculin expression was measured to allow normalization of viral protein levels. NP expression was measured to assess viral replication. Levels of M1 and M2 protein expression were assessed using an antibody (Mab E10) to a common epitope at the amino terminus of M1 and M2 proteins, allowing relative expression to be assessed. Levels of LC3B I and II were assessed using an antibody that detects both the precursor and the activated forms of LC3B protein. Data presented are representative of Western immunoblots from three independent experiments.(PDF)Click here for additional data file.S6 Fig, or 293T  cells were inoculated with the indicated viruses, encoding avian or human M segments, at a MOI of 5 PFU/cell. Cells were fixed at 12 hpi, permeabilised, and stained with anti-M2  and DAPI (blue) followed by imaging with confocal microscopy. Examples of optical sections are shown, either as merged 2-color images or the red and blue channels alone . (A) A549 cells with 63x magnification. (B) 3x magnification of the same images shown in A. (C) 293T cells with 63x magnification. (D) 3x magnification of the same images shown in C. Brightness was adjusted for optimal clarity, with all images treated equally.A549 (PDF)Click here for additional data file.S7 Fig(A) 63x magnification. (B) 3x magnification of the same images shown in A. Brightness was adjusted for optimal clarity, with all images treated equally. CQ: chloroquine.A549 cells were transduced with GFP-LC3 protein and inoculated 24 h later with the indicated IAVs, encoding avian- or human-derived M segments, at a MOI of 5 PFU/cell. Cells were fixed 8 h later and stained with anti-M2  and DAPI (blue) followed by imaging with confocal microscopy. Examples of optical sections are shown, either as merged 3-color images or the red, green, and blue channels alone . (PDF)Click here for additional data file.S8 Fig(A) Vinculin expression was measured to allow normalization of viral protein levels. NP expression was measured to assess viral replication. Levels of M1 and M2 protein expression were assessed using an antibody (Mab E10) to a common epitope at the amino terminus of M1 and M2 proteins, allowing relative expression to be assessed. Levels of LC3B I and II were assessed using an antibody that detects both the precursor and the activated forms of LC3B protein. Data presented are representative Western immunoblots from three independent experiments. LC3B I protein and LC3B II protein  were normalized, quantitated and displayed as a percentage of total LC3B protein in the absence (B) or presence (C) of 60 μM chloroquine. Data presented in B-C, show the means with SD from three independent experiments. For each experiment, two replicate radiograms were quantitated. Statistical significance was assessed using ordinary two-way ANOVA. CQ: chloroquine.A549 cells were inoculated at a MOI of 5 PFU/cell with PR8 viruses encoding avian or human-derived M segments and incubated in the presence or absence of 60 μM chloroquine from 1 hpi. Cells were lysed with whole cell lysis buffer following 8 h incubation at 37°C. Western immunoblot of virus-infected A549 cells: (PDF)Click here for additional data file.S9 Fig(A) Pandemic H1N1 influenza virus M1 and M2 proteins differ from the avian consensus sequences by 9 residues in M1 and 7 residues in M2. The pH1N1 amino acid identities are indicated in blue at their approximate positions within linear representations of M1 and M2 proteins. The pH1N1 M segment differs from the avian consensus by 8.3% at the nucleotide level. Here, pH1N1 RNA sequence is indicated by blue coloring. (B-D) Chimeric human-avian M segments were constructed in which only the non-synonymous changes in the avian consensus were introduced into the A/NL/602/09 (H1N1) M segment. (E) Avian consensus M1 and M2 proteins differ from the NL09 sequences by 9 residues in M1 and 7 residues in M2. The avian amino acid identities are indicated in red and avian RNA sequence is indicated by red coloring. (F-H) A second set of chimeric M segments was constructed in which the NL09 amino acid identities were introduced into the M segment of A/duck/Alberta/76 (H1N1) virus, yielding segments that encode avian consensus protein(s) but retain much of the nucleotide sequence of the NL09 M segment.(PDF)Click here for additional data file.S10 Fig293T cells were transduced with GFP-LC3 protein and inoculated 24 h later with the indicated IAVs, encoding avian, human or chimeric M segments, at a MOI of 5 PFU/cell. Cells were fixed 12 h later and stained with anti-M2  and DAPI (blue) followed by imaging with confocal microscopy. Examples of optical sections are shown, either as merged 3-color images or the red, green, and blue channels alone . 3x magnification of 63x images are shown. Brightness was adjusted for optimal clarity, with all images treated equally.(PDF)Click here for additional data file.S11 Fig(A) RT primer extension radiogram of virus-infected A549 cells. 5S rRNA levels were measured to allow normalization of viral RNA. Segment 7 vRNA expression was measured to assess viral replication. (B) Levels of mRNA7, (C) mRNA10, and (D) mRNA11 were quantitated and displayed as a percentage of total M gene expressed mRNA. Graphs in B-D show the means with SD from three independent experiments. For each experiment, two replicate radiograms were quantitated. Statistical significance was assessed using ordinary one-way ANOVA.A549 cells were inoculated at a MOI of 5 PFU/cell with PR8 NL09 M virus and PR8 Av M virus, along with six PR8-based viruses with chimeric M segments. Cells were incubated at 37°C for 8 h and then lysed with a RNeasy Kit. (PDF)Click here for additional data file.S12 FigGroups of four guinea pigs were inoculated with 10 PFU of each chimeric M-encoding virus, as indicated. Graphs show individual titers obtained from animals used in three independent experiments. Virus replication in nasal wash of inoculated animals was measured by plaque titration at days 2, 4, 6, and 8 post-infection and the titers at each time point were plotted (dotted lines). The differences between PR8 NL09 M 7 mut Av and avian M 9 mut NL encoding viruses were considered significant. Statistical significance in kinetics of growth was determined by assessing the interaction of time and virus using repeated measures, two-way, multiple comparisons ANOVA on mean values, with Bonferroni correction applied to account for comparison of a limited no of means.(PDF)Click here for additional data file.S13 Fig293T cells were inoculated with the indicated IAVs, encoding avian-, human- or PR8-derived M segments, at a MOI of 5 PFU/cell, or treated with 60 μM chloroquine. Cells were fixed 8 h later and stained with anti-M2  and DAPI (blue) followed by imaging with confocal microscopy. Examples of optical sections are shown, either as merged 2-color images or the red and blue channels alone . 63x magnification with 3x optical zoom. Brightness was adjusted for optimal clarity, with all images treated equally. CQ: chloroquine.(PDF)Click here for additional data file.S1 TableThe sequences analyzed to generate avian consensus sequences for M1 and M2 proteins are summarized.(DOCX)Click here for additional data file.


Tbx1, the gene for 22q11.2 deletion syndrome (22q11.2DS) and Foxi3, both required for segmentation of the pharyngeal apparatus (PA) to individual arches, genetically interact. We found that all Tbx1+/-;Foxi3+/- double heterozygous mouse embryos had thymus and parathyroid gland defects, similar to those in 22q11.2DS patients. We then examined Tbx1 and Foxi3 heterozygous, null as well as conditional Tbx1Cre and Sox172A-iCre/+ null mutant embryos. While Tbx1Cre/+;Foxi3f/f embryos had absent thymus and parathyroid glands, Foxi3-/- and Sox172A-iCre/+;Foxi3f/f endoderm conditional mutant embryos had in addition, interrupted aortic arch type B and retroesophageal origin of the right subclavian artery, which are all features of 22q11.2DS. Tbx1Cre/+;Foxi3f/f embryos had failed invagination of the third pharyngeal pouch with greatly reduced Gcm2 and Foxn1 expression, thereby explaining the absence of thymus and parathyroid glands. Immunofluorescence on tissue sections with E-cadherin and ZO-1 antibodies in wildtype mouse embryos at E8.5-E10.5, revealed that multilayers of epithelial cells form where cells are invaginating as a normal process. We noted that excessive multilayers formed in Foxi3-/-, Sox172A-iCre/+;Foxi3f/f as well as Tbx1 null mutant embryos where invagination should have occurred. Several genes expressed in the PA epithelia were downregulated in both Tbx1 and Foxi3 null mutant embryos including Notch pathway genes Jag1, Hes1, and Hey1, suggesting that they may, along with other genes, act downstream to explain the observed genetic interaction. We found Alcam and Fibronectin extracellular matrix proteins were reduced in expression in Foxi3 null but not Tbx1 null embryos, suggesting that some, but not all of the downstream mechanisms are shared.We investigated whether  Tbx1, the gene for 22q11.2 deletion syndrome and Foxi3, genetically interact in the third pharyngeal pouch endoderm during thymus and parathyroid gland development. When examining wildtype embryos, we found that each arch is surrounded by epithelial cells derived from the endoderm and ectoderm that undergo dynamic processes during PA segmentation. Invagination co-occurs with formation of multilayers of epithelia that become juxtaposed. The cells are then reorganized to form a dual-layer of tightly intercalated cells at the mature pouch-cleft junction. In Tbx1 and Foxi3 null mutant embryos, these processes are disrupted. Further, the endoderm cells form extensive multilayers in the region where cells should normally invaginate. When examining genes that may act downstream of Tbx1 and Foxi3 we found several including Notch pathway genes Jag1, Hes1, and Hey1 are downregulated in both mutant embryos. Together, we show that Tbx1 and Foxi3 are important for regulating PA segmentation through cellular and genetic mechanisms that may be critical in 22q11.2 deletion syndrome patients.The mechanisms required for segmentation of the pharyngeal apparatus (PA) to individual arches are not precisely delineated in mammalian species. Using mouse models, we found that two transcription factor genes,  The pharyngeal apparatus (PA) is an evolutionarily conserved structure that forms early in vertebrate embryos. The PA develops as a series of bulges, termed arches, found on the lateral surface of the head region of the embryo. During mammalian development, five pairs of pharyngeal arches numbered PA1, PA2, PA3, PA4, and PA6 (the fifth PA is transient) form subsequently, over time, from the rostral to caudal part of the head region of the embryo .–57.Foxi3endoderm  as compaendoderm . In cont embryos  in compa embryos . In zebr embryos . Fibrone- mutant  versus F embryos . Fibronencreased  as compancreased , which iTbx1 and Foxi3 in the formation of the thymus and parathyroid glands from the third pharyngeal pouch in mammals. Inactivation of Foxi3 in the Tbx1 domain resulted in absent thymus and parathyroid glands and inactivation in the endoderm resulted additionally in aortic arch defects that are similar as is observed in patients with 22q11.2DS. Expression of Jag1, Hey1 Fgf3, Pax8 and Pax9 was reduced in both Tbx1 and Foxi3 null mutant embryos, suggesting some shared downstream genes. We investigated the cellular mechanisms by which pharyngeal pouch-cleft junctions form in the process of pharyngeal segmentation. We found that the epithelial cells invaginate and form temporary multilayers in which cells become juxtaposed, repositioned and tightly intercalated to form junctions between the endoderm and ectoderm. Global inactivation of both genes resulted in failed invagination and excessive multilayers of endoderm cells. We identified autonomous and non-autonomous functions in this process. Together, this study adds new genetic, molecular and cellular insights into the process of pharyngeal segmentation in mammals.In this report, we found that there is a genetic interaction between Tbx1 and Foxi3. Our data indicates that there are a few major epithelial transitions required for morphogenesis, as shown in the model in During vertebrate embryonic development, the segmentation of the distal PA is needed to create individual arches that later form derivative structures including the thymus and parathyroid glands , 60. We A similar process has been described for branching morphogenesis to form pancreatic ducts from the distal foregut endoderm . In pancA few years ago, dynamic transitions of the pharyngeal endoderm were described in zebrafish . During Tbx1 is expressed in the mesoderm as well as the epithelia. In zebrafish, mesodermal tbx1 regulates expression of wnt11r  and fgf8a (Fibroblast growth factor 8a) morphogens that signal to the PE to promote pouch formation [Tbx1 in the mesoderm results failed segmentation of the distal PA [Tbx1 in pharyngeal segmentation. It was previously found that Tbx1 has autonomous roles in the PE for segmentation of the distal PA using the Sox172A-iCre/+ allele [Tbx1 was inactivated using this allele. In Tbx1 null mutant embryos, excessive multilayers formed by E9.5. We did not observe excessive multilayers when Tbx1 was inactivated in the endoderm at E9.5, although there were some additional layers by E10.5. We speculate that excessive multilayer formation may be partially suppressed in endodermal conditional mutant embryos because Tbx1 is still expressed in the mesoderm.ormation . Similaristal PA . Thus, t+ allele . We alsoFoxi class genes, Foxi1, Foxi2 and Foxi3. In zebrafish, foxi1 has a similar expression pattern and function to that of Foxi3 [foxi1 resulted in a failure to transition from a multilayered epithelium to a simple dual layered pharyngeal pouch and excessive multilayers formed [Foxi3 function in mammals, however we found that Foxi3 is also required in the invagination process. Further, in zebrafish, foxi1 appears to have its major role in the ectoderm to signal non-autonomously to the PE [foxi1 null mutant zebrafish, Alcama expression was normal. In contrast, in the mouse, loss of Foxi3 in the PE resulted in failed segmentation along with reduced Alcam expression. It is possible that ectodermal Foxi3 might have important signaling roles, but these would be independent to PE functions. Nevertheless, it appears that there are some differences in the function of these homologs in different vertebrates. There are also some differences in zebrafish in regards to tbx1 function. In zebrafish, the tbx1 gene does not have an autonomous role in the PE in pouch formation [Tbx1 has autonomous roles in the PE in the mouse.In mammals and zebrafish, there are three of Foxi3 . Recentls formed . This iso the PE . In foxiormation . In contFoxi3 and Tbx1 null mutant embryos, implicate some mechanistic differences in gene function on the extracellular milieu. Eph-ephrin signaling in adjacent cells is important for cell migration. In zebrafish, EphB2 and EphB3 are required to maintain E-cadherin expression during budding morphogenesis of the endoderm from the foregut [foxi1 mutant zebrafish, expression of EphrinB2a was not changed [Foxi3 mutant mouse embryos, and therefore is not directly implicated downstream of Foxi3.Differences in Alcam expression levels in  foregut . During  foregut . In foxi changed . Ephrin2foxi1 in the pharyngeal ectoderm initiates wnt4a signaling and that this is required for the second transition of the endoderm to form a final mature segment [Foxi3 conditional mutant mouse embryos, the ectoderm was able to invaginate properly, but segmentation failed. It is possible that endodermal cells might not have been able to properly respond to signals emanating from the ectoderm. Our data also indicates that invagination of the ectoderm is not dependent on invagination of the endoderm. Rather, the ectoderm can initiate invagination independently. This is consistent with studies performed in shark and chick embryos where the endoderm remains separate from the ectoderm throughout epithelial cell invagination [In zebrafish,  segment . In endogination . FurtherTbx1 and Foxi3 null mutant embryos. Fgf8 [Fgf8 in the pharyngeal epithelia in mouse embryos resulted in similar phenotypic defects in the distal pharyngeal apparatus as the Tbx1 null mutant embryos [Fgf8 expression is reduced in the pharyngeal pouches in Tbx1 null mutant mouse embryos and the two genes, Tbx1 and Fgf8 genetically interact [fgf3 and fgf8 are required for segmentation of the distal PA in zebrafish [Fgf8 expression was reduced Foxi3 null mutant mouse embryos and addition of exogenous fgf3 partially rescued defects in foxi1 morphants [Fgf3 is reduced in expression in Foxi3 mutant embryos. This suggests that Tbx1 and Foxi3 might act in the same genetic pathway as Fgf3 and Fgf8 as well as other FGF ligand genes. We found Pax8 and Pax9 expression was also reduced in the PE in Foxi3 and Tbx1 null mutant embryos, and it is possible that in particular, Pax9 is critical for PA segmentation [Tbx1 and Foxi3 null mutant embryos as well.The FGF signaling pathway was previously found to be disrupted in the pharyngeal epithelia of both os. Fgf8  is expreos. Fgf8  and mousos. Fgf8 –68. Inac embryos . Previouinteract . Both fgebrafish . Relevanorphants . As expeentation . In addiJagged1 (Jag1), encoding a cell surface Notch ligand, was reduced in expression the pharyngeal arch epithelia in Tbx1 null mutant embryos [Hes1, encoding a Notch downstream effector, was reduced in expression in Tbx1 null mutant embryos and further, Hes1 null mutant embryos had similar thymus and aortic arch artery defects as Tbx1 null mutant embryos [Eya1 in mouse models [Jag1, Hes1 and Hey1 expression was altered or reduced in the pharyngeal epithelia [The Notch pathway has many diverse roles in embryogenesis by regulating Notch effectors of the Hey/Her/Hes class of transcription factors. We previously noted that  embryos . In anot embryos . More ree models . In thatpithelia .Jag1, Hes1 and Hey1 were reduced in expression in Tbx1 and Foxi3 null mutant mouse embryos. Tbx1 is still expressed in the abnormal pharyngeal epithelia in Foxi3 null embryos, suggesting that perhaps the loss of expression that is observed might be due to downregulation of expression of Notch pathway genes and other genes described above. It is possible that some of the defects that were observed could be due to reduction of Notch signaling. Although our data support a possible role, it is not known if Notch pathway genes are required for segmentation of the PA. Therefore, more studies are needed to be done in the future to test a possible role for Notch signaling in this process.In this report, we found that TBX1 is the major candidate gene for these defects, and it is required for PA segmentation [Tbx1 may act upstream of Foxi3 in this process. One question is whether individuals might be identified that have mutations in FOXI3. There has been one report of a patient with a deletion of one allele of FOXI3 that had severe ear defects, mild craniofacial defects, and missing arteries derived from PA1 and PA2 [Patients with 22q11.2DS have defects within structures derived from the PA including craniofacial dysmorphism, T-cell deficiencies or dysfunction, hypocalcemia, as well as aortic arch and cardiac outflow tract defects . TBX1 isentation , 18, 20. and PA2 . These sFOXI3 or other downstream genes, such as FGF pathway genes, PAX9, or Notch pathway genes, might act as potential modifiers of phenotype in individuals with 22q11.2DS. Analysis of sequence from a large cohort of individuals with 22q11.2DS will be required to test this possibility.The phenotypic expression of 22q11.2DS varies extensively, implicating the existence of genetic or environmental modifiers. It would be interesting to determine whether DNA sequence variants in https://www.einstein.yu.edu/administration/animal-studies/), IACUC protocol # 2016–0507.All experiments using mice were carried out according to regulatory standards defined by the National Institutes of Health and the Institute for Animal Studies, Albert Einstein College of Medicine (Foxi3f/f (flox = f), Foxi3+/- [Tbx1Cre/+ [Sox172A-iCre/+ [Tbx1+/- [ROSA26GFPf/+ (RCE: loxP) [Foxi3-/- embryos were generated by inter-crossing Foxi3+/- mice. Double Tbx1 and Foxi3 heterozygous embryos were generated by inter-crossing Tbx1+/- and Foxi3+/- mice. Tbx1Cre/+;Foxi3f/f and Sox172A-iCre/+;Foxi3f/f embryos were generated by crossing male Tbx1Cre/+;Foxi3f/+ or Sox172A-iCre/+;Foxi3f/+ mice with Foxi3f/f females. Foxi3+/-, Tbx1+/-, Tbx1Cre/+;Foxi3f/+, Sox172A-iCre/+;Foxi3f/+, Sox172A-iCre/+;Tbx1f/+ and wildtype littermates were used as controls for the experiments, as indicated.The following mouse mutant alleles used in this study have been previously described: Foxi3+/- , Tbx1Crebx1Cre/+  Sox172A-A-iCre/+ , Tbx1+/-[Tbx1+/-  and ROSAE: loxP) . Foxi3-/Foxi3+/-, Sox172A-iCre/+, and Tbx1Cre/+ mice were backcrossed 10 generations to a Swiss Webster background from a mixed C57Bl/6, Swiss Webster background. The PCR strategies for mouse genotyping have been described in the original reports and are available upon request.The http://www.einstein.yu.edu/histopathology/page.aspx). A total of 80 embryos, including controls, at E15.5 were obtained from more than 10 independent crosses and analyzed morphologically using light microscopy. Fisher’s exact test was used to determine if parathyroid and thymus gland defects were significant in Tbx1+/-; Foxi3+/- compared to Tbx1+/- embryos.Mouse embryos were isolated in phosphate-buffered saline (PBS) and fixed overnight in 10% neutral buffered formalin (Sigma Corp.). Following fixation, the embryos were dehydrated through a graded ethanol series, embedded in paraffin and sectioned at 10 μm. All histological sections were stained with hematoxylin and eosin (H&E) using standard protocols in the Einstein Histopathology Core Facility  where each nuclei and mRNA signal dot were counted. Each probe was calculated separately. The ratios of cell number to number of signal dots was calculated for each embryo (n = 3). P-values were determined using the Student’s t-test.RNAscope escribed . Tissue in situ hybridization with non-radioactive probes was performed as previously described [Foxi3 [Tbx1 [Jag1 [and Hey1 [Hes1 was generated from a cDNA plasmid clone [in situ hybridization protocol, the embryos were fixed in 4% PFA and then dehydrated through a series of graded ethanol steps, embedded in paraffin, and sectioned at 10 μm thickness. Minimum of 2–4 embryos from 2–3 independent litters were analyzed for each experiment.Whole-mount RNA escribed , 77, usid [Foxi3 , Tbx1 [7i3 [Tbx1 , Jag1 [7x1 [Jag1 , and Heyand Hey1 . The proid clone , 81. FolEmbryos were collected at various stages: E8.5 (7–10 somite pairs), E9.0 (15–19 somite pairs), E9.5 (20–23 somite pairs) E10.0 (24–29 somite pairs), and E10.5 (30–33 somite pairs). Fixation was carried out in 4% PFA in PBS at 4°C for two hours. After fixation, tissue was washed in PBS and then cryoprotected in 30% sucrose in PBS overnight at 4°C. Embryos were embedded in OCT and cryosectioned at 10 μm. After fixation, frozen sections were obtained as described and then permeabilized in 0.5% Triton X-100 for 5 min. Blocking was performed with 5% goat serum in PBS/0.1% Triton X-100 (PBT) for 1 hour. Primary antibody was diluted in blocking solution and incubated for 1 hour. Primary antibodies used included: E-cadherin , ZO-1 , Fibronectin , Alcam , Ephrin b2  and GFP (ab6673 1:500 goat). Proliferation of cells was assessed by immunofluorescence using antibody anti-phospho Histone H3 (Ser10), which is a mitosis marker (06–570 Millipore). Sections were washed in PBT and incubated with a secondary antibody for 1 hour. Secondary antibodies used were Alexa Fluor 488 goat a-mouse IgG (Invitrogen A32723) at 1:500 and Alexa Fluor 568 donkey a-rabbit IgG (Invitrogen A11019) at 1:500. Slides were mounted in hard-set mounting medium with DAPI (Vector Labs H-1500). Images were then captured using a Zeiss Axio Observer microscope with an apotome.Tbx1-/-, Foxi3-/-, and Sox172A-iCre/+;Foxi3f/f embryos, which were collected and stained with an antibody for E-cadherin. To ensure that the cell quantification was accurate, we counted E-cadherin positive cells in the PA in every other section throughout each embryo. We did not count epithelial cells that were not part of the PA. When counting cells, we matched the embryos by stage using somite counts, and we matched the sections by position within the embryo. We also ensured that for each pair of control and mutant embryos, we counted the same number of sections (10–12 per embryo). We counted all phosphoH3 positive epithelial cells in each section and calculated the ratio of proliferating cells within the pharyngeal epithelium. For Tbx1-/- mutant embryos, since the PA is shorter in comparison to WT littermates, we also calculated the size of the PA using ImageJ. We then counted the E-cadherin positive cells of the PA, marking the epithelium, and divided this number by the size of the PA. The mean and standard error of the average cell counts for controls and mutant embryos were determined and they were compared using the t-test. Representations of the complete PA region from at least 3–6 embryos per genotype from at least 3 independent litters were used in each assay.To count epithelial cell number, we obtained 10 μm serial coronal sections of control, S1 FigFoxi3+/- (A-B), Tbx1+/- (C-D) and Foxi3+/-;Tbx1+/- (E-F) embryos at E15.5. Abbreviations: parathyroid (PT), thyroid (T) and thymus (Thy). (G-H) Tables summarizing parathyroid (G) and thymus (H) defects observed. Numbers of glands observed was indicated (two per embryo). Foxi3+/-, n = 8; Tbx1+/-, n = 13 and Foxi3+/-;Tbx1+/-, n = 14 embryos at E15.5. Normally parathyroid glands are adjacent to thyroid glands (A). Parathyroid glands were scored as being ectopic when they were more caudally located than the thyroid glands in mutant embryos . Thymus glands were noted as hypoplastic that were smaller in size (F) than normal . Ectopic thymus glands were scored as such that were more rostrally located in comparison to control embryos (F). (I-M) Coronal histology sections stained with H&E of WT (I-J) and Tbx1+/-;Foxi3+/- (K-M) embryos at E10.5. Black arrows indicate the third pharyngeal pouch (I-M). Related to Figs (A-F) Transverse histology sections stained with H&E of (TIF)Click here for additional data file.S2 FigFoxi3 mRNA probe on Tbx1Cre/+;Foxi3f/+ control (A-B), Tbx1Cre/+;Foxi3f/f conditional mutant embryos (C-D), Sox172A-iCre/+;Foxi3f/+ control (E-F) and Sox172A-iCre/+;Foxi3f/f conditional mutant embryos (G-H) with corresponding coronal sections. Black arrows indicate where Foxi3 mRNA expression is reduced within conditional mutant embryos. Related to Figs (A-H) WMISH using antisense (TIF)Click here for additional data file.S3 FigTbx1Cre/+;Foxi3f/f mutant (D-F) embryos. (G-H) India ink was injected into the ventricle of Tbx1Cre/+;Foxi3f/+ control (G) and mutant (H) embryos at E10.5 to visualize the aortic arches. (I-L) Transverse histology sections of Tbx1Cre/+;Foxi3f/- mutant embryos at E15.5. Abbreviations: right subclavian artery (R. sub), thymus (Thy), aorta (Ao), pulmonary trunk (PT), right atrium (RA), left atrium (LA), right ventricle (RV), left ventricle (LV), ventricular septum (VS). Related to Figs (A-F) Transverse histology sections stained with H&E of WT control (A-C) and (TIF)Click here for additional data file.S4 FigFoxi3+/- control (A), Foxi3-/- (B), and Sox172A-iCre/+;Foxi3f/f (C) embryos at E9.5. Arrow in A indicates PA3. Asterisks in B, indicate the hypoplastic first arch and in C, indicate the distal PA that failed to segment to arches. (D-F) Transverse histology sections stained with H&E of Sox172A-iCre/+;Foxi3f/f embryos at E15.5. RRSA is indicated (D), IAAB is present and indicated by the yellow arrow (E). Abbreviations: aorta (Ao), right ventricle (RV), left ventricle (LV), and retro-esophageal right subclavian artery (RRSA). (G-L) India ink was injected into the ventricle of WT control , Foxi3-/-  and Sox172A-iCre/+;Foxi3f/f  embryos at E10.5. The right and left side of these embryos are shown. Arrows indicate absent 4th aortic arch arteries in both genotypes of mutant embryos. Related to Figs (A-C) Whole mount images of (TIF)Click here for additional data file.S5 FigTbx1-/- mutant embryos (D-F) using a phospho-H3  antibody to mark cells undergoing mitosis. E-cadherin antibody (green) was utilized to visualize the epithelial cells. E8.5 (A and D) and E9.5 (B-C and E-F) staged embryos were analyzed. White boxes in B and E indicate area of magnification; n = 3 for both stages and genotypes. (G) Quantification of proliferation assay. The mitotic index is the ratio of proliferating cells to total cell counts of epithelium within the PA. The t-test was used to calculate P-values as shown. (H-K) DAPI (blue), E-cadherin (green), and ZO-1 (red) antibodies were used to visualize epithelial cells within the PA. Epithelial cells in WT (H-I) Tbx1-/- (J-K) coronal sections were visualized at E8.5 (H and J) and E9.5 (I and K); n = 3 each genotype. PE indicates pharyngeal endoderm. Arrows in H, indicate the position in the PA where cells are invaginating. (L) Quantification of cell numbers to proportion of the size of the PA. (M-P) Sox172A-iCre/+;Tbx1f/+ controls and Sox172A-iCre/+;Tbx1f/f conditional mutant embryos at E9.5 (M-N) and E10.5 . Related to (A-F) Proliferation assay on coronal sections of WT (A-C) and (TIF)Click here for additional data file.S6 FigFoxi3-/- (E-H), and Sox172A-iCre/+;Foxi3f/f (I-L) mutant embryos at E8.5 and E9.5. At E8.5 and E9.5; a total of n = 6 and n = 4, respectively, were analyzed for each control and mutant embryo. Related to (A-L) Proliferation assay was performed using a phospho-H3 (pH3) antibody on coronal sections of WT control (A-D), (TIF)Click here for additional data file.S7 FigIsl1 antisense probe on WT (A), Foxi3-/- (B), and Tbx1-/- (C) mutant embryos at E9.5. (D-E) WMISH was performed using an Fgf3 probe on WT (D) and Foxi3-/- (E) mutant embryos at E9.5. (F-H) WMISH was performed using a Pax8 probe on WT (F), Foxi3-/- (G) and Tbx1-/- (H) mutant embryos at E9.5. (I-K) Pax9 probe on WT control (I), Foxi3-/- (J) and Tbx1-/- (K) mutant embryos; n = 2–4 for each probe and genotype. (L-M) RNAscope in situ hybridization with an mRNA probe for Pax9 (green) on coronal sections in WT (L) and Foxi3-/- (M) embryos at E9.5; n = 2. PE indicates the pharyngeal endoderm. Related to (A-C) WMISH was performed using an (TIF)Click here for additional data file.


Z)-endoxifen (E) were quantified using LC-MS/MS. Genotyping for CYP2D6, CYP2C9, CYP2C19, CYP3A5, POR, and ABCB1 and UGT2B15 and copy number variation for CYP2D6 were done. The proportion of patients with low endoxifen level (<5.9 ng/mL) was 35.8% (median concentration 7.94 ng/mL). The allele frequency of CYP2D6 gene deletion (*5) and duplication (*1×N or *2×N) was 4.3% and 14.8%, respectively. Twenty-six percent of the patients carried duplicated or multiplicated CYP2D6 gene. An increase in CYP2D6 activity score was associated with increased endoxifen concentration and MRE/NDM (p < 0.001). The IIV in endoxifen concentration and MRE/NDM was 74.6% and 59%, respectively. CYP2D6 diplotype explained 28.2% and 44% of the variability in absolute endoxifen concentration and MRE/NDM, respectively. The explanatory power of CYP2D6 diplotype was improved among ABCB1c.4036G carriers  compared to A/A genotype. CYP2C9, CYP2C19, and CYP3A5 genotypes had no significant influence on endoxifen concentration or MRE/NDM. In conclusion, we report a high rate of low endoxifen level as well as large IIV in tamoxifen and its metabolite concentrations. CYP2D6 is significant predictor of plasma endoxifen level in a gene-dose dependent manner.Tamoxifen displays wide inter-individual variability (IIV) in its pharmacokinetics and treatment outcome. Data on tamoxifen pharmacokinetics and pharmacogenetics from black African breast cancer patient populations is lacking. We investigated the pharmacokinetic and pharmacogenetic profile of tamoxifen and its major active metabolite, endoxifen, in Ethiopian breast cancer patients. A total of 81 female breast cancer patients on adjuvant tamoxifen therapy were enrolled. Tamoxifen (Tam) and its major metabolites, N-desmethyltamoxifen (NDM), 4-hydroxy-tamoxifen (4-HT), and ( Tamoxifen is widely used as adjuvant therapy for women with estrogen receptor-positive breast cancer . Its useCYP2D6, CYP3A4, CYP3A5, CYP2C9, and CYP2C19, with key roles for CYP2D6 and CYP3A   mid-level QC [QCM] (8 ng/mL of E and 4-HT and 80 ng/mL of NDM and Tam), and (iii) high level QC [QCH] (80 ng/mL of E and 4-HT and 800 ng/mL of NDM and Tam) were also included at each run.Blood samples were collected in EDTA tube, centrifuged at 2500 CYP2D6 and tamoxifen therapy [CYP2D6 activity score (AS) was then determined as the sum of the values assigned to each allele of the diplotype. Accordingly, CYP2D6 AS was translated into patient’s phenotype status as poor metabolizer, PM (AS = 0), intermediate metabolizer, IM (AS = 0.5), normal metabolizer, NM (AS = 1.5–2) and ultra-rapid metabolizer, UM (AS > 2). Patients with AS of 1 were classified as a CYP2D6 normal metabolizer or intermediate metabolizers (NM or IM).Phenotype classification from genotype was based on the recent Clinical Pharmacogenetics Implementation Consortium (CPIC) guideline for  therapy . FunctioChi-square test was used to compare the observed and expected genotype frequency according to Hardy–Weinberg equilibrium. The plasma concentrations of tamoxifen and its active metabolites were described as median with interquartile range (IQR). Interpatient variability (IIV) of plasma endoxifen and endoxifen/N-desmethyltamoxifen metabolic ratio (MRE/NDM), a marker for CYP2D6 metabolic activity, was described by coefficient of variation (%COV). ANOVA or independent t-test was used to determine the association of log transformed concentrations of tamoxifen and its metabolites and MRE/NDM across CYP2D6 diplotypes, phenotypes, non CYP2D6 genotypes , and other patient specific factors . Pearson correlation coefficient was determined to assess the magnitude of association between tamoxifen and its metabolites. The impact of CYP2D6 diplotype and phenotype to explain the absolute concentration of endoxifen or MRE/NDM was assessed using linear regression. Adjusted coefficient of determination (adjusted R2) was used to define the proportion of the variance in endoxifen concentration and MRE/NDM that could be predictable from CYP2D6 diplotype and phenotype. The data were analyzed using SPSS for Windows . Graphs were prepared using ggplot2 package in R (version 3.5.1) [Descriptive statistics were computed to explore the demographic characteristics, clinical profiles, genotype frequencies of participants. n 3.5.1) .CYP2D6 genotype and its activity score is a significant predictor of plasma endoxifen exposure. Tamoxifen therapy guided by CYP2D6 genotyping in clinical practice may assist treatment decision-making for optimal patient benefit. Therapeutic drug monitoring is also recommended for those with normal metabolizer phenotypes. The marginal effect of POR and ABCB1 on tamoxifen and its metabolites PK found in this study needs further investigation in a larger sample size. Despite the simplified AS system recommended by CPIC for genotype-phenotype translation, our study signals further refinement of the approach, probably considering other sources of variability such as POR and ABCB1. Future studies are also recommended to investigate the impact of the CYP2D6 genetic variations on the clinical outcome of breast cancer in Ethiopian patients.In conclusion, we report a high rate of low plasma endoxifen level and wide between-patient variability in plasma concentration of endoxifen, the main therapeutically active metabolites of tamoxifen.