Expression and function of Dlx genes in the osteoblast lineage

Our laboratory and others have shown that overexpression of Dlx5 stimulates osteoblast differentiation. Dlx5^−/−/Dlx6^−/− mice have more severe craniofacial and limb defects than Dlx5^−/−, some of which are potentially due to defects in osteoblast maturation. We wished to investigate the degree to which other Dlx genes compensate for the lack of Dlx5, thus allowing normal development of the majority of skeletal elements in Dlx5^−/− mice. Dlx gene expression in cells from different stages of the osteoblast lineage isolated by FACS sorting showed that Dlx2, Dlx5 and Dlx6 are expressed most strongly in less mature osteoblasts, whereas Dlx3 is very highly expressed in differentiated osteoblasts and osteocytes. In situ hybridization and Northern blot analysis demonstrated the presence of endogenous Dlx3 mRNA within osteoblasts and osteocytes. Dlx3 strongly upregulates osteoblastic markers with a potency comparable to Dlx5. Cloned chick or mouse Dlx6 showed stimulatory effects on osteoblast differentiation. Our results suggest that Dlx2 and Dlx6 have the potential to stimulate osteoblastic differentiation and may compensate for the absence of Dlx5 to produce relatively normal osteoblastic differentiation in Dlx5 knockout mice, while Dlx3 may play a distinct role in late stage osteoblast differentiation and osteocyte function.     

Cdk5 is required for the positioning and survival of GABAergic neurons in developing mouse striatum

Cyclin‐dependent kinase 5 (Cdk5) is a serine/threonine kinase, and its activity is dependent upon an association with a neuron‐specific activating subunit. It was previously reported that Cdk5^−/− mice exhibit perinatal lethality and defective neuronal positioning. In this study, they focused on the analysis of neuronal positioning of GABAergic neurons in the forebrain. Defective formation of the ventral striatum, nucleus accumbens, and olfactory tubercles was found in Cdk5^−/− embryos. To further study this abnormal development, we generated and analyzed Dlx5/6‐Cre p35 conditional KO (cKO); p39^−/− mice in which forebrain GABAergic neurons have lost their Cdk5 kinase activity. Defective formation of the nucleus accumbens and olfactory tubercles as well as neuronal loss in the striatum of Dlx5/6‐Cre p35cKO; p39^−/− mice was found. Elevated levels of phosphorylated JNK were observed in neonatal striatal samples from Dlx5/6‐Cre p35cKO; p39^−/− mice, suggestive of neuronal death. These results indicate that Cdk5 is required for the formation of the ventral striatum in a cell‐autonomous manner, and loss of the kinase activity of Cdk5 causes GABAergic neuronal death in the developing mouse forebrain. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 419–437, 2017     

Betaine (N,N,N-trimethylglycine) averts photochemically-induced thrombosis in pial microvessels in?vivo and platelet aggregation in?vitro

Betaine (N,N,N-trimethylglycine) is an important food component with established health benefits through its homocysteine-lowering effects, and is used to lower total homocysteine concentration in plasma of patients with homocystinuria. It is well established that hyperhomocysteinemia is an established risk factor for cardiovascular disease and stroke. However, the possible protective effect of betaine on coagulation events in vivo and in vitro has thus far not been studied. Betaine was given to mice at oral doses of either 10 mg/kg (n = 6) or 40 mg/kg (n = 6) for seven consecutive days, and control mice (n = 6) received water only. The thrombotic occlusion time in photochemically induced thrombosis in pial arterioles was significantly delayed in mice pretreated with betaine at doses of 10 mg/kg (P < 0.001) and 40 mg/kg (P < 0.01). Similar effects were observed in pial venules with 10 mg/kg (P < 0.05) and 40 mg/kg (P < 0.05) betaine. In vitro, in whole blood samples collected from untreated mice (n = 3–5), betaine (0.01–1 mg/mL) significantly reversed platelet aggregation induced by adenosine diphosphate (5 µM). The number of circulating platelets and plasma concentration of fibrinogen in vivo were not significantly affected by betaine pretreament compared with the control group. Lipid peroxidation (LPO) in mice pretreated with betaine was significantly reduced compared with the control group. Moreover, betaine (0.01–1 mg/mL) caused a dose-dependent and significant prolongation of PT (n = 5) and aPTT (n = 4–6). In conclusion, our data show that betaine protected against coagulation events in vivo and in vitro and decreased LPO in plasma.     

Enrichment and physiological characterization of a novel comammox Nitrospira indicates ammonium inhibition of complete nitrification

The recent discovery of bacteria within the genus Nitrospira capable of complete ammonia oxidation (comammox) demonstrated that the sequential oxidation of ammonia to nitrate via nitrite can also be performed within a single bacterial cell. Although comammox Nitrospira exhibit a wide distribution in natural and engineered ecosystems, information on their physiological properties is scarce due to the limited number of cultured representatives. Additionally, most available genomic information is derived from metagenomic sequencing and high-quality genomes of Nitrospira in general are limited. In this study, we obtained a high (90%) enrichment of a novel comammox species, tentatively named “Candidatus Nitrospira kreftii”, and performed a detailed genomic and physiological characterization. The complete genome of “Ca. N. kreftii” allowed reconstruction of its basic metabolic traits. Similar to Nitrospira inopinata, the enrichment culture exhibited a very high ammonia affinity (K_{m(app)_NH3} ≈ 0.040 ± 0.01 µM), but a higher nitrite affinity (K_{m(app)_NO2}- = 12.5 ± 4.0 µM), indicating an adaptation to highly oligotrophic environments. Furthermore, we observed partial inhibition of ammonia oxidation at ammonium concentrations as low as 25 µM. This inhibition of “Ca. N. kreftii” indicates that differences in ammonium tolerance rather than affinity could potentially be a niche determining factor for different comammox Nitrospira.Subject terms: Bacterial genomics, Environmental microbiology, Bacterial physiology     

Ectopic expression of the Dlx genes induces glutamic acid decarboxylase and Dlx expression.

The expression of the Dlx homeobox genes is closely associated with neurons that express gamma-aminobutyric acid (GABA) in the embryonic rostral forebrain. To test whether the Dlx genes are sufficient to induce some aspects of the phenotype of GABAergic neurons, we adapted the electroporation method to ectopically express DLX proteins in slice cultures of the mouse embryonic cerebral cortex. This approach showed that ectopic expression of Dlx2 and Dlx5 induced the expression of glutamic acid decarboxylases (GADs), the enzymes that synthesize GABA. We also used this method to show cross-regulation between different Dlx family members. We find that Dlx2 can induce Dlx5 expression, and that Dlx1, Dlx2 and Dlx5 can induce expression from a Dlx5/6-lacZ enhancer/"reporter construct.     

Genetic inhibition of RIPK3 ameliorates functional outcome in controlled cortical impact independent of necroptosis

Traumatic brain injury (TBI) is a leading cause of death and disability with no specific effective therapy, in part because disease driving mechanisms remain to be elucidated. Receptor interacting protein kinases (RIPKs) are serine/threonine kinases that assemble multi-molecular complexes that induce apoptosis, necroptosis, inflammasome and nuclear factor kappa B activation. Prior studies using pharmacological inhibitors implicated necroptosis in the pathogenesis of TBI and stroke, but these studies cannot be used to conclusively demonstrate a role for necroptosis because of the possibility of off target effects. Using a model of cerebral contusion and RIPK3 and mixed lineage kinase like knockout (MLKL^−/−) mice, we found evidence for activation of RIPK3 and MLKL and assembly of a RIPK1-RIPK3-MLKL necrosome complex in pericontusional brain tissue. Phosphorylated forms of RIPK3 and MLKL were detected in endothelium, CD11b + immune cells, and neurons, and RIPK3 was upregulated and activated in three-dimensional human endothelial cell cultures subjected to CCI. RIPK3^−/− and MLKL^−/− mice had reduced blood-brain barrier damage at 24 h (p < 0.05), but no differences in neuronal death (6 h, p = ns in CA1, CA3 and DG), brain edema (24 h, p = ns), or lesion size (4 weeks, p = ns) after CCI. RIPK3^−/−, but not MLKL^−/− mice, were protected against postinjury motor and cognitive deficits at 1–4 weeks (RIPK3^−/− vs WT: p < 0.05 for group in wire grip, Morris water maze hidden platform trials, p < 0.05 for novel object recognition test, p < 0.01 for rotarod test). RIPK3^−/− mice had reduced infiltrating leukocytes (p < 0.05 vs WT in CD11b + cells, microglia and macrophages), HMGB1 release and interleukin-1 beta activation at 24–48 h (p < 0.01) after CCI. Our data indicate that RIPK3 contributes to functional outcome after cerebral contusion by mechanisms involving inflammation but independent of necroptosis.Subject terms: Molecular neuroscience, Brain injuries     

Synergism therapeutic and immunoregulatory effects of Albendazole + rAd-mIL-28B against Echinococcosis in experiment-infected mice with protoscoleces

The metacestode stage of Echinococcus granulosus can cause cystic echinococcosis (CE), which still widely occurs around the world. Since the early 1970s, benzimidazoles have been shown to inhibit the growth of cysts and used to treat CE. However, benzimidazoles are still ineffective in 20%-40% of cases. In order to explore the new agents against CE, we have investigated the therapeutic effect of the recombinant adenoviral vector expressing mouse IL-28B (rAd-mIL-28B) on protoscoleces-infected mice. In our study, we successfully established the model mice which infected with protoscoleces intraperitoneally. At 18 weeks post-infection, the mice received rAd-mIL-28B (1×10⁷ PFU) weekly by intramuscular injection for 6 weeks. Compared with the untreated control (13.1 ± 2.2 g), there was a significant reduction in cysts wet weight in rAd-mIL-28B group (8.3 ± 3.5 g) (P < 0.05), especially in Albendazole (ABZ) + rAd-mIL-28B group (5.8 ± 1.4 g) (P < 0.01). We also observed the severe damage of the germinal layer and the laminated layer of cysts after treatment. rAd-mIL-28B group showed a prominent increase in the level of Th1 type cytokines (such as IFN-γ, IL-2 and TNF-α). Meanwhile, the frequency of Foxp3⁺ T cells was decreased in the rAd-mIL-28B group (4.83 ± 0.81%) and ABZ + rAd-mIL-28B group (4.60 ± 0.51%), comparing with the untreated group (8.13 ± 2.60%) (P < 0.05). In addition, compared with the untreated control (122.14 ± 81.09 pg/ml), the level of IFN-γ significantly increased in peritoneal fluid in the rAd-mIL-28B group (628.87 ± 467.16 pg/ml) (P < 0.05) and ABZ + rAd-mIL-28B group (999.76 ± 587.60 pg/ml) (P < 0.001). Taken together, it suggested that ABZ + IL-28B may be a potential therapeutic agent against CE.     

Retinol-binding protein,retinol, and modified-relative-dose response in Ugandan children aged 12–23 months and their non-pregnant caregivers

Retinol-binding protein (RBP), retinol, and modified-relative-dose response (MRDR) are used to assess vitamin A status. We describe vitamin A status in Ugandan children and women using dried blood spot (DBS) RBP, serum RBP, plasma retinol, and MRDR and compare DBS-RBP, serum RBP, and plasma retinol. Blood was collected from 39 children aged 12–23 months and 28 non-pregnant mothers aged 15–49 years as a subsample from a survey in Amuria district, Uganda, in 2016. DBS RBP was assessed using a commercial enzyme immunoassay kit, serum RBP using an in-house sandwich enzyme-linked immunosorbent assay, and plasma retinol/MRDR test using high-performance liquid chromatography. We examined (a) median concentration or value (Q1, Q3); (b) R² between DBS-RBP, serum RBP, and plasma retinol; and (c) Bland-Altman plots. Median (Q1, Q3) for children and mothers, respectively, were as follows: DBS-RBP 1.15 µmol/L (0.97, 1.42) and 1.73 (1.52, 1.96), serum RBP 0.95 µmol/L (0.78, 1.18) and 1.47 µmol/L (1.30, 1.79), plasma retinol 0.82 µmol/L (0.67, 0.99) and 1.33 µmol/L (1.22, 1.58), and MRDR 0.025 (0.014, 0.042) and 0.014 (0.009, 0.019). DBS RBP-serum RBP R² was 0.09 for both children and mothers. The mean biases were −0.19 µmol/L (95% limits of agreement [LOA] 0.62, −0.99) for children and −0.01 µmol/L (95% LOA −1.11, −1.31) for mothers. DBS RBP-plasma retinol R² was 0.11 for children and 0.13 for mothers. Mean biases were 0.33 µmol/L (95% LOA −0.37, 1.03) for children, and 0.29 µmol/L (95% LOA −0.69, 1.27) for mothers. Serum RBP-plasma retinol R² was 0.75 for children and 0.55 for mothers, with mean biases of 0.13 µmol/L (95% LOA −0.23, 0.49) for children and 0.18 µmol/L (95% LOA −0.61, 0.96) for mothers. Results varied by indicator and matrix. The serum RBP-retinol R² for children was moderate (0.75), but poor for other comparisons. Understanding the relationships among vitamin A indicators across contexts and population groups is needed.     

Combined In Silico,In Vivo,and In Vitro Studies Shed Insights into the Acute Inflammatory Response in Middle-Aged Mice

We combined in silico, in vivo, and in vitro studies to gain insights into age-dependent changes in acute inflammation in response to bacterial endotoxin (LPS). Time-course cytokine, chemokine, and NO₂ ^−/NO₃ ⁻ data from “middle-aged” (6–8 months old) C57BL/6 mice were used to re-parameterize a mechanistic mathematical model of acute inflammation originally calibrated for “young” (2–3 months old) mice. These studies suggested that macrophages from middle-aged mice are more susceptible to cell death, as well as producing higher levels of pro-inflammatory cytokines, vs. macrophages from young mice. In support of the in silico-derived hypotheses, resident peritoneal cells from endotoxemic middle-aged mice exhibited reduced viability and produced elevated levels of TNF-α, IL-6, IL-10, and KC/CXCL1 as compared to cells from young mice. Our studies demonstrate the utility of a combined in silico, in vivo, and in vitro approach to the study of acute inflammation in shock states, and suggest hypotheses with regard to the changes in the cytokine milieu that accompany aging.     

The liver X receptor agonist TO901317 protects mice against cisplatin-induced kidney injury

Liver X receptors are in the nuclear receptor superfamily and are contained in the regulation of lipid and cholesterol metabolism. Besides, liver X receptors are considered crucial regulators of the inflammatory response and innate immunity. The current study evaluates the in vivo effects that the synthetic liver X receptor agonist TO901317 protects against cisplatin-induced kidney injury in mice. Mice received cisplatin administration through a single intraperitoneal injection (20 mg/kg in saline). And then the mice were treated with the TO901317 by daily gavage (10 mg/kg/day) 12 h postcisplatin administration, and cisplatin nephrotoxicity was evaluated. At 72 h after cisplatin treatment, elevated plasma urea and creatinine levels (P < 0.05) were evidenced which indicates the renal dysfunction of the vehicle-treated mice, consistent with tubular necrosis, protein cast, dilation of renal tubules, and desquamation of epithelial cells in renal tubules. In contrast, the severity of renal dysfunction and histological damage was reduced in TO901317 treated mice (P < 0.05). In accordance, circulating tumor necrosis factor alpha levels, renal tumor necrosis factor alpha, p47^phox, gp91^phox, and protein expression levels and COX-2 mRNA, renal monocyte chemoattractant protein 1, VACAM-1 mRNA and intercellular adhesion molecule-1 contents, and renal prostaglandin E₂ amounts, were higher in samples from cisplatin-treated mice in comparison with controls (P < 0.05) but attenuated in the TO901317 treatment group (P < 0.05). Taken together, treatment with the liver X receptor agonist TO901317 ameliorated the inflammatory response and oxidative stress in cisplatin-induced kidney injury in mice.     

Modulation of Recombinant Antigenic Constructs Containing Multi-Epitopes towards Effective Reduction of Atherosclerotic Lesion in B6;129S-Ldlrtm1HerApobtm2Sgy/J Mice

Atherosclerosis is increasingly recognized as a complex chronic inflammatory disease. Many more studies have extended vaccination against atherosclerosis by using epitopes from self-antigens or beyond and demonstrated that vaccination with antigens or derivatives could reduce the extent of the lesions in atherosclerosis-prone mice. Our previous study has demonstrated that construct AHHC [ApoB100^688-707 + hHSP60^303-312 + hHSP60^153-163 + Cpn derived peptide (C)] significantly reduced atherosclerotic lesion. The aim of this study was to investigate whether AHHC can be modulated towards increased lesion reduction in mice by creating two other derivatives with a sequential epitope-substitution named RHHC in which A was replaced by an “R” (C5aR^1-31) and RPHC with a further “H” (hHSP60^303-312) conversion into “P” (protease-activated receptor-1^42-55) in mice. Antigenic epitopes were incorporated into a dendroaspin scaffold. Immunization of B6;129S-Ldlr^tm1HerApob^tm2Sgy/J mice with three constructs elicited production of high levels of antibodies against each epitope (apart from hHSP60^153-163 and P which induced a low antibody response). Histological analyses demonstrated that the mice immunized with either RPHC or RHHC showed significant reductions in the size of atherosclerostic lesions compared to those with AHHC (69.5±1.1% versus 55.7±3.4%, P<0.01 or 65.6±1.3% versus 55.7±3.4%, P<0.01). Reduction of plaque size in the aortic sinus and descending aorta correlated with alterations in cellular immune responses when compared with controls. We conclude that a recombinant construct RPHC may provide new antigenic and structural features which are favorable for significant reduction in atherosclerotic lesion formation. This approach offers a novel strategy for developing anti-atherosclerotic agents.