Notch signaling pathway is one of the most important pathways to regulate intercellular signal transduction and is crucial in the regulation of bone regeneration. Nephroblastoma overexpressed (NOV or CCN3) serves as a non-canonical secreted ligand of Notch signaling pathway and its role in the process of osteogenic differentiation of mesenchymal stem cells (MSCs) was undefined. Here we conducted a comprehensive study on this issue. In vivo and in vitro studies have shown that CCN3 significantly inhibited the early and late osteogenic differentiation of mouse embryonic fibroblasts (MEFs), the expression of osteogenesis-related factors, and the subcutaneous ectopic osteogenesis of MEFs in nude mice. In mechanism studies, we found that CCN3 significantly inhibited the expression of BMP9 and the activation of BMP/Smad and BMP/MAPK signaling pathways. There was also a mutual inhibition between CCN3 and DLL1, one of the classic membrane protein ligands of Notch signaling pathway. Additionally, we further found that Hey1, the target gene shared by BMP and Notch signaling pathways, partially reversed the inhibitory effect of CCN3 on osteoblastic differentiation of MEFs. In summary, our findings suggested that CCN3 significantly inhibited the osteogenic differentiation of MEFs. The inhibitory effect of CCN3 was mainly through the inhibition of BMP signaling and the mutual inhibition with DLL1, so as to inhibit the expression of Hey1, the target gene shared by BMP and Notch signaling pathways. In the process of mammalian growth and cell differentiation, proliferation, and apoptosis, Notch signaling pathway plays an irreplaceable role2. It is also crucial in the process of bone regeneration6. The ligands  and receptors  of canonical Notch signaling pathway are cell membrane proteins that can interact to activate Notch signaling pathway only when cells adhere to each other8, thus regulating the expression of the target genes, such as Hey112. The current study found that, in addition to the canonical membrane protein ligands, there is also a secreted ligand for the Notch signaling pathway: nephroblastoma overexpressed (NOV/CCN3). It can affect the Notch signaling pathway when cells do not adhere to each other15. CCN3 is a member of the CCN family and is widely expressed in the nervous system, musculoskeletal system, and so on. It is found in high expression levels both during bone development and fracture healing21.Notch signaling pathway is one of the highly conserved signaling pathways in the process of biological evolution24. However, as a non-canonical secreted ligand of the Notch signaling pathway, how does CCN3 affect the osteogenic differentiation of MSCs? How does it coordinate with cell membrane ligand DLL1 to influence the osteogenic differentiation of MSCs? These are the primary coverage of our research.Our previous research has confirmed that the Notch signal activated by canonical membrane protein ligand DLL1 can promote bone morphogenetic protein (BMP)  induced osteogenic differentiation of mesenchymal stem cells (MSCs) by affecting BMP signaling25. MEFs have the characteristics of MSCs in vitro and in vivo25 and are considered to be an important cell line for MSC-related experiments28. So, in this study, we used the overexpressing CCN3 adenoviruses (Ad-CCN3) and small interfering CCN3 adenoviruses (Ad-siCCN3) to upregulate and downregulate CCN3 levels, respectively, in MEFs and explored the effects and mechanisms of CCN3 on osteoblastic differentiation of MEFs, so as to provide theoretical basis for the clinical application of CCN3-related proteins and factors in bone tissue engineering. Our results showed that CCN3 significantly inhibited the osteogenic differentiation of MEFs mainly through the inhibition of BMP signaling and the mutual inhibition with DLL1, so as to inhibit the expression of Hey1.Mouse embryonic fibroblasts (MEFs) are multipotent progenitor cells with the capacity of differentiating into tissues of both mesenchymal and non-mesenchymal origin5 (at a density of 80%), the average cycle threshold (Ct) value of the CCN3 expression was 22.42. The results of ELISA assay showed that, when the number of MEFs reached 2.8 × 105 (at a density of 40%), the concentration of CCN3 in the supernatant of cell culture was 0.27 ng/mL, while the concentration of CCN3 in the supernatant of cell culture was 1.01 ng/mL when the number of MEFs reached 5.8 × 105 (at a density of 80%)  and enzyme-linked immunosorbent assay (ELISA). The results of qRT-PCR showed that when the number of MEFs reached 5.8 × 10We sought to explore whether CCN3 had any effect on early and late osteogenic indicators or not. Alkaline phosphatase (ALP) assays was used to detect the changes of ALP activity, one of the early osteogenic indicators, and Alizarin Red S staining was used to detect the changes of calcium deposition, one of the late osteogenic indicators. We examined the effectiveness of Ad-CCN3 Fig.  and Ad-sRunx2 is crucial in the process of osteogenesis. Runx2, osteopontin (OPN), and osteocalcin (OCN) are key indicators for osteogenic differentiation of MSCs. Thus we investigated the effects of CCN3 on the expression of Runx2, OPN, and OCN. Our results revealed that the expression levels of Runx2, OPN, and OCN in the BMP9+CCN3 group was significantly downregulated compared to the BMP9+GFP group Fig. ; oppositBased on the conclusion that CCN3 inhibited osteogenic differentiation of MEFs in vitro, we further confirmed it by subcutaneous ectopic osteogenesis in nude mice. The general observation and the results of micro-CT three-dimensional (3D) reconstruction showed that, compared with the BMP9 group, the volume of osteogenic masses was decreased in the BMP9+CCN3 group, while increased in the BMP9+siCCN3 group. The siCCN3 group also showed a strong ability to promote the formation of osteogenic masses Fig. . Further24. Mitogen-activated protein kinases (MAPKs) are key branch of non-Smad BMP pathway. Zhao had demonstrated that BMP9 was able to activate extracellular signal–regulated kinase 1/2 (ERK1/2) and p38 MAPKs in mycobacterial phagosomal compartments and MAPKs ) play positive roles in BMP2-induced osteogenic differentiation29. Then does CCN3 also exert any impact on this signaling pathway? We further examined the effects of CCN3 on canonical and non-canonical BMP9 signaling pathways. The results of qRT-PCR showed that CCN3 downregulated the mRNA level of BMP9 predominantly  through BMP signaling pathwaytly Fig.  and siCCtly Fig. . Howevertly Fig. , and thetly Fig. , while Dtly Fig. . The restly Fig.  and hightly Fig.  than in tly Fig.  and incrtly Fig. . Taken tWe have confirmed that both non-canonical ligand CCN3 (secretory protein) and canonical ligand DLL1 (membrane protein) of Notch signaling pathway affected osteogenic differentiation of MEFs by regulating BMP signaling, and CCN3 inhibited osteogenic differentiation while DLL1 promoted osteogenic differentiation. We hypothesized that there was a competitive relationship between CCN3 and DLL1, and they coordinate with each other to regulate osteogenic differentiation of MEFs.To confirm this hypothesis, we used qRT-PCR to determine the mRNA levels of the canonical receptors and ligands of Notch signaling pathway. The results showed that the mRNA levels of all canonical receptors were higher in the CCN3 group than in the GFP group, prompting the activation effect of CCN3 on Notch signaling pathway. Conversely, lower mRNA levels of the ligands such as DLL1 and DLL3 were found in the CCN3 group, as compared with the GFP group Fig. ; siCCN3 30 had demonstrated that Hey1 played an indispensable role in BMP9-induced osteogenic differentiation of MSCs. Thus we investigated the role Hey1 played in the process of CCN3-induced inhibition of osteogenic differentiation of MSCs. First, we found that, for the expression of Hey1, CCN3 exerted a significant negative regulatory effect , which was consistent with the results of Katsube and others37. Matsushita et al.17 upregulated and downregulated CCN3 levels in vivo and found that CCN3 had the ability to significantly inhibit osteogenic differentiation, which was similar to the results of Rydziel and others39. However, there are also some authors who found that CCN3 can promote osteogenic differentiation of MSCs: Tan et al.40 pointed out that CCN3 could promote the osteogenic differentiation of rat primary osteoblasts; in the process of studying odontogenic differentiation of dental pulp stem cells (DPSCs), which was similar to the biological pathway of osteogenic differentiation of MSCs, Wang et al.41 found that CCN3 could promote odontogenic differentiation of DPSCs in vitro and in vivo.CCN3/NOV is a member of the CCN family that was originally found in the study of finding the specific integration of myeloblastosis-associated virus type 1 in avian nephroblastoma37. Additionally, our in vivo experiments indicated that CCN3 could inhibit subcutaneous ectopic osteogenesis in nude mice, reduce the volume of osteogenic mass, and the values of BV/TV, Tb.N, and Tb.Th. Meanwhile, CCN3 also increased the value of SMI and decreased the maturation level of osteogenic differentiation of MEFs, which were in accordance with the results of Rydziel and others39. Taken together, through in vitro and in vivo experiments, we confirmed the inhibitory effect of CCN3 on osteogenic differentiation of MEFs, and we systematically and comprehensively discussed the possible mechanisms of CCN3 affecting the osteogenic differentiation of MEFs afterwards.To investigate the exact role of CCN3 in osteogenic differentiation of MSCs, we used MEFs with MSC characteristics in this study, upregulated and downregulated CCN3 levels by Ad-CCN3 and Ad-siCCN3, and detected the effects of CCN3 on ALP activity (one of the early osteogenic indicators), calcium deposition (one of the late osteogenic indicators), expression of osteogenic-related factors, and subcutaneous ectopic osteogenesis of MEFs in nude mice through in vitro and in vivo experiments. Our results demonstrated that CCN3 significantly inhibited the early and late osteogenic differentiation of MEFs and the expression of osteogenic factors Runx2, OPN, and OCN. Meanwhile, CCN3 also inhibited the osteogenic differentiation of MEFs induced by BMP9. These results were consistent with the in vitro findings of Katsube and others38 or promoting the expression of gremlin, a BMP2 antagonist42. Simultaneously, CCN3 also exerts a regulatory effect on Notch and Wnt/β-Catenin signaling pathways38.Recent studies have indicated that CCN3 inhibits the osteogenic differentiation mainly by binding and neutralizing BMP243, whereas Notch signaling alone exerted no effect but could significantly promote BMP9-induced osteogenic differentiation24 during this process, we hypothesized that CCN3 plays an inhibitory role by affecting BMP9 and Notch signaling pathway. We first examined the effect of CCN3 on BMP9 expression. The results of qRT-PCR showed that CCN3 could reduce the mRNA level of BMP9, which was different from the findings that CCN3 only had a direct/indirect inhibitory effect on BMP2 protein reported by Rydziel and others42. However, BMP9 could upregulate the expression of CCN3, which suggested that there may be some negative feedback between CCN3 and BMP9. Our further study showed that CCN3 could significantly inhibit the phosphorylation levels of Smad1/5/8, which was similar to that of Rydziel and others38. Additionally, CCN3 could also inhibit the phosphorylation levels of Erk1/2, p38, and JNK, which was different from the perspective pointed out by Rydziel et al.36 that CCN3 had an effect on BMP2/MAPK pathway, but mainly inhibited the phosphorylation levels of Erk1/2. In summary, we conclude that CCN3 inhibits the osteogenic differentiation of MEFs by inhibiting the gene expression of BMP9 and downregulating the BMP/Smad and BMP/MAPK signaling pathways.Since BMPs and Notch signaling pathway play an important role in osteogenic differentiation, and our previous studies have confirmed that BMP9 had a strong stimulative abilityWe further explored the effects of CCN3 on the canonical ligands and receptors of Notch signaling pathway. Our results revealed that CCN3 could promote the expression of Notch1, Notch2, Notch3, and Notch4, suggesting the activation effect of CCN3 on Notch signaling pathway. However, we found that CCN3 could inhibit the expression of DLL1, and in turn, DLL1 inhibited the expression of CCN3. Since our previous studies have confirmed that the Notch signaling activated by DLL1 could promote BMP9-induced osteogenic differentiation of MSCs, it can be speculated that CCN3 inhibited BMP9-induced osteogenic differentiation by downregulating the DLL1 expression. Meanwhile, CCN3 promoted the expression of Notch receptors, suggesting that it may also exert some effects through binding to Notch receptors, and the specific mechanism still needs further study.30. We further explored the exact role of Hey1 in the process of osteogenic differentiation of MEFs inhibited by CCN3. The results of qRT-PCR confirmed that CCN3 could significantly downregulate the mRNA level of Hey1, which was consistent with the research of Rydziel et al.36. Then we detected the effects of Hey1 on CCN3-inhibited osteogenic differentiation of MEFs by ALP staining and quantitative assays, micro-CT scanning, and analysis of subcutaneous bony masses in nude mice, hematoxylin& eosin (H&E) staining, Masson’s Trichrome staining, and immunohistochemical testing. Our results showed that Hey1 alone did not have the ability to induce osteogenic differentiation, which was similar to the results of Sharff et al.30 in BMP9-induced osteogenic differentiation. However, Hey1 significantly promoted BMP9-induced osteogenic differentiation, which was consistent with the results of Sharff et al.30 and Wang et al.44. Moreover, Hey1 could partially reverse the downregulation effect of CCN3 on BV/TV, Tb.N, and Tb.Th, inhibit the upregulation of SMI, and reverse the inhibitory effect of CCN3 on osteogenic differentiation of MEFs. These results suggested that CCN3 inhibits Hey1 through both BMP and Notch signaling pathways. Hey1 plays an important role in the osteogenic differentiation of MEFs inhibited by CCN3, and it is a key intermediary factor affecting the osteogenic differentiation of MEFs, but the mechanism needs further clarification.Our previous studies have confirmed that Hey1 plays an indispensable role in BMP9-induced osteogenic differentiation of MSCs. Hey1 is an important and direct target of BMP9 signaling and is upregulated at the immediate early stage of osteogenic differentiation. Silencing Hey1 expression reduced BMP9-stimulated osteogenic differentiation both in vitro and in vivoAs ligands of the Notch signaling pathway, DLL1 and CCN3 play different roles in osteogenic differentiation. CCN3 is a kind of secretory ligand, it also works when there are fewer cells; but DLL1 is a membrane ligand, only fuctions when the cell number is big enough to contact with each other. So we suspect that, when the amount of cells is little, CCN3 inhibits BMP signaling and Notch signaling protein ligand DLL1, thereby inhibiting the expression of Hey1, the common target gene of BMP and Notch signaling pathways, and inhibiting osteogenic differentiation. As the number of cells increases, DLL1 activates the Notch signaling pathway when cells are in contact with each other, thus regulating the expression of the target genes such as Hey1 and promoting osteogenic differentiation. In this process, the effect of CCN3 on cell proliferation and apoptosis needs further research and confirmation.36 while has promoted effect at low concentration 40. In our study, only adenovirus were used to upregulate and downregulate CCN3 levels, but no dose–effect studies were performed. Moreover, the way that CCN3 regulates the expression of BMP9 and DLL1 remains unclear and needs further study.In summary, this study identified the inhibitory effect of CCN3 on osteogenic differentiation of MEFs and further revealed that the inhibitory effect of CCN3 is mainly through the inhibition of BMP signaling and the mutual inhibition with DLL1 , so as to inhibit the expression of Hey1, the target gene shared by BMP and Notch signaling pathways. However, there are also some researchers who have put forward such views that CCN3 inhibits osteogenic differentiation at high concentration45, and maintained in complete Dulbecco’s modified Eagle’s medium (DMEM)  complemented by 10% fetal bovine serum (FBS)  and antibiotics . Cells were incubated at 37 °C in 5% CO2. Human colorectal cancer cells (HCT116) were obtained from the ATCC  and cultured in the same condition as MEFs. All experimental protocols were approved by the Ethics committee of Chongqing Medical University Approval.MEFs were isolated from post-coitus day 12.5 mice, as described previously30.DMEM high sugar medium was purchased from Hyclone Corporation (USA); FBS was obtained from Gibco Corporation (USA); ALP staining kit and substrate, vitamin C, β-phosphoglycerol, and Alizarin Red S were ordered from Sigma Corporation (USA); Anti-β-actin antibodies was obtained from Biyuntian Company ; Anti-Runx2, anti-OPN, anti-OCN antibodies, and the secondary antibodies were ordered from Cell Signaling Technology ; Anti-Smad1/5/8, anti-p38, anti-Erk1/2, and anti-JNK antibodies were obtained from Zhongshan Golden Bridge Biotechnology ; Anti- phospho-Smad1/5/8, anti-phospho-p38, anti-phospho-Erk1/2, and anti-phospho-JNK antibodies were ordered from Santa Cruz Corporation (USA). The recombinant adenoviruses including Ad-CCN3, Ad-siCCN3, Ad-GFP, Ad-RFP, Ad-BMP9, Ad-DLL1, and Ad-Hey1 were kindly provided by Dr Tong-Chuan He . GFP and RFP were used as tags for tracking the viruses, and Ad-GFP and Ad-RFP were used as vector controls. The effectiveness of the above viruses was confirmed in our and Professor He’s previous experiments2 cell culture dishes) were infected with an optimal titer of Ad-BMP9 or Ad-GFP control, exchanged the medium with DMEM medium (without serum and antibiotics) 4 h later, and collected the culture medium after 1 day and 2 days, respectively. The culture medium was centrifuged at 1000 rpm for 5 min and then placed at 4 °C for use.Subconfluent HCT116 cells  and reverse transcription was performed using the Takara Prime Script RT Reagent Kit. The first-strand cDNA products were further diluted five-fold and used as PCR templates. RT-PCR reactions were performed using a SYBR Premix Ex Taq Kit  on a PCR machine . All samples were normalized with the expression level of GAPDH and primers used are listed in Table MEFs were inoculated (at a density of 30%) in 24-well plates for 6 h and then infected with specific adenoviruses, followed by induced osteogenesis by BMP9 conditioned medium for 3/5/7 days after the fluorescence appeared. ALP staining and quantitative assays were measured at the specified time points. For ALP staining, MEFs were fixed with 4% formaldehyde, and the prepared ALP dyeing solution (250 μL/well) was added afterwards. Plates were protected from light and the staining results could be observed after 20 min. For ALP quantitative assay, MEFs were lysed with cell lysis buffer for 10 min and centrifuged at 13,000 rpm for 5 min. In another EP tube, 20 μL of the prepared ALP substrate was added, and 5 μL of cell lysate was pipetted into the tube, mixed well, and left standing for 60 min. The results were tested on Hitachi 7060C Automatic Biochemical Analyzer. Each assay condition was performed in triplicate and the results were repeated in three independent experiments. ALP activity was normalized by total cellular protein concentrations among the samples.2O. The results were recorded under microscope and repeated in at least three independent experiments.MEFs were inoculated (at a density of 30%) in 24-well plates for 6 h and then infected with specific adenoviruses, followed by induced osteogenesis by BMP9 conditioned medium, Vitamin C, and β-phosphoglycerol for 14 days after the fluorescence appeared. Alizarin Red S staining was used for matrix mineralization assay. MEFs were fixed with 0.05% (v/v) glutaraldehyde for 10 min and the mineralized matrices were stained with 0.4% Alizarin Red S for 5 min, followed by extensive washing with ddHg for 10 min to remove debris. Supernatants were collected and diluted five-fold. Expression levels of CCN3 protein were assayed using specific ELISA kits according to the manufacturer’s instructions .MEF cell culture supernatants were collected and cell culture media were centrifuged at 2000 × 2 cell culture dishes for 6 h and then infected with specific adenoviruses, followed by induced osteogenesis by Ad-BMP9 for 3 days after the fluorescence appeared. Cells were harvested and washed with cold phosphate-buffered saline at least three times. Proteins were extracted with cell lysis buffer  supplemented with proteinase inhibitor and phosphatase inhibitor. The cell lysates were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride membranes. The blots was blocked in 5% bovine serum albumin for 2 h at 37 °C, incubated in the primary antibody (diluted 1:1000) overnight at 4 °C, and then incubated in a secondary antibody-conjugated to horseradish peroxidase  for 1 h at 37 °C. Finally, the membrane was exposed with ECL .MEFs were inoculated (at a density of 30%) in 100 cm6 cells per injection) into the flanks of athymic nude (nu/nu) mice  until the fluorescence could been seen. At 4 weeks after injection, animals were euthanized, and the bony masses were collected for micro-CT imaging and histologic evaluation.MEFs were infected with specific adenoviruses and harvested for subcutaneous injection  . N-Recon software was used for 3D image reconstruction and all image data analysis was performed using the CT-AN software. Retrieved bony masses were decalcified with EDTA and then processed for paraffin embedding. Serial sections of embedded tissue were collected for H&E staining, Masson’s Trichrome staining, and immunohistochemical detection.x) ± standard deviation (SD). One-way analysis of variance was used to compare multiple groups. The t test was performed between two groups. P < 0.05 was considered as statistically significant.Statistical analysis was performed by the SPSS 17.0 software. Each experiment was repeated three times independently. The data was expressed as mean (Supplementary fig 1Supplementary fig 2Supplementary figure legends


Antidepressant drugs are a standard biological treatment for various neuropsychiatric disorders, yet relatively little is known about their electrophysiologic and synaptic effects on mood systems that set moment-to-moment emotional tone. In vivo electrical recording of local field potentials (LFPs) and single neuron spiking has been crucial for elucidating important details of neural processing and control in many other systems, and yet electrical approaches have not been broadly applied to the actions of antidepressants on mood-related circuits. Here we review the literature encompassing electrophysiologic effects of antidepressants in animals, including studies that examine older drugs, and extending to more recently synthesized novel compounds, as well as rapidly acting antidepressants. The existing studies on neuromodulator-based drugs have focused on recording in the brainstem nuclei, with much less known about their effects on prefrontal or sensory cortex. Studies on neuromodulatory drugs have moreover focused on single unit firing patterns with less emphasis on LFPs, whereas the rapidly acting antidepressant literature shows the opposite trend. In a synthesis of this information, we hypothesize that all classes of antidepressants could have common final effects on limbic circuitry. Whereas NMDA receptor blockade may induce a high powered gamma oscillatory state via direct and fast alteration of glutamatergic systems in mood-related circuits, neuromodulatory antidepressants may induce similar effects over slower timescales, corresponding with the timecourse of response in patients, while resetting synaptic excitatory versus inhibitory signaling to a normal level. Thus, gamma signaling may provide a biomarker  of the therapeutic effects of all classes of antidepressants. In vivo electrophysiology has been crucial for elucidating important properties of many neural systems, yet these electrical approaches have not yet been broadly applied to the actions of antidepressants. Antidepressant drugs are a standard treatment in a range of neuropsychiatric disorders, including but not limited to major depression, anxiety disorders, obsessive–compulsive disorder, and post-traumatic stress disorder , which are voltage waves in the brain that correspond closely with electroencephalographic (EEG) signals, and single neuron action potential firing patterns (spiking) is indeed an effective tool for elucidating circuit-specific neural mechanisms through which antidepressants and other centrally acting drugs produce their therapeutic effects  neurons], locus coeruleus [norepinephrine (NE) neurons], and ventral tegmental area (VTA) and substantia nigra [dopamine (DA) neurons] , serotonin–norepinephrine reuptake inhibitors (SNRIs), tricyclic antidepressants (TCAs), monoamine oxidase inhibitors (MAOIs), norepinephrine–dopamine reuptake inhibitors (NDRIs), miscellaneous antidepressants, novel compounds and potential antidepressants, and NMDA receptor (NMDAR) antagonists. Throughout the review, we describe how these recording data synergize with other techniques, such as in vivo microdialysis and behavioral assays, since these additional techniques were also often used in these studies. As a general rule, the existing studies on monoaminergic drugs appear to have focused on recording in the serotonergic, noradrenergic, and dopaminergic brainstem nuclei, with much less known about their effects on prefrontal or sensory cortex. A number of the studies include data from subregions of hippocampus, whereas there appears to be less known about how antidepressants affect amygdala electrophysiology  “Single + unit/neuron/cell + recording(s)” + antidepressant(s)/(see LIST below). (2) “Single + unit/neuron/cell + electrophysiology/electrophysiological” + antidepressant(s)/LIST. (3) “In vivo/awake + recording(s)” + antidepressant(s)/LIST. (4) “In vivo/awake + electrophysiology/electrophysiological” + antidepressant(s)/LIST. (5) “local field potential(s)” + antidepressant(s)/LIST. This LIST comprised the following terms : escitalopram, fluoxetine, citalopram, sertraline, paroxetine, fluvoxamine, bupropion, reboxetine, duloxetine, venlafaxine, desvenlafaxine, desipramine, nortriptyline, imipramine, clomipramine, amitriptyline, tricyclic, monoamine oxidase inhibitor, MAO inhibitor, tranylcypromine, phenelzine, moclobemide, selegiline, SSRI, SNRI, mirtazapine, ketamine, MK-801. These searches had some degree of redundancy and overlap, to minimize the chances of missing relevant hits. We included all relevant hits in this review.A number of in vivo recording studies in the presence of SSRIs show that these drugs acutely dampen firing of neurons in the dorsal raphe nucleus, a primary site of origin of serotonergic forebrain projection neurons  signaling. Acute treatment with fluoxetine in wild type mice did not affect mPFC firing, but did increase spike rates in 5-HT2A knockout mice, where these across-group differences may correspond to poorer novel object recognition in the wild type mice when given this drug  fluoxetine treatment normalized this elevated locus coeruleus firing  administration of SSRIs  to rats enhanced the firing rates of spontaneously active neurons in the ventral tegmental area (VTA), a major dopaminergic nucleus. Acute injection of 1 mg/kg paroxetine or fluoxetine (2.5 or 5 mg/kg) also increased the number of spontaneously active VTA cells, and an acute injection of 2.5 mg/kg fluoxetine increased firing in substantia nigra pars compacta neurons as well , has been shown to normalize suppressed dorsal raphe firing and increase tonic activation of 5-HT1A receptors in the hippocampus, unlike the SSRI paroxetine  de Boer  appears The NE reuptake inhibitor reboxetine appears to have opposite effects from SSRIs since it acutely enhances dorsal raphe firing as well as mPFC extracellular 5-HT, whereas citalopram reduced mPFC 5-HT , while having little effect on firing rate   regarding these studies: 1) most antidepressants acutely dampen firing in monoaminergic brainstem nuclei due to autoreceptor mediated inhibition, which typically desensitizes upon chronic drug administration; (2) the NMDAR antagonists ketamine and MK-801 acutely enhance gamma and high frequency oscillations. These two hypotheses  on dorsal raphe, locus coeruleus, and VTA, neural firing patterns. These studies consistently found that acute administration of drugs that boost 5-HT, NE, or DA, respectively, inhibit dorsal raphe, locus coeruleus, and VTA firing Crespi . SomatodThe data from the first major conclusion may also suggest that 5-HT and NE are functionally opposed in a number of brain circuits, which may be a general principle describing many of their interactions in vivo. A number of studies reviewed above contribute to this hypothesis. For example, release of these two transmitters is regulated in an opposing fashion by the VTA  when acutely administered. This was demonstrated in a number of the studies reviewed above, where many of these experiments were testing these drugs in the NMDAR hypofunction model of schizophrenia  gamma power induced by all therapeutically effective antidepressants corresponds to long-term neural changes that reset synaptic excitatory versus inhibitory signaling to a normal level (Fee et al. To summarize, here we have reviewed the literature on in vivo electrophysiologic studies in the presence of antidepressant drugs. While a number of pioneering studies have already been carried out, most prominently by Blier et al., our understanding of the effects of these drugs throughout the brain is not yet well developed. Underdeveloped areas for future inquiry include more recordings in awake, freely moving animals with a focus on neocortical areas, although subcortical structures such as amygdala, hippocampus, thalamus, and the brainstem monoaminergic nuclei are also not well characterized in the awake state. Studying neocortical areas in animals, including with respect to ketamine, has a particularly high degree of translational relevance since human EEG and TMS studies are best suited for monitoring or manipulating neocortex rather than deeper structures. A recent study in humans with treatment-resistant epilepsy, however, used intracranial EEG recordings to identify an amygdala-hippocampus subnetwork, where increased variability in beta-frequency coherence correlated with worsening mood (Kirkby et al. Additional lines of inquiry for future studies include comparing awake recordings with the asleep state, in the presence of these drugs. The potential interaction between psychological stress and antidepressants is also not well characterized, electrophysiologically. An understanding of the electrophysiological, circuit-based correlates of individual differences in response to antidepressants is also largely missing at this time. Since human subjects with major depression are in many cases therapeutically resistant to a number of antidepressant drugs, determining neural correlates of individual response in rodents would facilitate translational research toward precision medicine. Future recording studies of antidepressants should address these neglected aspects in the existing literature, especially the poorly understood neocortical effects of these drugs, and thereby elucidate all aspects of the circuitry underlying antidepressant drug response with the high degree of spatiotemporal precision afforded by in vivo electrophysiology, particularly when complemented by optogenetics. Studies of ketamine are especially amenable to electrophysiological investigation in animal models, in that its rapid therapeutic onset mirrors that seen in human subjects (Berman et al. An important issue raised by this literature review comprises the extent to which in vivo electrophysiological findings in rodents can be used to improve human pharmacotherapy, including through our understanding of human pharmaconeuroimaging data. We would suggest, for example, that our understanding of ketamine has been reinforced through parallel studies in rodents and humans, where this drug has been shown to boost the power of gamma oscillations across these species (Hakami et al. In conclusion, much of neuroscience is centered around the idea that action potential generation and synaptic communication are the final common language of nearly all neural functioning, which would include mood, mood regulation, anxiety and motivation. We suggest here that electrophysiologic techniques and understanding have now evolved to the point that they can be applied to problems with less well-defined inputs and outputs than the traditional sensory and motor studies performed by electrophysiologists. In vivo electrophysiology should be used more broadly now to enhance our basic understanding of neuropharmacology, cellular physiology, and both macro- and microcircuitry, with translational relevance for drug discovery and development, while also increasing the clinical effectiveness of antidepressant treatment in major depression and a range of other neuropsychiatric disorders.


Pocillopora damicornis and Vibrio coralliilyticus is commensal. An increase in temperature, or in the abundance of V. coralliilyticus, can turn this association pathogenic, causing tissue lysis, expulsion of the corals’ symbiotic algae (genus Symbiodinium), and eventually coral death. Using a combination of microfluidics, fluorescence microscopy, stable isotopes, electron microscopy and NanoSIMS isotopic imaging, we provide insights into the onset and progression of V. coralliilyticus infection in the daytime and at night, at the tissue and (sub-)cellular level. The objective of our study was to connect the macro-scale behavioural response of the coral to the micro-scale nutritional interactions that occur between the host and its symbiont. In the daytime, polyps enhanced their mucus production, and actively spewed pathogens. Vibrio infection primarily resulted in the formation of tissue lesions in the coenosarc. NanoSIMS analysis revealed infection reduced 13C-assimilation in Symbiodinium, but increased 13C-assimilation in the host. In the night incubations, no mucus spewing was observed, and a mucus film was formed on the coral surface. Vibrio inoculation and infection at night showed reduced 13C-turnover in Symbiodinium, but did not impact host 13C-turnover. Our results show that both the nutritional interactions that occur between the two symbiotic partners and the behavioural response of the host organism play key roles in determining the progression and severity of host-pathogen interactions. More generally, our approach provides a new means of studying interactions  between partners involved in complex holobiont systems, under both homoeostatic and pathogenic conditions.Under homoeostatic conditions, the relationship between the coral  Symbiodinium) has become emblematic of mutualistic symbiosis in the marine environment. Symbiodinium cells live intracellularly within coral cells and provide the coral animal with a reliable food source via translocation of photosynthates and their immediate derivatives . Concno acids –14 and dno acids .Symbiodinium and their host  prior to analysis, in order to test whether the rate of bacterial-derived enrichment was constant between fragments fixed at different time-points. Note that Symbiodinium cells with a diameter of less than 3 μm were excluded from the dataset because these were deemed unlikely to be representative of the response of whole cells.The resulting Symbiodinium and host datasets were analysed separately. Non-parametric Wilcoxon signed-rank tests were used to compare 13C-enrichment levels  in V. coralliilyticus-challenged corals with non-challenged control corals, because the data did not fulfil the criteria required for normality. Non-parametric Wilcoxon signed-rank tests were also used to test if the hourly rate of bacterial-derived 15N-enrichment was constant between fragments and within colonies, fixed at different time points.Fragments fixed at different incubation time-points were considered as independent replicates. V. coralliilyticus challenge modifies autotrophic C-assimilation in Symbiodinium and the coral host, coral fragments were challenged with 15N-labelled V. coralliilyticus suspended in 13C-labelled seawater, in the light. Their performance was compared to non-challenged fragments, which received 13C-labelled seawater only. Videos of both the non-challenged incubations and the V. coralliilyticus inoculations are provided as Supplementary Information  was similar in all fragments . The exception , showed a similar reduction in symbiont performance, but the difference was non-significant  and then challenged with 15N-labelled V. coralliilyticus and turnover of 13C monitored in non-labelled seawater in the dark. Again, their performance was compared to that of non-challenged corals, which received non-labelled seawater only. Videos of the non-challenged incubations and the V. coralliilyticus inoculations are provided as Supplementary Information  retained more ing Fig. .V. coralliilyticus in the host tissue  had 28% more , allowed us to identify pathogens in situ and test whether or not they obtained host-derived material (i.e. were labelled in 13C). Because the pathogens contained host-derived 13C (0.19 ± 0.11%), we tested whether there was an overall linear relationship between the enrichments in carbon and nitrogen, respectively, between pathogens and the host across time. A significant positive correlation  was observed between the APE 13C of V. coralliilyticus and the host  existed between the APE 15N of the bacteria and the host . The source of this phenomena cannot be attributed solely to light/dark dynamics, as some experiments conducted by Gavish et al. under dark conditions were more similar to the light infections observed here [The observed absence of mucus spewing by polyps in the second experiment resulted in the formation of a disperse mucus film on the surface of the fragments. As a result, coral epithelia (inner and outer surfaces) are likely exposed to ved here . The difVibrios are known to develop biofilms on both inanimate and living surfaces [V. coralliilyticus motility is unimpeded by anoxia [Vibrio virulence is not weakened, and may even be enhanced, in the dark. Because circadian rhythmicity exists in many biochemical pathways and in coral behaviour [Since surfaces , 52, it y anoxia , which oehaviour –56 this V. coralliilyticus in the light tended to display higher levels of carbon assimilation in the host tissue, than the control  that were challenged in the light, allocated less carbon to anabolic processes than Symbiodinium in control corals, despite remaining structurally intact  and/or the numerous proteins secreted by V. coralliilyticus during the infection process [Vibrio strain, such as V. fisheri .Symbiodinium cells in fragments of the infected colony replaced their structural carbon at a slower rate than Symbiodinium in the corresponding control  or indirectly, after the internal recycling of ammonia [15N-assimilation by Symbiodinium that we observed in the dark suggests 15N-fixation is coupled with photosynthesis [15N assimilated in the dark however, confirms that both metabolic pathways are active and used during infection.A strong nitrogen . The 15Nproducts . This mall Figs. , 5. Ther ammonia , 60. Theynthesis  and is tIn the present study, we show that coral/pathogen infection dynamics  depends on both the behavioural response of the host organism and the intricate nutritional interactions that occur between the symbiont and its host. It is tempting to propose that the observed defence responses (increased mucus production and the active spewing of mucus) are fuelled by an increased amount of photosynthates translocated from the symbiont to the host. However, at this point our results should be viewed within the context of the limited number of colonies tested here, the high inoculum density, and the contrasting observations from previous experiments.Future studies should combine our approach with other physiological parameters such as photosynthesis, respiration and symbiont density. Developing microfluidic chips fitted with integrated oxygen sensors is certainly a feasible target for the field of coral research in the near-future, given that such chips already exist for algae , 62 and It is important to highlight here, that this approach is not limited to corals. It can be applied to study broadly the biochemical signalling that occurs between bacteria and their host  or to inSupplementary Information


Escherichia coli DH5α injection in mice. TSLP levels were measured in human subjects, mice, and macrophages. TSLP deficiency or murine double minute 2 (MDM2) deficiency was induced using siRNA or an MDM2 inhibitor, nutlin-3a. We found that TSLP levels were elevated in serum of patients and mice with sepsis. TSLP deficiency lowered liver damage and inflammatory cytokine levels in mice with sepsis. TSLP was produced by the MDM2/NF-κB signaling pathway in LPS-stimulated macrophages. TSLP downregulation by an MDM2 inhibitor, nutlin-3a, alleviated clinical symptoms and septic inflammatory responses. Pharmacological inhibition of TSLP level by cisplatin reduced the septic inflammatory responses. Altogether, the present results show that TSLP exacerbates septic inflammation via the MDM2 signaling pathway, suggesting that TSLP may be a potential target for the treatment of sepsis. Thymic stromal lymphopoietin (TSLP) is crucial for Th2-mediated inflammation. Sepsis is a serious systemic inflammatory reaction with organ dysfunction by infection. However, the function of TSLP during sepsis is poorly understood. Thus, we investigated a role and regulatory mechanism of TSLP during sepsis. Sepsis was induced by lipopolysaccharides (LPS) or  Escherichia coli (E. coli) which is responsible for about 16% according to the European Prevalence of Infection in their Intensive Care study [Sepsis is a systemic inflammatory reaction syndrome with infection and is a major source of morbidity and mortality . Approxire study . Macrophre study . Gram-nere study . Thymic stromal lymphopoietin (TSLP) is produced by various cell types and is known to mainly promote allergic and inflammatory diseases . TSLP acMurine double minute 2 (MDM2) is well known to limit p53-mediated cell cycle arrest and apoptosis and to be a potential therapeutic target in cancer therapy . HoweverIn this study, we investigated a regulatory mechanism of TSLP in sepsis using LPS-stimulated macrophages and an LPS-induced sepsis model. Our findings show that TSLP is produced through the MDM2 signaling pathway in LPS-stimulated macrophages and TSLP causes organ dysfunction and an overwhelming systemic inflammatory reaction in septic mice. n = 20; 10 males, 10 females) and patients with sepsis . The patient samples were taken within 24 h after sepsis diagnosis. The patient characteristics are listed in Human study was approved by the Bioethics Committee of Kyung Hee University (KHSIRB-14-013(EA)). The Biospecimens and data used in this study were provided by the Biobank of Gyeongsang National University Hospital, a member of the Korea Biobank Network. All samples derived from the National Biobank of Korea were obtained with informed consent under institutional review board-approved protocols. All subjects provided written informed consent under the Helsinki Declaration. The biospecimens were taken from healthy volunteers . The homozygous TSLP−/− was identified by reverse transcription PCR. All experiments were conducted in accordance with internationally accepted principles for laboratory animal use and care, as found in the United States guidelines  and approved by the Animal Care Committee of Kyung Hee University (No. KHUASP(SE)-14-023). Sepsis was induced in male C57BL/6 mice (7–9 weeks old) by intraperitoneal injection of LPS  or E. coli DH5α (1 × 106 CFU). Serum from the heart was obtained 4 h after LPS or E. coli injection. At selected time points, peritoneal cavities were washed with Dulbecco’s Modified Eagle’s Medium  including heparin and lavage fluid was then centrifuged. Supernatants were stored at −70 °C before evaluation of cytokines by enzyme-linked immunosorbent assay (ELISA). Tissues were obtained 12 h after intraperitoneal injection of LPS or E. coli according to several previously published studies [E. coli (1 × 108 CFU). Mice were intravenously injected with recombinant mouse TSLP  or PBS as a control (Sigma-Aldrich Co.) by referring to the studies of Piliponsky et al. [Male C57BL/6 mice of 7–9 weeks old were obtained from Dae-Han Experimental Animal Center . TSLP studies ,18. Micey et al. . Serum a2. Thioglycolate (TG)-elicited macrophages were harvested from peritoneal lavage of male C57BL/6 mice 3–4 days after intraperitoneal injection of 2.5 mL of TG. Briefly, nonadherent cells were removed by washing with PBS. Adherent peritoneal macrophages were cultured overnight in DMEM supplemented with 10% FBS and penicillin/streptomycin. Human monocyte-like cell lines, THP-1  and HL-60 cells (Korean Cell Line Bank) derived from blood of patients with acute monocytic/promyelocytic leukemia were cultured in Roswell Park Memorial Institute (RPMI) 1640 containing 10% FBS and penicillin/streptomycin at 37 °C in a humidified atmosphere of 5% CO2. To induce differentiation into macrophage-like cells, THP-1 cells were differentiated with phorbol 12-myristate 13-acetate  for 24 h. HL-60 cells were differentiated with PMA (16 nM) for 72 h. The cells were washed with PBS and rested in fresh RPMI cell culture medium (without PMA) for 24 h, as reported previously [RAW 264.7  was cultured in DMEM with 10% fetal bovine serum (FBS) and penicillin (100 U/mL)/streptomycin (100 µg/mL) at 37 °C in a humidified atmosphere of 5% COeviously ,22,23. R5 cells) by ELISA for human and mouse TSLP , IL-6 , vascular endothelial growth factor , intercellular adhesion molecule-1 , macrophage inflammatory protein 2  ligand 2, CXCL2, R&D Systems), and tumor necrosis factor-α  following the manufacturer’s protocols. Total protein concentrations in tissue homogenates were determined using a bicinchoninic acid  protein assay kit. These biochemical markers were analyzed using an ELISA microplate reader  at 405 nm. Aspartate aminotransferase , alanine aminotransferase , blood urea nitrogen , and creatine kinase  levels were measured in serum by the manufacturer’s protocols. Nitric oxide concentration in cell-free culture supernatants from RAW 264.7 cells was measured by the Griess method. Briefly, 100 μL of cell-free culture supernatant was mixed with 50 μL of 0.1% N-(1-naphtyl)-ethylenediamine dihydrochloride in distilled water and 50 μL of 1% sulfanilic acid in 5% phosphoric acid in a 96-well plate. The absorbance was measured at 540 nm using an ELISA microplate reader. We evaluated serum, tissue homogenates, and cell-free culture supernatants (3 × 106 cells), peritoneal macrophages (1 × 106 cells), THP-1 (1 × 106 cells), and HL-60 (1 × 106 cells) using an easy-BLUETM RNA extraction kit . The isolated total RNA was dissolved in 50 μL RNase-free water. Each concentration of the total RNA was measured by NanoDrop spectrophotometry . The total RNA was incubated at 70 °C for 5 min, placed on ice, and reverse-transcribed to cDNA for 60 min at 42 °C and 5 min at 94 °C using a cDNA synthesis kit . Real-time PCR was performed with primers as in the ΔΔCT method. RNA isolation was performed on liver, lung, kidney, and large intestine tissues, RAW 264.7 , and peritoneal macrophages (5 × 106 cells). Tissues were homogenized in homogenization buffer. The cells were lysed in cell lysis buffer (R&D Systems) containing protease inhibitor. These were subjected to separation on SDS-PAGE gels and transferred onto nitrocellulose membranes. The following antibodies were used: anti-MDM2 , NF-κB , HIF-1α , p53 , actin , GAPDH , and histone  antibodies. The membranes were incubated with peroxidase-conjugated second antibodies (Santa Cruz Biotechnology). Signals were developed with an enhanced chemiluminescence solution . Samples for immunoblotting were obtained from liver, lung, kidney, and large intestine tissues, RAW 264.7 cells  for 15 min on ice, lysed with 0.6 μL of 10% Nonidet P-40. After centrifuge, the nuclei pellets were resuspended in 20 μL of buffer A and 40 μL of buffer B  for 20 min on ice. After centrifuge, the supernatant (nuclear extract) was used to analyze expression levels of NF-κB, HIF-1α, and histone. RAW 264.7 cells were suspended in 40 μL of buffer A  were plated on a 96-well plate and subjected to LPS, E. coli, nutlin-3a, or cisplatin for 24 h. MTT reagent (5 mg/mL) was added to each well and incubated at 37 °C for 4 h. The insoluble formazan product was dissolved in dimethyl sulfoxide. Optical density was analyzed with an ELISA microplate reader at 540 nm.Cell viability was analyzed by a 3--2,5-diphenyltetrazolium bromide  assay. For this assay, RAW 264.7 cells . The relative luciferase activity was determined by the ratio of t-test, while multiple comparisons were made using one-way ANOVA analysis with Tukey’s post hoc test using IBM SPSS statistics 23 . A p value of less than 0.05 was considered statistically significant. Data are presented as the mean ± standard error of the mean (SEM). Pilot experiments were performed to estimate the sample size . In vitro data are representative of three independent experiments (n = 5/group). Pairwise comparisons were made using independent p < 0.05). However, there were no significant differences in the TSLP levels between males and females, older and younger patients, patients who died and patients who survived, or patients with a specific bacterial species and others. We performed a time-course study to determine TSLP levels in serum and peritoneal lavage of mice following LPS or E. coli injection according to previous reports [E. coli injection and reached a maximum at around 4 h after the injection (p < 0.05). TSLP level began to decline gradually around 4 h after the injection. At the same time, the serum IL-6 and TNF-α levels which are early biomarkers for sepsis [p < 0.05; E. coli injection , suggesting a synergistic effect of TSLP on LPS-induced septic responses. However, the TSLP plus LPS-injected septic mice did not show significant changes in VEGF, ICAM-1, MIP2, and TNF-α levels. ALT levels were not significantly different between TSLP plus LPS-injected septic mice and LPS-injected septic mice . IL-6 and AST levels significantly increased in serum of TSLP plus LPS-injected septic mice vs. LPS-injected septic mice . Also, LPS or E. coli significantly induced increases in the production (p < 0.05). Furthermore, we confirmed that the findings obtained from mouse macrophages also occur in human by using THP-1 and HL-60 cells, which are commonly used as surrogates of monocytes isolated from human peripheral blood mononuclear cells [E. coli significantly increased the production and mRNA expression of TSLP in PMA-differentiated THP-1 and HL-60 macrophage-like cells . To directly show that HIF-1α potentiates TSLP production from macrophages, we performed HIF-1α siRNA silencing experiments. After LPS stimulation, we confirmed HIF-1α mRNA reduction using HIF-1α siRNA by real-time PCR  and mRNA expression of TSLP in HIF-1α siRNA-transfected RAW 264.7 cells were downregulated as compared with those of control siRNA-transfected RAW 264.7 cells after LPS stimulation  and protein expressions of MDM2 increased in liver , VEGF (p < 0.05), ICAM-1 (p < 0.05), and TNF-α  and mRNA expression of TSLP (p < 0.05), and expression of MDM2 in LPS-stimulated RAW 264.7 cells. TSLP (p < 0.05) and MDM2 levels were suppressed by cisplatin in serum or liver of septic mice  [−/− mice had a liver with lower basal cytokine expression. Ashrin et al. [Macrophage is a key cell that leads to overwhelming production of cytokines and chemokines, and regulates an intense proinflammatory response during sepsis . Here, we (MODS) . Patiente (MODS) . An incre (MODS) . In thise (MODS) . High ILe (MODS) . Thus, tn et al.  reportedn et al.  reportedE. coli (3 × 108 CFU) [E. coli (1 × 106 CFU) or LPS (10 mg/kg) was used in this study. LPS or E. coli injection increased serum TSLP levels up to about 1750 pg/mL or 368 pg/mL, respectively. In a cecal ligation and puncture (CLP) model which is a sepsis experimental model, TSLP levels were elevated up to about 70 pg/mL in serum or 200 pg/mL in plasma [E. coli-induced sepsis model. The dose of LPS which leads to death in half of mice (LD50) is about 1–25 mg/kg . In the 108 CFU)  or LPS (Kuethe et al.  describeMoon and Kim demonstrated that TSLP is expressed and produced via NF-κB pathway in mast cells . Jang etHere, we found that cisplatin effectively protects against severe sepsis in mice. The study of Ishikawa et al.  presentsE. coli-stimulated macrophages. TSLP causes organ dysfunction and an overwhelming systemic inflammatory reaction in septic mice (In summary, this study provides evidence that TSLP is produced via TLR4 signaling in macrophages during sepsis. The MDM2/NF-κB signaling pathway regulates the production of TSLP in tic mice . Therefo


Mimusops caffra, Ilex paraguarieni, and Glechoma hederacea, have been reported as major sources of UA. The chemistry of UA has been studied extensively based on the literature, with modifications mostly having been made at positions C-3 (hydroxyl), C12-C13 (double bonds) and C-28 (carboxylic acid), leading to several UA derivatives  with enhanced potency, bioavailability and water solubility. This article comprehensively reviews the information that has become available over the last decade with respect to the sources, chemistry, biological potency and clinical trials of UA and its derivatives as potential therapeutic agents, with a focus on addressing NCDs.Non-communicable diseases (NCDs) such as cancer, diabetes, and chronic respiratory and cardiovascular diseases continue to be threatening and deadly to human kind. Resistance to and side effects of known drugs for treatment further increase the threat, while at the same time leaving scientists to search for alternative sources from nature, especially from plants. Pentacyclic triterpenoids (PT) from medicinal plants have been identified as one class of secondary metabolites that could play a critical role in the treatment and management of several NCDs. One of such PT is ursolic acid , which possesses important biological effects, including anti-inflammatory, anticancer, antidiabetic, antioxidant and antibacterial effects, but its bioavailability and solubility limits its clinical application.  Non-communicable diseases (NCDs), including cancers, cardiovascular diseases, pulmonary illnesses, and diabetes, account for nearly 71 percent of all fatalities globally, as reported by the World Health Organization. The rapid increase of NCDs has been influenced by a series of flexible behavioural threat variables, including unhealthy eating habits, physical inactivity, cigarette smoke exposure and damaging use of alcoholic drinks, as well as natural air pollutants, metabolic  and occupational  risks . UA. UA51]. 6.7g/mol ,52,53,58It is well known that low aqueous solubility of a drug may seriously affect its medication effectiveness. It has also been documented that certain treatments have a low solubility and few side effects. Ursolic acid, however, has low water solubility, which limits bioavailability in the human body . The lim1) structure-activity relationship synthesized a total of twenty-three analogues through modification at position C-3 and C-28. 3-O-acetylursolic acid was obtained from acetylation of UA, which was later treated with bromo-diolefine to yield fatty esters as shown in The chemical modifications to date have focused mainly on the hydroxyl group at position C-3, the unsaturated double bond at position C12-C13, and the carboxylic acid at position C-28 at rings A, C and E, respectively, as displayed in 1) from Ocimum sanctum, which led to the preparation of three derivatives, namely, 3β-acetoxy-urs-12-en-28-oic acid (25), [N-(3β-acetoxy-urs-11-oxo-12-en-28-acyl) aniline] (26) and [Methyl N-(3β-butyryloxyl-urs-12-en-28-oyl)-2-amine acetate] (27) as shown in Batra and Sastry (2013) reported isolation of UA (19) and 3β-acetoxy-urs-12-en-28-oic acid (20) ..47–61) i62–78) as displayed in O-acetate derivative was developed by the addition of acetic anhydride in anhydrous pyridine, which was later treated with oxalyl chloride to yield the 28-acyl-chloride intermediate. They further dissolved the intermediate in dichloromethane to give a series of amino compounds including aminobenzene, p-fluoroaniline, p-chloroaniline, p-bromobenzenamine, p-methoxylaniline o-fluoroaniline, o-chloroaniline and o-bromobenzenamine through condensation in the presence of triethylamine. After this, saponification analogues (62–69) were produced, which were later hydrolysed to give more derivatives (70–77). Lastly, the 3-oxo anlogue (78) was developed through oxidation with pyridinium chlorochromate. All the structure of these UA derivatives were elucidated by a series of spectroscopic techniques, such as nuclear magnetic resonance (NMR) including 13C-NMR and 1H-NMR with melting point, high resolution mass spectrometry, and electrospray ionization mass spectrometry (ESI-MS)  with high in vitro anticancer potency when compared to the ursolic acid parent compound. The modifications of compound 1 occurred at hydroxyl C3, C-11, and carboxylic C-28, as shown in 2, 3 and 4 showed potent anti-proliferation activity in cancer cells, with compound 3 displaying higher significance  and compound 16  were more significant because of their evident high antitumor inhibition against cancer cells. This led to a belief that the antitumor properties of the mentioned compounds could be attributed to the substitution made during the synthesis modification of UA; electron withdrawing, alkanoyloxy imino chain at position C-2, and alkyl side chains at position C-3 -amino-1-hydroxyethylethoxy piperazine and N-[3b-acetoxy-urs-12-en-28-oyl]-amino-4-piperidineethanol exhibited better in vitro antiproliferative activity. The N-[3β-acetoxy-urs-12-en-28-oyl]-2-aminodiethanol derivative was reported to have anticancer activity (45 ± 4.3%) on Kunming mice compared to the control group [In a different investigation, Shao and co-authors (2011) explored the in vivo and in vitro anticancer activity of a number of synthesized UA derivatives. The synthesized compounds were evaluated against various cancer cells of BGC-823, SH-SY5Y, HELF, HeLa and HepG2 by the assay called MTT. ol group . Rashid ol group . More au79–82, 91, 97) against at least one of the cancer cell lines  (IC50 < 10µM). Compound 79 showed a higher potency compared to the positive control, with IC50 values of 12.49 ± 0.08, 0.36 ± 0.05, and 0.61 ± 0.07 µM against SMMC-7721, HeLa and MDA-MB-231 cell lines, respectively. The bioactive derivatives  did not show any significant cytotoxicity against QSG-7701 (IC50 > 40µM) [In an attempt to develop new anticancer agents, Gu and co-authors (2017) proposed a series of quinoline and oxidiazole derivatives of UA. These researchers evaluated the cytotoxicity of synthesized derivatives against cancer cell lines such as human breast (MDA-MB-231), heptacarcinoma (SMMC-7721), HeLa and normal hepatocyte cell lines (QSG-7701) using MTT assay. The cancer drug atoposide was used as a positive control. The results from this particular study showed interesting significant effects of UA derivatives ( > 40µM) .1) suppressed the proliferation of androgen-independent DU145 and androgen-dependent human prostate cancer cells (LNCaP) through inhibition of NF-κB and STAT3 activation. Tumour growth was suppressed significantly when four-week-old athymic BALB/c male nude mice inoculated with DU145 cells were treated with (+)-UA  for 6 weeks. No significant effects on body weight were observed in mice [Ursolic acid caused bax up-regulation and down-regulation of Bcl-2 and discharge from mitochondria of cytochrome C to the cytosol. In addition, UA cleaved caspase-9 and reduced mitochondrial membrane ability. Thus, UA induces apoptosis in MDA-MB-231 cells through both the mitochondrial death pathway and a mechanism dependent on the extrinsic death receptor. Therefore, ursolic acid could be used as a promising anti-cancer drug in treatment of breast cancer . UA (1)  in mice . Moreove in mice .2 MTD toxicity. The DLTs were predominantly diarrhoea and hepatotoxicity. Furthermore, a linear pharmacokinetic profile was found in the ursolic acid liposomal model [Many clinical trials using chemical, subcutaneous, orthotopic human xenograft and randomly transgenic tumour development designs have provided extensive proof that naturally existing and synthetic UA products have chemopreventive and therapeutic effects. Liposomal ursolic acid (LUA) was used as a fresh drug in normal young volunteers and in individuals with developed solid tumours in order to determine the highest permitted level (MTD), dose-limiting toxicities (DLTs), and UA pharmacokinetics. A total sample of LUA  was given to 63 subjects . For the first season, clinical information revealed that LUA had manageable 98 mg/mal model . UA is aal model . TherefoSambucus australis plant. Modifications of the UA structure were made at C-3 (hydroxyl group) to yield 3β-formyloxy-urs-12-en-28-oic acid (19) and 3β-acetoxy-urs-12-en-28-oic acid (20), as shown in Staphylococcus aureus, Bacillus cereus, Shigella flexneri, two strains of Escherichia coli, Aeromonas caveae, Pseudomonas aeruginosa, Klebsiella pneumonia, and Vibrio colareae) using the microdilution method. Compound 19 (64 μg/mL) when combined with kanamycin showed significant activity towards Escherichia coli compared to the multidrug resistance observed from sputum, which reduced MIC from 128 μg/mL to 8 μg/mL [Public health is faced with many challenges, including the resistance of many already-available antibiotics. There is hope in the use of naturally occurring products as a substitute that can produce a better and more promising therapeutic effect through the discovery of new compounds and derivatives against bacterial pathogens. Nascimento and colleagues (2014) semi-synthesized two analogues of UA isolated from the pound 19  μg/mL whStaphylococcus aureus (MRSA), which is resistant to methicillin. Researchers in this research noted that with no haemolytic consequences, UA produced a reduction in staphylococcal membrane integrity. UA-treated protein type relative to ordinary MRSA cells showed that UA impacted translation efficiency, chaperon subunits, ribonuclease, oxidative reactions, and glycolysis of a variety of proteins engaged in the transformation method [Ilex hainanensis Merr. These authors assessed seven UA derivatives against Fusobacterium nucleatum (Gram-ve) and Streptococcus mutans (Gram + ve) for their antibacterial activity. Only three derivatives displayed important activity against Gram positive at distinct levels and displayed substantial activity against Gram negative at a minimum inhibitor level (MIC) of 625 μg/mL [Streptococcus mutans) development in their quest for powerful antimicrobial drugs [Wang and co-authors (2016), in their investigation, explored the biological antibacterial properties of UA against n method . Zhao an25 μg/mL . Park anal drugs .Diabetes mellitus is a chronic disease that is triggered by insulin resistance or deficiency. This disease is one of the major risks to human well-being worldwide . Long-te50 of 2.66 ± 0.84, 1.01 ± 0.44, 3.26 ± 0.21 and 3.24 ± 0.21 µM [50 = 1.27 ± 0.27 and IC50 = 1.28 ± 0.27 µM, compared to the other derivatives and the positive control [107) showed a greater efficiency against α-glucosidase with an IC50 value of 115.1 μM; 3,5 times more potent than the standard drug (acarbose) [Wu and colleagues (2014) produced a series of UA derivatives and evaluated their potency as an antidiabetic agent against α-glucosidase. Most of the derivatives showed activity, except for one compound with four derivatives showing significant inhibition at IC 0.21 µM . Wu and  control . Khusnutcarbose) .108) was used as hypoglycaemic agents to evaluate their therapeutic effects against non-obese type 2 diabetic mice for two weeks. In this study, biomarkers in serum and urine were measured. In addition, protein expression, gene, and histomorphology analysis were measured from mice tibias. Moreover, femurs were taken for bone Ca measurement and trabecular bone three-dimensional architecture. These authors revealed a reduced testosterone level in the STZ serum of mice. UA analogues showed increased bone Ca, BMD, significantly increased Fibroblast growth factor 23 (FGF-23) and osteocalcin (OCN), and reduced diabetic mice parathyroid hormone (PTH) levels and crosslaps (CTX). UAD reversed the trabecular deleterious effects caused by STZ and stimulated remodelling of the bone. Treatment with UAD for the STZ group significantly increased the osteoprotegerin (OPG)/nuclear factor (NF)-κB ligand (RANKL) ratio. It has been shown that UA derivatives can improve STZ-induced bone deterioration by improving the dysfunction of mesenchymal stem cells [Yu and others (2015) reported the antidiabetic effects of UA derivatives in bone deteriorations (BMD) of diabetic mice (6 weeks old) induced by streptozotocin (STZ). In this research study, UA derivative (em cells .Chinese hamster ovary (CHO-K1) neurons with the TGR5 gene were transfected by Lo and others (2017) to test the antidiabetic effects of UA. Using a fluorescent marker, the features of the transfected cells were verified through glucose uptake. In addition, NCI-H716 cells that secreted incretin were also explored, and ELISA sets were used to quantify the glucagon-like protein (GLP-1) concentrations. In fact, Type 1-like diabetic rats induced by STZ were used to define the impact of in vitro ursolic acid. The level of UA dependently enhanced glucose uptake in TGR5 producing CHO-K1 cells. UA caused a concentration-dependent increase in GLP-1 secretion of NCI-H716 cells, which was inhibited by triamterene at efficient levels to inhibit TGR5. Ursolic acid also improved the amount of GLP-1 plasma by activating TGR5, which was further described in vitro with diabetic rats of type 1 .−), nitric oxide (NOO), superoxide anion (•O2), hydroxyl radical (•OH), and hydrogen peroxide (H2O2), play a crucial role after SAH [Neurological disorders include anxiety, depression, stroke and Alzheimer’s disease, among others . Long-tefter SAH . Severalfter SAH .Sahni and co-workers (2016) reported that UA derivatives of C-6 (C-17 propyl amide) and C-2 (C-3 methyl ester) displayed important neuroprotective activity in an in vivo model of D-galactose-induced neurotoxicity in rats. Thus, C-2 and C-6 could be advantageous in cognitive disorder therapy, for instance, in the treatment of Alzheimer’s disease and dementia . Li and p < 0.001), and protects the MPTP-intoxicated mouse’s dopaminergic neurons (p < 0.01). Among three distinct doses, the most efficient dose for PD was 25 mg/kg body wt. [Another disease, known as Parkinson’s disease (PD), is a chronic progressive neurodegenerative disorder that is defined both by motor and nonmotor characteristics. Through its progressive degenerative impacts on mobility and muscle control, it has a major clinical effect on patients, families, and caregivers . PD is tbody wt. . The lit50 values of UA range between 75 and 700 μM for drastic growth of Lactuca sativa (lettuce) [118) from Castanea crenata (Japanese chestnut). These authors reported the herbicidal effects of compound 118 in root and shoot growth of Echinochloa crus-galli (barnyard grass), L. sativa, and Raphanus sativus (radish) p-hydroxybenzoic acid. Compound 118 inhibition (IC50 = 2.62 and 0.41 mM) was >5 times higher than p-hydroxybenzoic acid (IC50 = 15.33 and 2.11 mM) in the Echinochloa crus-galli shoot and root growth, respectively. These results indicate that the isolated compound 118 has the ability to create natural herbicides for Echinochloa crus-galli management [Compounds with allelophatic potency are denoted as allelochemicals. Allelochemicals can cause inhibition of photosynthesis, decreases in chlorophyll content, inhibition of enzymatic activity, and disruption of the membrane and structure of cells. These effects can also be beneficial for host plants, inhibiting the development of pathogenic fungus organisms as a result of their antibacterial, antifungal and growth-inhibiting activities . Phenolilettuce) ,116. Tuylettuce)  explorednagement .Sambucus nigra L. is a major berry crop plant globally that has been commonly converted into jams, jellies and drinks commonly used in gastronomy, and which has previously been used for its prospective health advantages [S. nigra by means of Gas Chromatography with Electron Impact Mass Spectrometry (GC-EIMS). UA  was most abundant in the leaves and flowers of S. nigra  [Citharexylum spinosum L. including UA in germination and seedlings growth of plants such as R. sativus, L. sativa and Phalaris canariensis (canary grass). The roots were the most adversely affected; UA (1) was the most phytotoxic and caused 91.74 ± 0.24% and 89.55 ± 0.31% inhibition of roots and shoots, respectively [The elder vantages ,118. Basctively)  studied ectively .Salvia syriaca L. methanol extract. Significant growth inhibition (GI) of wheat seedlings was shown by UA, decreasing shoot lengths and Triticum aestivum cv (wheat) seedling roots when evaluated at 0.2 mg/L (200 ppm) as reported by Abu-Irmaileh and Abu-Zarga (2015). These authors reported UA GI (p = 0.05) on seed germination, root length and shoot height of T. aestivum at 70%, 9.1 and 6 cm, respectively [UA was isolated and characterized from the ectively .It is evident that the therapeutic activity of ursolic acid improves with modifications when compared with various current used standard drugs. A majority of reports described increases in the solubility, bioavailability, and potency of UA through modifications at different positions. Many authors report modifications at the hydroxyl position C-3, hydrogen C-2, and carboxylic acid position C-28. Although several synthetic ursolic acid inhibitors have already been submitted, fresh derivatives still need to be developed and synthesized to further enhance their medicinal impacts on non-communicable diseases. Therefore, UA and its derivatives provide hope as potential therapeutic agents for non-communicable diseases through in vivo, in vitro, preclinical and clinical trials. Moreover, UA and its derivatives possess herbicidal effects in the growth of various plants.


High fertility rate has been consistently reported in Nigeria. The three major ethnic groups in Nigeria, Hausa/Fulani, Igbo, and Yoruba have different socio-cultural identities particularly those that relate to fertility but fertility index is often reported at the national level. This paper examined ethnic differences in fertility and identified its determinants in Nigeria.This cross-sectional design study focused on 23,140 women aged 15–49 years. Fertility was measured from information on the full birth history of women of reproductive age. Fertility was assessed using descriptive statistics, parity progression ratio(PPR) and negative binomial model (α = 0.05).p < 0.001) times higher among Hausa/Fulani than Yoruba women, but Igbo and Yoruba women exhibited a similar pattern. Controlling for other factors barely changes this pattern.The total fertility rate was 8.02, 4.91 and 4.43 among women in Hausa/Fulani, Igbo and Yoruba ethnic group respectively. The proportion of women with ≥5 children was highest among the Hausa/Fulani (40%), followed by Igbo (21.6%) and Yoruba (17.5%). For women aged 45–49 years; the PPR was highest among Hausa/Fulani while Igbo and Yoruba exhibited a similar pattern. The mean fertility was 1.725(C.I = 1.661–1.792, Variation existed in fertility across the main ethnic groups in Nigeria, but highest among Hausa/Fulani. Fertility reduction strategies that target improvement in women’s education will reduce the fertility rate in Nigeria, particularly among Hausa/Fulani women. Ethnicity is important in fertility reduction strategies in Nigeria. Regardlglobally  and if tglobally , 8. The Fertility differentials have been widely reported across socio-economic classes in Nigeria –11, but The people from Hausa/Fulani ethnic origin reside predominantly in the Northern part of Nigeria, while the Igbo and Yoruba dominate the South . People The socio-economic characteristics described above have been previously identified as important predictors of fertility in Nigeria –11, but A cross-sectional population-based study which involved secondary data analyses of the weighted sample of 2013 Nigeria Demographic Health and Survey (NDHS) was used. The sample was selected using a stratified 3-stage cluster design consisting of 904 clusters, 372 in urban areas and 532 in rural areas. A representative sample of 40,680 households was selected for the survey and a fixed sample take of 45 households was selected per cluster. However, in the current study, a sub-sample of 23,140 women which comprises of 12,409 Hausa/Fulani, 5557 Igbo and 5174 Yoruba were used. All women of reproductive age (15–49 years) irrespective of whether they have had at least a child or not were included in the study. Every woman with missing information on fertility was excluded from the analysis. At the point of data collection, the data originators excluded women who were seriously ill or mentally imbalance from the interview.The dependent variable was fertility measured by children ever born (V201). This variable was derived from the detailed birth histories collected from women. The variable was re-categorized into the following groups; 0, 1–2, 3–4 and 5+ to examine the percentage of women in each tribe who belong to a specific class of childbearing. The main independent variable was ethnicity. In the questionnaire for the study, a question was asked on the ethnic group the respondent belongs (what is your ethnic group?). The question was left as open ended and as such, many ethnic groups were reported in the original data, but the variable (V131) was however re-coded by sieving out other tribes leaving only the three largest, Hausa/Fulani, Igbo and Yoruba in this study. To avoid the effect of collinearity in the regression model used, the highly correlated predictors were removed from the model. In the case where two or more factors have high variance inflated factor, one was removed from the model. Categorical principal component analysis was further used to reduce the number of predictors to a set of uncorrelated factors thus retaining the independent variables below;The computation of wealth index was based on information received on ownership of the household’s consumable goods, dwelling characteristics, drinking water source, toilet facilities, and other characteristics that are related to household’s socioeconomic status. The assets were independently assigned a weight (factor score) generated with the use of principal component analysis (PCA). The obtained asset scores were standardized. A score was assigned to each asset and a total score was obtained for each household. The data originator  had geneThe data were weighted due to complex sampling procedures used during the data collection. In order to provide a 3-year trend in ASFR and TFR, the direct method based  on full The TFR and mean CEB among women aged 45–49 years were compared for the evaluation of long-term change in fertility. The mean CEB among women aged 45–49 years is an indication of average completed fertility for women who commenced childbearing three decades prior to the survey. If fertility is stable over time in a population, there would be a similarity in TFR and the mean number of CEB by women age 45–49. However, for declining fertility levels, lower TFR than the mean number of CEB is the case. The number of CEB to all women of reproductive age is useful for observing primary infertility level and variation in family size across age groups. The parity progression ratio (PPR) was determined based on the designed procedure in the tool for demographic estimation (page 70) ;A negative binomial regression model was fitted for the data. The nature of fertility data for this study informed the use of the model. The dependent variable exhibited a skewed distribution with mean strikingly lower than the variance termed as over dispersion. Over dispersion thus occurs as a result of neglected unobserved heterogeneity.The conditional variance of the negative binomial distribution surpasses its conditional mean and this makes it suitable for the analysis of this data.The multivariate analysis was conducted to examine the relationship between fertility and ethnicity and more importantly to identify the predictors of fertility in each of the tribes. Further analysis involved the pooling of all the data for the three ethnic groups together to examine how ethnicity relates to fertility. In this instance, three models were generated. The first model involved only the ethnicity as the explanatory variable, while the second included the demographic variables. In the third model, both direct and indirect factors were included in the model. The selection of the variables into the regression model was based on Bongaarts theoretical model  as earliFormal approval to use data-set for this study was obtained from the DHS program. The ethical approval was granted by the Nigeria National Ethics Committee (NHREC/2008/07). Informed consent was obtained from the respondents, and they were assured of the confidentiality and anonymity of the information they provided.The characteristics of the women in the sample are shown in Table In Fig. Figures Figures In Fig. The age-specific fertility rate in a single year and 5-year age group are presented in Fig. In Fig. As shown in Table p < 0.001), 0.515(C.I = 0.480–0.552), and 0.846(C.I = 0.786–0.910) times respectively likely to bear children than those in the age group 45–49 years and this pattern was found among Igbo and Yoruba women.The multivariate analysis shows that the predictors of fertility among the Hausa/Fulani women were; age, education, age at first and fertility preference and these are common predictors among the three ethnic groups. For Igbo and Yoruba ethnic groups, similar variables like age, education, sex preference, ever used any contraceptive method, age at first birth fertility preference and marital status were identified as predictors of fertility except sex preference which was not statistically significant in Yoruba ethnic group. In all the three ethnic groups, the incidence rate ratio (IRR) of fertility increases as the age group of women increases but falls consistently with the increasing level of education. For example, Hausa/Fulani women in age group 15–24, 25–34 and 35–44 years were 0.212 and 1.369(IRR = 1.185–1.583) times higher among women that have no formal education and primary education respectively than those with higher education. Igbo women who have no sex preference compared to those who have sex preference were expected to have a rate 1.106(C.I = 1.018–1.201) times greater for fertility. Likewise, Igbo women who began childbearing at ages below 18 years compared to those who began theirs at ages ≥18 years were expected to have a greater fertility rate of 1.358(C.I = 1.229–1.501) and this was the pattern exhibited across the Yoruba and Hausa/Fulani women. Across the three tribes, women who have preference for bearing at least 5 children experienced higher fertility than those who preferred less. Lower IRR was found among the Yoruba women who have a preference for less than 5 children than those who prefer to have at least 5 children , however, the gap was narrower among Hausa/Fulani women than Yoruba and Igbo women.The first model in Table Fertility determines the population size, structure, and composition of any society, while ethnicity is a social cluster of individuals that shares a unique culture, religion, language, beliefs or certain characteristics that distinguish them from their neighboring communities. Ethnicity may not be genetic but it describes people by a group different from their birth identity if they live for a long period in a new locality and they imbibe the culture, symbols, and relationships of their host community . CentralAbout half of the studied women belong to the Hausa/Fulani ethnic group, while Igbo and Yoruba women shared a quarter and slightly above one-fifth respectively. The distribution of women by ethnic group found in this study reflects true ethnic composition in Nigeria . It is sThe study revealed that in the 3-year period 2010–2012) prior the survey year, the TFR obtained for Hausa/Fulani women was 7.26, while that of Igbo and Yoruba was 4.56 and 4.37 respectively. Thus, the estimated TFR for Hausa/Fulani ethnic group was above the national figure in Nigeria (5.5) 010–2012 .The findings from the multivariate analysis show that the likelihood of fertility was higher among the Hausa/Fulani ethnic group than the Yoruba but no difference was observed between Igbo and Yoruba. In the literature, higher fertility has been consistently reported in the regions where the majority of the people are Hausa/Fulani than the regions where people from Yoruba or Igbo ethnic background dominate , 10, 17 The study further showed that all the identified determinants of fertility among Hausa/Fulani like age, education, age at first and fertility preference were common to other ethnic groups but in addition, other factors like sex preference, ever used any contraceptive method and marital status were found among Igbo and Yoruba ethnic groups. As expected, the fertility incidence increases with age, and age at first birth. This is primarily because early commencement of childbearing predisposes women to increase the number of years of childbearing exposure within reproductive age range . This paEthnic diversity has the tendency to enhance societal values, customs and the attainment of suitable demographic change, but must be planned for through the formulation of intercultural policies and programs development. Examining the relationship between ethnicity and fertility has a direct consequence on population policies and programmes. The goal of the National Policy on Population for Sustainable Development (NPSD) in Nigeria is to achieve a reduction in the TFR of at least 0.6 children every 5 years . The ferNigeria is currently facing the challenge of rapid population growth driven by high fertility rate. If the future generations of Nigerians must have a good living standard, the high fertility rate must be checked and the ethnic traditional customs that are inimical to fertility controls must be critically addressed through policies and programs. Otherwise, by 2050, a huge number of Nigerians will live to see more dreadful consequences of overpopulation like acute poverty, widespread unemployment, high crime rate, environmental degradation, congestion than what is being witnessed currently. In the quest to reduce the level of fertility in Nigeria, research that targets men will be a welcome development towards the accomplishment of this goal.The data may be susceptible to bias often resulted from misclassification of birth timing due to recall bias. In the African context, there is the likelihood of omitting dead children from the full birth history list, thus there might be under-estimation of fertility level. Also, distortions as a result of displacement and omission of births might lead to underestimation of fertility. However, the data originator ensures that these biases were minimized at the points of the study design and data collection. It is also necessary to note that some covariates at the date of the interview may not have applied to the time the birth of a specific child took place. Due to the nature of the data (secondary) used for this study, there is an extent to which some theoretical frameworks can work effectively in the building of the model. However, the choice of the variables for this study was based on the availability of the variables in the data as reconciled with those in the existing theoretical frameworks.Fertility remains high in all the three main ethnic groups in Nigeria, but more prominent among Hausa/Fulani women. The Igbo and Yoruba exhibited similar fertility pattern. Fertility rates and behavior were more homogeneous within the three ethnic groups than national groups and the state of fertility transition is not the same across the groups. While the Igbo and Yoruba ethnic groups are currently undergoing fertility transition, such is yet to commence among Hausa/Fulani. Thus, the national data hide the Igbo and Yoruba sub-groups for whom the fertility transition is more advanced. Although, there were differences between the predictors of fertility among the three tribes, the common predictors of fertility among the tribes were; age, education, age at first and fertility preference. Improving female education and school enrollment will be an important fertility reduction intervention in Nigeria, particularly among Hausa/Fulani women.