Direct Regulation of Osteocytic Connexin 43 Hemichannels through AKT Kinase Activated by Mechanical Stimulation

Connexin (Cx) 43 hemichannels in osteocytes are thought to play a critical role in releasing bone modulators in response to mechanical loading, a process important for bone formation and remodeling. However, the underlying mechanism that regulates the opening of mechanosensitive hemichannels is largely unknown. We have recently shown that Cx43 and integrin α5 interact directly with each other, and activation of PI3K appears to be required for Cx43 hemichannel opening by mechanical stimulation. Here, we show that mechanical loading through fluid flow shear stress (FFSS) increased the level of active AKT, a downstream effector of PI3K, which is correlated with the opening of hemichannels. Both Cx43 and integrin α5 are directly phosphorylated by AKT. Inhibition of AKT activation significantly reduced FFSS-induced opening of hemichannels and disrupted the interaction between Cx43 and integrin α5. Moreover, AKT phosphorylation on Cx43 and integrin α5 enhanced their interaction. In contrast to the C terminus of wild-type Cx43, overexpression of the C-terminal mutant containing S373A, a consensus site previously shown to be phosphorylated by AKT, failed to bind with α5 and hence could not inhibit hemichannel opening. Together, our results suggest that AKT activated by FFSS directly phosphorylates Cx43 and integrin α5, and Ser-373 of Cx43 plays a predominant role in mediating the interaction between these two proteins and Cx43 hemichannel opening, a crucial step to mediate the anabolic function of mechanical loading in the bone.     

Effect of Partial H2O-D2O Replacement on the Anisotropy of Transverse Proton Spin Relaxation in Bovine Articular Cartilage

Anisotropy of transverse proton spin relaxation in collagen-rich tissues like cartilage and tendon is a well-known phenomenon that manifests itself as the “magic-angle” effect in magnetic resonance images of these tissues. It is usually attributed to the non-zero averaging of intra-molecular dipolar interactions in water molecules bound to oriented collagen fibers. One way to manipulate the contributions of these interactions to spin relaxation is by partially replacing the water in the cartilage sample with deuterium oxide. It is known that dipolar interactions in deuterated solutions are weaker, resulting in a decrease in proton relaxation rates. In this work, we investigate the effects of deuteration on the longitudinal and the isotropic and anisotropic contributions to transverse relaxation of water protons in bovine articular cartilage. We demonstrate that the anisotropy of transverse proton spin relaxation in articular cartilage is independent of the degree of deuteration, bringing into question some of the assumptions currently held over the origins of relaxation anisotropy in oriented tissues.     

Comparing Glaucoma Progression on 24-2 and 10-2 Visual Field Examinations

PurposeTo compare the rate of mean deviation (MD) change on 24-2 versus 10-2 VFs in treated glaucomatous eyes with 5 or more examinations.MethodsIn a retrospective study, 24-2 and 10-2 VFs of 131 glaucoma patients (167 eyes) who had undergone at least 5 VFs examinations during their follow-up were analyzed. All these patients had VF defects both on 24-2 and 10-2 VFs. Rates of MD change were calculated using best linear unbiased predictions (BLUP).ResultsMedian age, MD on 24-2 VF at baseline, number of VFs performed during follow-up and follow-up duration were 55 years, -16.9 dB, 9 and 9 years respectively. Median rate of MD change was significantly greater (p<0.001) on 10-2 VF (-0.26 dB/year; interquartile range [IQR]: -0.47, -0.11) compared to 24-2 VFs (-0.19 dB/year; IQR: -0.41, -0.03). Comparing the rates of MD change in eyes with different severities of VF loss (early [MD better than -6 dB], moderate [-6 dB to -12 dB], advanced [-12 to -20 dB] and severe [MD worse than -20 dB]) at baseline (based on the MD on 24-2 VF), median rate of MD change was comparable between 10-2 and 24-2 VFs in mild (-0.45 dB/year vs. -0.40 dB/year, P = 0.42) and moderate (-0.32 dB/year vs. -0.40 dB/year, P = 0.26) VF loss categories, while the same were significantly greater on 10-2 VFs in advanced (-0.28 dB/year vs. -0.21 dB/year, P = 0.04) and severe (-0.18 dB/year vs. -0.06 dB/year, P<0.001) VF loss categories.ConclusionsIn patients with VF defects both on 24-2 and 10-2 VFs, evaluating the rate of MD change on 10-2 VFs may help in better estimation of glaucoma progression, especially so in eyes with advanced glaucoma at baseline.     

Targeting head and neck squamous cell carcinoma using a novel fusion toxin-diphtheria toxin/HN-1

The current treatment strategies, chemotherapy and radiation therapy being used for the management of cancer are deficient in targeted approach leading to treatment related toxicities and relapse. Contrarily, fusion toxins exhibit remarkable tumor specificity thus emerging as an alternative therapy for the treatment of cancer. Diphtheria toxin-HN-1 peptide (DT/HN-1) is a fusion toxin designed to target the head and neck squamous cell carcinoma (HNSCC). The aim of this study was to construct, characterize, and evaluate the cytotoxicity and specificity of DT/HN-1 fusion toxin against the HNSCC cells. The purified DT/HN-1 fusion toxin was characterized by SDS-PAGE and western blotting. Refolding of purified fusion toxins was monitored by fluorescence spectra and circular dichroism spectra. The activity of DT/HN-1 fusion toxin was demonstrated on various HNSCC cell lines by cell viability assay, cell proliferation assay, protein synthesis inhibition assay, apoptosis and cell cycle analysis. The fusion toxin DT/HN-1 demonstrated remarkably high degree of cytotoxicity specific to the HNSCC cells. The IC₅₀ of DT/HN-1 fusion toxin was ~1 to 5 nM in all the three HNSCC cell lines. The percentage apoptotic cells in DT/HN-1 treated UMB-SCC-745 cells are 16% compared to 4% in untreated. To further demonstrate the specific toxicity of DT/HN-1 fusion toxin towards the HNSCC cells we constructed, characterized and evaluated the efficacy of DT protein. The DT protein coding for only a fragment of diphtheria toxin without its native receptor binding domain failed to exhibit any cytotoxicity on all the cell lines used in this study thus establishing the importance of a ligand in achieving targeted toxicity. To evaluate the translocation ability of HN-1 peptide, an additional construct DTΔT/HN-1 was constructed, characterized and evaluated for its cytotoxic activity. The fusion toxin DTΔT/HN-1 deficient of the translocation domain of diphtheria toxin showed no cytotoxicity on all the cell lines clearly indicating the inability of HN-1 peptide to translocate catalytic domain of the toxin into the cytosol.     

Prolonged Amphetamine Treatments Cause Long-Term Decrease of Dopamine Uptake in Cultured Cells

Neurochemical Research - Amphetamine (AMPH) is a systemic stimulant used to treat a variety of diseases including Attention Deficit Hyperactive Disorder, narcolepsy and obesity. Previous data...     

Outpatient Healthcare Settings and Transmission of Clostridium difficile

Background

Recent reports suggest that community-associated Clostridium difficile infection (CDI) (i.e., no healthcare facility admission within 90 days) may be increasing in frequency. We hypothesized that outpatient clinics could be an important source for acquisition of community-associated CDI.

Methods

We performed a 6-month prospective study of CDI patients to determine frequency of and risk factors for skin and environmental shedding during outpatient visits and to derive a prediction rule for positive cultures. We performed a point–prevalence culture survey to assess the frequency of C. difficile contamination in outpatient settings and evaluated the frequency of prior outpatient visits in patients with community-associated CDI.

Results

Of 67 CDI patients studied, 54 (81%) had 1 or more outpatient visits within 12 weeks after diagnosis. Of 44 patients cultured during outpatient visits, 14 (32%) had skin contamination and 12 (27%) contaminated environmental surfaces. Decreased mobility, fecal incontinence, and treatment with non-CDI antibiotics were associated with positive cultures, whereas vancomycin taper therapy was protective. In patients not on CDI therapy, a prediction rule including incontinence or decreased mobility was 90% sensitive and 79% specific for detection of spore shedding. Of 84 clinic and emergency department rooms cultured, 12 (14%) had 1 or more contaminated environmental sites. For 33 community-associated CDI cases, 31 (94%) had an outpatient visit during the 12 weeks prior to onset of diarrhea.

Conclusions

Patients with recent CDI present a significant risk for transmission of spores during outpatient visits. The outpatient setting may be an underappreciated source of community-associated CDI cases.     

Effect of an interspinous implant on loads in the lumbar spine.

Interspinous process implants are increasingly used to treat canal stenoses. Little information exists about the effects of implant height and stiffness on the biomechanical behavior of the lumbar spine. Therefore, a three-dimensional nonlinear finite element model of the osseoligamentous lumbar spine (L1 to L5) was created with a slightly degenerated disc at L3/L4. An interspinous implant was inserted at that segment. Implants with different heights and stiffnesses were studied. The model was loaded with the upper body weight and muscle forces to simulate walking and 25 degrees extension. Implant forces are influenced strongly by the height and negligibly by the elastic modulus of the implant. Intersegmental rotation at implant level is markedly reduced, while intradiscal pressure is slightly increased. Implant size and stiffness have only a minor effect on intradiscal pressure. The maximum von Mises stress in the vertebral arch is strongly increased by the implant.     

Alkaline phosphatase and acid phosphatase levels in saliva and serum of patients with healthy periodontium,gingivitis, and periodontitis before and after scaling with root planing: A clinico-biochemical study

Periodontitis is commonly diagnosed based on clinical parameters. However, the analysis of a few unique biomarkers of the disease process present in the saliva and blood can further assist the estimation of the rate of disease progression.AimThe present study attempted to correlate the alkaline phosphatase (ALP) and acid phosphatase (ACP) levels in saliva and serum between patients with healthy periodontium, gingivitis, and chronic periodontitis.Materials and methodsThe present study was conducted in 135 subjects between 20 and 55 years of age. The subjects were divided into three groups, namely healthy (Group A), gingivitis (Group B), and chronic periodontitis (Group C). The clinical parameters were recorded using the plaque index (PI), gingival index (GI), and probing depth (PD). Saliva and serum were analyzed for ALP and ACP levels using an auto analyzer. All patients underwent scaling and root planning (SRP) along with oral hygiene instructions. Patients were then recalled after four weeks, and blood and saliva samples were collected to estimate ALP and ACP levels prior to clinical examination.ResultsThe clinical parameters exhibited a statistically significant decrease in the PI and GI in both group B and group C after SRP. A significant change in the PD and attachment levels (AL) was observed in the periodontitis group after SRP. The mean salivary & serum ALP levels exhibited a statistically significant decrease in group B & C after SRP. The mean serum ACP levels exhibited a statistically significant decrease in group B & C after SRP However, the salivary ACP levels decrease after SRP was only statistically significant in group C.ConclusionSerum and salivary ALP and ACP levels were markedly decreased in the gingivitis and periodontitis groups after SRP and were positively correlated with the clinical parameters.     

Factors Affecting the Ability of the Spectral Domain Optical Coherence Tomograph to Detect Photographic Retinal Nerve Fiber Layer Defects

Purpose

To evaluate the ability of normative database classification (color-coded maps) of spectral domain optical coherence tomograph (SDOCT) in detecting wedge shaped retinal nerve fiber layer (RNFL) defects identified on photographs and the factors affecting the ability of SDOCT in detecting these RNFL defects.

Methods

In a cross-sectional study, 238 eyes (476 RNFL quadrants) of 172 normal subjects and 85 eyes (103 RNFL quadrants with wedge shaped RNFL defects) of 66 glaucoma patients underwent RNFL imaging with SDOCT. Logistic regression models were used to evaluate the factors associated with false positive and false negative RNFL classifications of the color-coded maps of SDOCT.

Results

False positive classification at a p value of <5% was seen in 108 of 476 quadrants (22.8%). False negative classification at a p value of <5% was seen in 16 of 103 quadrants (15.5%). Of the 103 quadrants with RNFL defects, 64 showed a corresponding VF defect in the opposite hemisphere and 39 were preperimetric. Higher signal strength index (SSI) of the scan was less likely to have a false positive classification (odds ratio: 0.97, p = 0.01). Presence of an associated visual field defect (odds ratio: 0.17, p = 0.01) and inferior quadrant RNFL defects as compared to superior (odds ratio: 0.24, p = 0.04) were less likely to show false negative classifications.

Conclusions

Scans with lower signal strengths were more likely to show false positive RNFL classifications, and preperimetric and superior quadrant RNFL defects were more likely to show false negative classifications on color-coded maps of SDOCT.