Organ printing: the future of bone regeneration?

In engineered bone grafts, the combined actions of bone-forming cells, matrix and bioactive stimuli determine the eventual performance of the implant. The current notion is that well-built 3D constructs include the biological elements that recapitulate native bone tissue structure to achieve bone formation once implanted. The relatively new technology of organ/tissue printing now enables the accurate 3D organization of the components that are important for bone formation and also addresses issues, such as graft porosity and vascularization. Bone printing is seen as a great promise, because it combines rapid prototyping technology to produce a scaffold of the desired shape and internal structure with incorporation of multiple living cell types that can form the bone tissue once implanted.     

Effects of Venlafaxine and Escitalopram Treatments on NMDA Receptors in the Rat Depression Model

Depression may relate to neurocognitive impairment that results from alteration of N-methyl-D: -aspartate receptor (NMDAR) levels. Venlafaxine and escitalopram are two drugs commonly used to treat depression. The drugs may affect expression of NMDARs, which mediate learning and memory formation. The aim of the study was to examine whether the effects of venlafaxine and escitalopram treatments are associated with NMDARs in a rat model of depression. Forty male Wistar albino rats were randomly divided into four groups (n?=?10) as follows: control group, chronic mild stress group (CMS), venlafaxine (20?mg/kg body weight per day)?+?CMS, and escitalopram (10?mg/kg body weight per day)?+?CMS. After induction of depression, a decrease in the concentration of NR2B was observed; venlafaxine treatment prevented the reduction of NR2B expression. Escitalopram treatment did not effect the reduced levels of NR2B resulting from depression. There was no significant difference in NR2A concentration among groups. The present data support the notion that venlafaxine plays a role in maintaining NR2B receptor in experimental depression. It may be possible that treatment with escitalopram has no effect on NMDARs in experimental depression.     

Higher Seroprevalence of Hepatitis B Virus Antigen in Patients with Cystic Hydatid Disease than in Patients Referred to Internal Medicine Clinics in Turkey

Turkey remains an intermediate area for prevalence of hepatitis B virus (HBV) surface antigenemia. The sheep-raising areas of Turkey also pose a high risk for cystic hydatid disease (CHD). Both HBV infection and CHD are major public health issues particularly in eastern parts of Turkey; however, there is no data regarding HBV infection in patients who have had CHD. The aims of this study were to evaluate the association between HBV infection and CHD and suggest ways to reduce HBV infection which is still widespread in Turkey. A retrospective study was conducted with 94 adult patients with active CHD referred to the hepatology department, Yuzuncuyil University School of Medicine from December 2010 to December 2012. All subjects came from rural areas of the region and underwent ultrasonography of abdomen which detected CHD of the liver. All the patients were serologically positive for Echinococcus granulosus. The control group consisted of 500 patients (300 men and 200 women) referred to the internal medicine clinics for other reasons. The patients with CHD and in the control group were tested for the existence of HBs antigen according to the standard procedures. The seroprevalence of HBs antigen was significantly higher in patients with active CHD than those in the control group (12.7% vs 5.2%; P=0.0017). Our data indicate that there is significant association between HBV infection and CHD. All patients with CHD should be screened for HBV infection.     

Amyloid Beta Peptides Affect Pregnenolone and Pregnenolone Sulfate Levels in PC-12 and SH-SY5Y Cells Depending on Cholesterol

Increased amyloid beta (AB) peptide concentration is one of the initiating factors in the neurodegeneration process. It has been suggested that cholesterol induces the synthesis of AB peptide from amyloid precursor protein or facilitates the formation of amyloid plaque by lowering the aggregation threshold of the peptide. It is also shown that AB peptides may affect cholesterol metabolism and the synthesis of steroid hormones such as progesterone and estradiol. Pregnenolone (P) and pregnenolone sulfate (PS) are the major steroids produced from cholesterol in neural tissue. In toxicity conditions, the effect of AB peptides on P and PS levels has not yet been determined. Furthermore, it has not been clearly defined how changes in cellular P and PS levels affect neuronal cell survival. The aim of this study was to determine the effects of AB peptides on cellular changes in P and PS levels depending on the level of their main precursor, cholesterol. Cholesterol and toxic concentrations of AB fragments (AB 25–35, AB 1–40 and AB 1–42) were applied to PC-12 and SH-SY5Y cells. Changes in cellular cholesterol, P and PS levels were determined simultaneously in a dose—and time-dependent manner. The cell viability and cell death types were also evaluated. AB peptides affected both cell viability and P/PS levels. Steroid levels were altered depending on AB fragment type and the cholesterol content of the cells. Treatment with each of the AB fragments alone increased P levels by twofold. However, combined treatment with AB peptides and cholesterol increased P levels by approximately sixfold, while PS levels were increased only about 2.5 fold in both cell lines. P levels in the groups treated with AB 25–35 were higher than those in AB 1–40 and AB 1–42 groups. The cell viabilities were significantly low in the group treated by AB and cholesterol (9 mM). The effect of AB peptides on P levels might be a result of cellular self-defense. On the other hand, the rate of P increase might be playing a key role in the cell death mechanism of AB toxicity depending on cellular cholesterol levels.     

Estrogen and non‐genomic upregulation of voltage‐gated Na+ channel activity in MDA‐MB‐231 human breast cancer cells: Role in adhesion

External (but not internal) application of β‐estradiol (E2) increased the current amplitude of voltage‐gated Na⁺ channels (VGSCs) in MDA‐MB‐231 human breast cancer (BCa) cells. The G‐protein activator GTP‐γ‐S, by itself, also increased the VGSC current whilst the G‐protein inhibitor GDP‐β‐S decreased the effect of E2. Expression of GPR30 (a G‐protein‐coupled estrogen receptor) in MDA‐MB‐231 cells was confirmed by PCR, Western blot and immunocytochemistry. Importantly, G‐1, a specific agonist for GPR30, also increased the VGSC current amplitude in a dose‐dependent manner. Transfection and siRNA‐silencing of GPR30 expression resulted in corresponding changes in GPR30 protein expression but only internally, and the response to E2 was not affected. The protein kinase A inhibitor, PKI, abolished the effect of E2, whilst forskolin, an adenylate cyclase activator, by itself, increased VGSC activity. On the other hand, pre‐incubation of the MDA‐MB‐231 cells with brefeldin A (a trans‐Golgi protein trafficking inhibitor) had no effect on the E2‐induced increase in VGSC amplitude, indicating that such trafficking (‘externalisation’) of VGSC was not involved. Finally, acute application of E2 decreased cell adhesion whilst the specific VGSC blocker tetrodotoxin increased it. Co‐application of E2 and tetrodotoxin inhibited the effect of E2 on cell adhesion, suggesting that the effect of E2 was mainly through VGSC activity. Pre‐treatment of the cells with PKI abolished the effect of E2 on adhesion, consistent with the proposed role of PKA. Potential implications of the E2‐induced non‐genomic upregulation of VGSC activity for BCa progression are discussed. J. Cell. Physiol. 224: 527–539, 2010. © 2010 Wiley‐Liss, Inc.     

Role of the nitric oxide pathway in ischemia-reperfusion injury in isolated perfused guinea pig lungs

We examined the role of the nitric oxide (NO) pathway on ischemia-reperfusion injury via the use of isolated perfused guinea pig lungs. We administered both L-Arginine and N-nitro-L-arginine methyl ester (L-NAME) to the lungs in or after 3 h of ischemia. We observed pulmonary artery pressures as well as tissue and perfusate malondialdehyde (MDA) and glutathione (GSH) levels. We observed that L-NAME significantly increased both tissue and perfusate GSH levels and pulmonary artery pressures, but it decreased both tissue and perfusate MDA levels. On the other hand, L-arginine significantly decreased pulmonary artery pressure and both tissue and perfusate glutathione levels, but it increased both tissue and perfusate MDA levels. Electron microscopic evaluation supported our findings by indicating the preservation of lamellar bodies of type II pneumocytes. We concluded that L-NAME administration during reperfusion improves lung recovery from ischemic injury.     

SH2 Modified STAT1 Induces HLA-I Expression and Improves IFN-γ Signaling in IFN-α Resistant HCV Replicon Cells

Background

We have developed multiple stable cell lines containing subgenomic HCV RNA that are resistant to treatment with interferon alpha (IFN-α. Characterization of these IFN-α resistant replicon cells showed defects in the phosphorylation and nuclear translocation of STAT1 and STAT2 proteins due to a defective Jak-STAT pathway.

Methodology/Principal Findings

In this study, we have developed an alternative strategy to overcome interferon resistance in a cell culture model by improving intracellular STAT1 signaling. An engineered STAT1-CC molecule with double cysteine substitutions in the Src-homology 2 (SH2) domains of STAT1 (at Ala-656 and Asn-658) efficiently phosphorylates and translocates to the nucleus of IFN-resistant cells in an IFN-γ dependent manner. Transfection of a plasmid clone containing STAT1-CC significantly activated the GAS promoter compared to wild type STAT1 and STAT3. The activity of the engineered STAT1-CC is dependent upon the phosphorylation of tyrosine residue 701, since the construct with a substituted phenylalanine residue at position 701 (STAT1-CC-Y701F) failed to activate GAS promoter in the replicon cells. Intracellular expression of STAT1-CC protein showed phosphorylation and nuclear translocation in the resistant cell line after IFN-γ treatment. Transient transfection of STAT1-CC plasmid clone into an interferon resistant cell line resulted in inhibition of viral replication and viral clearance in an IFN-γ dependent manner. Furthermore, the resistant replicon cells transfected with STAT1-CC constructs significantly up regulated surface HLA-1 expression when compared to the wild type and Y to F mutant controls.

Conclusions

These results suggest that modification of the SH2 domain of the STAT1 molecule allows for improved IFN-γ signaling through increased STAT1 phosphorylation, nuclear translocation, HLA-1 surface expression, and prolonged interferon antiviral gene activation.     

Localization of type IV collagen a 1 to a 6 chains in basement membrane during mouse molar germ development.

The dental basement membrane (BM) putatively mediates epithelial-mesenchymal interactions during tooth morphogenesis and cytodifferentiation. Type IV collagen alpha chains, a major network-forming protein of the dental BM, was studied and results disclosed distinct expression patterns at different stages of mouse molar germ development. At the dental placode and bud stage, the BM of the oral epithelium expressed alpha 1, alpha 2, alpha 5 and alpha 6 chains while the gubernaculum dentis, in addition to the above four chains, also expressed a 4 chain. An asymmetrical expression for alpha 4, alpha 5 and alpha 6 chains was observed at the bud stage. At the early bell stage, the BM associated with the inner enamel epithelium (IEE) of molar germ expressed alpha 1, alpha 2 and alpha 4 chains while the BM of the outer enamel epithelium (OEE) expressed only alpha 1 and a 2 chains. With the onset of dentinogenesis, the collagen a chain profile of the IEE BM gradually disappeared. Howeverfrom the early to late bell stage, the gubernaculum dentis consistently expressed alpha 1, alpha 2, alpha 5 and a 6 chains resembling fetal oral mucosa. These findings suggest that stage- and position-specific distribution of type IV collagen alpha subunits occur during molar germ development and that these changes are essential for molar morphogenesis and cytodifferentiation.