Cardiac-specific inducible and conditional gene targeting in mice

Mouse genetic engineering has revolutionized our understanding of the molecular and genetic basis of heart development and disease. This technology involves conditional tissue-specific and temporal transgenic and gene targeting approaches, as well as introduction of polymorphisms into the mouse genome. These approaches are increasingly used to elucidate the genetic pathways underlying tissue homeostasis, physiology, and pathophysiology of adult heart. They have also led to the development of clinically relevant models of human cardiac diseases. Here, we review the technologies and their limitations in general and the cardiovascular research community in particular.     

Overexpression of FoxM1 offers a promising therapeutic target in diffuse large B-cell lymphoma

Background

FoxM1 has been shown to play a critical role in the pathogenesis of various epithelial malignancies. However, its role in lymphoid malignancies has not been fully clarified. We, therefore, investigated the role of FoxM1 expression in a large cohort of diffuse large B-cell lymphoma samples and panel of cell lines.

Design and Methods

FoxM1 expression was investigated in a large series of diffuse large B-cell lymphoma tissues in a tissue microarray format by immunohistochemistry. Apoptosis was measured by flow cytometry and protein expression was detected by immunoblotting using diffuse large B-cell lymphoma cell lines following treatment with either pharmacological inhibitor of FoxM1 or small interference RNA knockdown strategy. Invasion/migration and soft agar colony assays were also performed following treatment with FoxM1 inhibitor.

Results

FoxM1 expression was detected in 84.6% of diffuse large B-cell lymphoma tumors and found to be significantly associated with proliferative tumor marker Ki67 (P<0.0001), matrix metalloproteinases-2 (P=0.0008), matrix metalloproteinases-9 (P=0.0002), S-phase kinase associated protein-2 (P<0.0001) and inversely associated with p27 expression (P=0.0215). Expression of small interference RNA targeted against FoxM1 or treatment of diffuse large B-cell lymphoma cells with thiostrepton caused its downregulation accompanied by decreased expression of matrix metalloproteinases-2 and matrix metalloproteinases-9. Inhibition of FoxM1 in diffuse large B-cell lymphoma cells also decreased invasive and migratory capability, and induced caspase dependent apoptosis via activation of the mitochondrial apoptotic pathway. Finally, combined thiostrepton and bortezomib at sub-toxic doses led to efficient apoptosis in diffuse large B-cell lymphoma cells.

Conclusions

Altogether, these results suggest that FoxM1 is over-expressed in the majority of diffuse large B-cell lymphoma samples. These data also indicate that targeting FoxM1 signaling can serve as a potential therapeutic modality in the management of diffuse large B-cell lymphoma.     

Serum neutrophil gelatinase-associated lipocalin and cystatin C as markers of renal function in patients with stages 2–5 chronic kidney disease

Individuals with reduced glomerular filtration rate (GFR) are at greater risk for cardiovascular disease and other comorbidities. Creatinine-based equations are used to estimate GFR, identify patients with potential kidney disease, and classify them into different stages since serum creatinine is insensitive to changes in the GFR. The aim of our work was to evaluate diagnostic performance of serum cystatin C and neutrophil gelatinase-associated lipocalin (NGAL) as markers of kidney function in patients with reduced GFR. Fifty cases at different stages of renal impairment and 30 healthy control subjects were tested. Only serum NGAL and cystatin C were higher in stage 2 chronic kidney disease (CKD) when compared to the control group (p?<?0.05). For stages 3–5 the median levels of cystatin C, NGAL, and creatinine were found to be significantly higher than the control group. ROC curve constructed to differentiate stage 2 patients from the controls showed AUC for NGAL 0.795, sensitivity 86%, and specificity 63.3%; AUC for cystatin C 0.957, sensitivity 86%, and specificity 90%; and AUC for creatinine 0.738. Frequency of cases that tested positive for NGAL and cystatin C in stage 2 was higher than those in control group (p?<?0.05) with an OR of 10.364 (95% CI 1.099–97.686) for NGAL and OR 54 (95% CI 4.7–613) for cystatin C. NGAL and cystatin C exhibited higher sensitivity than creatinine for diagnosis of stage 2 CKD. Their use as adjunctive diagnostic tools in patients with mildly reduced GFR may be justified on the long term to diagnose early renal insult.     

Hyperactive Transforming Growth Factor‐β1 Signaling Potentiates Skeletal Defects in a Neurofibromatosis Type 1 Mouse Model

Dysregulated transforming growth factor beta (TGF‐β) signaling is associated with a spectrum of osseous defects as seen in Loeys‐Dietz syndrome, Marfan syndrome, and Camurati‐Engelmann disease. Intriguingly, neurofibromatosis type 1 (NF1) patients exhibit many of these characteristic skeletal features, including kyphoscoliosis, osteoporosis, tibial dysplasia, and pseudarthrosis; however, the molecular mechanisms mediating these phenotypes remain unclear. Here, we provide genetic and pharmacologic evidence that hyperactive TGF‐β1 signaling pivotally underpins osseous defects in Nf1^flox/?;Col2.3Cre mice, a model which closely recapitulates the skeletal abnormalities found in the human disease. Compared to controls, we show that serum TGF‐β1 levels are fivefold to sixfold increased both in Nf1^flox/?;Col2.3Cre mice and in a cohort of NF1 patients. Nf1‐deficient osteoblasts, the principal source of TGF‐β1 in bone, overexpress TGF‐β1 in a gene dosage–dependent fashion. Moreover, Nf1‐deficient osteoblasts and osteoclasts are hyperresponsive to TGF‐β1 stimulation, potentiating osteoclast bone resorptive activity while inhibiting osteoblast differentiation. These cellular phenotypes are further accompanied by p21‐Ras–dependent hyperactivation of the canonical TGF‐β1–Smad pathway. Reexpression of the human, full‐length neurofibromin guanosine triphosphatase (GTPase)‐activating protein (GAP)‐related domain (NF1 GRD) in primary Nf1‐deficient osteoblast progenitors, attenuated TGF‐β1 expression levels and reduced Smad phosphorylation in response to TGF‐β1 stimulation. As an in vivo proof of principle, we demonstrate that administration of the TGF‐β receptor 1 (TβRI) kinase inhibitor, SD‐208, can rescue bone mass deficits and prevent tibial fracture nonunion in Nf1^flox/?;Col2.3Cre mice. In sum, these data demonstrate a pivotal role for hyperactive TGF‐β1 signaling in the pathogenesis of NF1‐associated osteoporosis and pseudarthrosis, thus implicating the TGF‐β signaling pathway as a potential therapeutic target in the treatment of NF1 osseous defects that are refractory to current therapies. © 2013 American Society for Bone and Mineral Research.     

Tracheal intubation with size 1 Airtraq in a 3 month old child with occipital meningocele: A novel experience

Meningocele is a neural tube defect where meninges protrude through a skeletal defect. Occipital meningocele compounded with the problems of paediatric airway itself pose challenges to anaesthesiologist in securing and maintaining airway. We present a case report of a 3 month-old-child with occipital meningocele posted for ventriculo-peritoneal shunt for hydrocephalus, who was successfully intubated using a size 1 Airtraq optical laryngoscope.     

In vitro kinetic release study,antimicrobial activity and in vivo toxicity profile of a kojic acid ester-based nanoemulsion for topical application

Nanoemulsions have emerged as novel vehicles for drug delivery that allow sustained or controlled release for topical application. In this study, kojic acid ester-based nanoemulsion (KAE-NA) was analyzed for in vitro permeation evaluation, kinetic release study, in vitro antimicrobial activity and in vivo toxicity profile on embryonic zebrafish (Danio rerio). Based on KAE-NA in vitro permeation evaluation, the percentage of permeation was significantly improved from 4.94% at 1 h to 59.64% at 8 h of application. The permeation rate of KAE-NA at 8 h was 4659.50 μg cm⁻² h⁻¹ (initial concentration, C₀ = 2000 μg mL⁻¹) with a permeability coefficient (K_p) value of 0.48 cm h⁻¹. The kinetic release analysis showed the Korsmeyer–Peppas model was the best fitted kinetic model with high linearity [R² = 0.9964]. Antimicrobial activity of KAE-NA was studied against the skin pathogen bacteria Staphylococcus aureus ATCC 43300. The results indicated that the inhibition zone size of the KAE-NA (8.00 ± 0.0 mm) was slightly bigger than that of its active ingredient, kojic acid ester (6.5 ± 0.0 mm). The toxicity profile of KAE-NA on embryonic zebrafish revealed less toxicity with LC₅₀ (50% lethal concentration) more than 500 μg mL⁻¹. The survival rate of the embryonic zebrafish was more than 80% when treated at doses ranging from 7.81–250 μg mL⁻¹ and showed normal development throughout the experiment without any observed deformation. Hence, KAE-NA proved to be less toxic on the embryonic zebrafish.

Nanoemulsions have emerged as novel vehicles for drug delivery that allow sustained or controlled release for topical application.     

Carnosic acid ameliorates depressive‐like symptoms along with the modulation of FGF9 in the hippocampus of middle carotid artery occlusion‐induced Sprague Dawley rats

The increased survival rate of stroke patients has led to the higher incidences of post‐stroke depression. Carnosic acid has the ability to cross blood brain barrier with good neuro‐modulatory actions. Recently, inclined level of fibroblast growth factor 9 (FGF9) in the postmortem brain of the depressed patients was noted. Therefore, in the present study, the effect of carnosic acid on post‐stroke depression‐like behavior, and the expression of FGF9 were evaluated. After 3 weeks of middle carotid artery occlusion in Sprague Dawley rats, carnosic acid (20 and 40 mg/kg) was administered for 2 weeks. Sucrose preference test, forced swimming test, and open field test were performed and hippocampi were analyzed for FGF9 and FGFR‐3. In comparison to post‐stroke depressed rats, carnosic acid increased the sucrose preference, and reduced the immobility time of the rats by ~2×. The speed and distance‐covered were also increased. At 40 mg/kg, FGF9 was reduced by ~3× while FGFR‐3/Actin was increased by ~1.5×. Altogether results suggest anti‐depressant‐like activity of carnosic acid in post‐stroke depressed rats with decreased expression of hippocampal FGF9.     

Formation and Investigation of Physicochemical,Biological and Bacteriostatic Properties of Nanocomposite Foils Containing Silver Nanoparticles and Graphene Oxide in Hyaluronic Acid Matrix

Natural polysaccharides, including hyaluronic acid, find a wide range of applications in biomedical sciences. There is a growing interest in nanocomposites containing hyaluronic acid and nanoparticles such as nanometals or graphene. In this study, we prepared foils of pure sodium hyaluronate and sodium hyaluronate containing nanosilver, graphene oxide, nanosilver/graphene oxide and characterized their properties. UV-vis spectroscopy and scanning electron microscopy (SEM) confirmed the formation of 10–20 nm silver nanoparticles. The structural changes were investigated using Fourier transforms infrared (FTIR) spectra and size exclusion chromatography. The obtained results suggest changes in molecular weights in the samples containing nanoparticles, which was highest in a sample containing nanosilver/graphene oxide. We also assessed the mechanical properties of the foils (thickness, tensile strength and elongation at break) and their wettability. The foils containing nanosilver and nanosilver/graphene oxide presented bacteriostatic activity against E. coli, Staphylococcus spp. and Bacillus spp., which was not observed in the control and sample containing graphene oxide. The composites containing graphene oxide and nanosilver/graphene oxide exhibited a cytotoxic effect on human melanoma WM266-4 cell lines (ATCC, Manassas, VA, USA).