Mariano Aguilar

ASEP/MD: A program for the calculation of solvent effects combining QM/MM methods and the mean field approximation ☆

ASEP/MD is a computer program designed to implement the Averaged Solvent Electrostatic Potential/Molecular Dynamics (ASEP/MD) method developed by our group. It can be used for the study of solvent effects and properties of molecules in their liquid state or in solution. It is written in the FORTRAN90 programming language, and should be easy to follow, understand, maintain and modify. Given the nature of the ASEP/MD method, external programs are needed for the quantum calculations and molecular dynamics simulations. The present version of ASEP/MD includes interface routines for the GAUSSIAN package, HONDO, and MOLDY, but adding support for other programs is straightforward. This article describes the program and its usage.

Crosstalk between PARP-1 and NF-jB modulates the promotion of skin neoplasia

Poly (ADP-ribose) polymerase-1 (PARP-1)-deficient mice are protected against septic shock, type I diabetes, stroke and inflammation. It is now accepted that inflammation and related events, such as activation of NF-κB, are key components in the initiation and progression of epithelial cancer and in particular in the neoplastic transformation of keratinocytes and skin carcinogenesis. Here, we report that PARP-1-deficient mice display a strikingly reduced susceptibility to skin carcinogenesis. In parp-1−/− mice, development of papilloma-like premalignant lesions induced with DMBA and TPA, is strongly delayed and the final number of tumor-bearing mice and total tumor number were significantly reduced. In addition, epidermis of parp-1−/− mice did not show increased proliferation rates after treatment with carcinogen. Deregulated NF-κB is a hallmark for tumorigenesis together with the concomitant release of early inflammatory mediators. In the absence of PARP-1, NF-κB activation and induction κB-target genes did not take place during the promotion of tumor development. These results suggest that PARP-1 abolition impairs the promotion of skin carcinogenesis interfering with the activation of NF-κB and might have an important implication in targeting PARP-1 as a new antineoplastic therapeutic approach.

P Glycoprotein: A New Mechanism to Control Drug-Induced Nephrotoxicity

The role of P glycoprotein (P-gp) in kidney is now being explored, and under physiological conditions, this protein is thought to be an excretory pump of cationic xenobiotics and metabolites. Functionally, two different types of P-gp have been described, but only the class I has been related to drug transport, and its overexpression confers the multidrug resistance phenotype in tumoral cells. It has been proposed that P-gp is involved in the energy-dependent transport of substrates through the cell membrane (toxic metabolites, toxins, nutrients, ions, peptides, etc.) – like a ‘hydrophobic molecule vacuum cleaner’. Several physiological functions have been attributed to P-gp: defense against xenobiotic aggression and transmembrane transport of prenylcysteine methyl esters, removing these cytotoxic metabolites from cells. A variety of substrates ranging from chemotherapeutics to steroid hormones, antibiotics, and calcium channel blockers can be transported by P-gp, suggesting the possible involvement of this protein in other unknown functions. Results from our group and others have suggested that overexpression of P-gp in renal tubular and mesangial cells prevents pharmacological nephrotoxicity by cyclosporin A (CsA). On the other hand CsA, a substrate of the pump, could act as a blocker in tubular cells by competitive inhibition. One relevant aspect in kidney is the possible relationship between P-gp and protein kinase C. Several reports suggest that protein kinase C may play a role in inducing the P-gp overexpression in cells under xenobiotic pressure, through activation of the ras oncoprotein family. This could be mediated directly by angiotensin II as a ras activator. This way, the detoxicant function of P-gp against products of the ras catabolism could mediate their accumulation when the ‘vacuum cleaner’ function is blocked by CsA or tacrolimus, contributing to the initial development of fibroblastic activation that leads to interstitial fibrosis associated with nephrotoxicity by these immunosuppressor drugs. In conclusion, P-gp expression could be an important component of a complex detoxifying system in kidney against xenobiotics or regulating the traffic of endogenous metabolites responsible for the susceptibility of subjects to the development of nephrotoxicity against different drugs.

Histomorphometric comparison of maxillary pristine bone and composite bone graft biopsies obtained after sinus augmentation

INTRODUCTION Sinus grafting is a technique oriented to facilitate implant placement in posterior atrophic maxillae. Several modifications of the original technique and a wide variety of materials have been proposed; most of them associated with implant survival rates. However, the quality of the bone obtained after the application of certain grafting materials has not been fully elucidated yet. The aims of this multicenter study were to analyse histomorphometrical samples obtained 6 months after sinus grafting using a composite graft consisting of anorganic bovine bone (ABB)+ autologous bone (AB), and to compare these samples with maxillary pristine bone biopsies. MATERIAL AND METHODS Ninety maxillary sinus augmentations were performed for delayed implant placement (N = 90) in 45 consecutive patients (test group). Bone cores were harvested 6 months after grafting for histomorphometric and ultrastructural study. Control pristine bone biopsies were taken from the posterior maxilla of 10 patients (control). Bone radiographic changes were assessed up to 24 months after implant loading. RESULTS The total mean values after analysis of test cores revealed a proportion of 46.08 + or - 16.6% of vital bone, 42.27 + or - 15.1% of non-mineralized connective tissue, and 37.02 + or - 25.1% of the remaining ABB particles. Significant bone remodeling activities were noticed in sinus grafting samples when compared with pristine bone. A statistically significant difference was observed in the number of osteoid lines between two groups, with higher values in the test one (15.1 + or - 11.48% vs. 2.5 + or - 2.2%, P = 0.0005). Ultrastructural study showed that vital trabecular bone was in intimal contact with ABB particles. Radiographic analysis revealed that the higher the proportion of remaining ABB, the lower the total vertical resorption of the graft. CONCLUSION Sinus grafting constitutes an excellent model for the study of de novo bone formation patterns and graft consolidation, when a combination of different bone substitutes is applied. The combination of ABB+AB yields highly satisfactory outcomes from both a clinical and a histologic perspective.

Role of Intrarenal Endothelin 1, Endothelin 3, and Angiotensin II Expression in Chronic Cyclosporin A Nephrotoxicity in Rats

Endothelin 1 (Et1) is widely expressed in the kidney and is related to several functions and to pathological conditions with progression towards sclerosis. The function of endothelin 3 (Et3) at the renal level is debatable, but it could have an important regulatory function in the reabsorption of water through its action on tubular type B receptors. Angiotensin II has recently been implicated as the principal factor responsible for the progression of interstitial fibrosis induced by cyclosporin A (CsA). We investigated this relationship in vivo and analyzed the modifications induced by CsA toxicity in Sprague-Dawley rats treated with 25 mg/kg/day of CsA for 28 and 56 days. Immunohistochemical methods and molecular analysis were used to study the expression of Et1 and Et3 and immunohistochemistry alone to determine the intrarenal expression of angiotensin II. Rats treated with CsA developed chronic nephrotoxicity lesions; semiquantitative analyses of hyaline arteriolopathy revealed that the passage of time affected the extent of this lesion and led to the diminution of the total glomerular area. Immunohistochemical results showed that chronic CsA treatment induced moderate secretion of Et1 and Et3 at tubular and glomerular levels and that the local expression of angiotensin II in the treatment groups was more evident than in control animals. Besides, the mRNA levels of preproEt3 showed a dramatic increase from 28 days after CsA treatment (control group 0.07 ± 0.11 vs. CsA group 0.48 ± 0.11, p < 0.01), while the mRNA levels of preproEt1 increased from 56 days (control group 0.15 ± 0.05 vs. CsA group 0.34 ± 0.09, p < 0.05). At 28 days, renal lesions correlated strongly with the mRNA levels of Et3 (r > 0.50, p < 0.01). However, at 56 days, the key finding was the strong correlation of the most important analytical, histological, and immunohistochemical parameters of CsA nephrotoxicity with Et1 mRNA levels (r > 0.50, p < 0.01). These results support the hypothesis that the clinical and morphological phenomena linked with CsA nephrotoxicity are related to hypersecretion of endothelins and local expression of angiotensin II in the outer medulla and medullary rays; Et3 and angiotensin II are the first to act, followed subsequently by Et1.

Slow Resorption of Anorganic Bovine Bone by Osteoclasts in Maxillary Sinus Augmentation

PURPOSE Different biomaterials have been suggested for guided bone regeneration (GBR). These might show the ideal properties to let a new bone formation in the grafted area. Among these ideal features, it is essential their controlled resorption in order to be replaced for new vital bone. Bovine bone has been used widely as a good biomaterial for GBR, however there is still an interesting controversy about its resorbable capacity. In this sense, the objective of this study was to examine the behavior of anorganic bovine bone (ABB) in long-term maxillary sinus graft healing and study its relationship with morphological and morphometrical variables. MATERIALS AND METHODS Seventeen maxillary sinus augmentation procedures were performed in patients. Bone cores were obtained from implant receptor sites at 6 months, 3 years, and 7 years of implant placement for histological, morphometric, and immunohistochemical (tartrate resistant acid phosphatase [TRAP]/cathepsin K/CD68) studies. RESULTS The percentages of bone, ABB particles, connective tissue, osteocytes, and osteoblasts in maxillary sinus grafts were similar at 6 months, 3 years, and 7 years. A progressive and significant decrease was detected in osteoclasts (p = .05, Kruskal-Wallis test), TRAP and cathepsin K expression (p = .014 and p = .021, respectively), and osteoid lines (p = .038). CONCLUSION According to these data, a decrease in osteoclasts over time may, partially, explain the ABB persistence observed in core biopsies. Further studies with more cases and different graft maturation times are required to elucidate the resorption rates and cell events underlying these phenomena.

Role of P-glycoprotein in chronic cyclosporine nephrotoxicity and its relationship to intrarenal angiotensin II deposits.

CYCLOSPORINE (CsA), among other actions in the kidney, induces P-glycoprotein (P-gp) overexpression in tubular cells. P-gp expels hydrophobic xenobiotics from the cytoplasm, including chemotherapeutic and immunosuppressive agents such as CsA, tacrolimus, and rapamycin. One of the main secondary effects of CsA treatment is nephrotoxicity. Morphologic findings of CsA nephrotoxicity include vacuolar isometric degeneration of kidney tubules, hyaline arteriopathy, and stripped and diffuse interstitial fibrosis. Experimental models in rats have shown that low sodium (LS) diets decrease renal blood flow and intensify histopathologic lesions of CsA nephrotoxicity, especially hyaline arteriopathy and stripped tubulointerstitial fibrosis, parallel with the upregulation of the renin-angiotensin system (RAS). In this context, some studies have found that blockers of angiotensin II receptors, as well as angiotensin II–converting enzyme inhibitors: (a) prevent the development of interstitial fibrosis in experimental models of chronic CsA nephrotoxicity; and (b) reduce the accumulation of TGF-b1 and proteins of the extracellular matrix in CsA-induced chronic nephropathy. Our aim was to analyze the alterations induced by CsA in P-gp expression in kidney tubular cells of Sprague–Dawley rats and to characterize the relationship of these changes with the angiotensin II deposits and chronic CsA nephrotoxicity lesions.

Cyclooxygenase-2 Expression in Gingival Biopsies From Periodontal Patients Is Correlated With Connective Tissue Loss

BACKGROUND The objective of this study is to compare cyclooxygenase-2 (COX-2) protein expression in gingival biopsies from patients with chronic periodontitis (CP), patients with gingivitis (GV), and individuals with no periodontal disease (control group) and to establish its relationship with clinical variables and connective tissue loss in the lamina propria. METHODS A cross-sectional and analytic study was conducted in 108 gingival biopsies from 52 patients with CP, 39 with GV, and 17 controls. All biopsies were processed for conventional histopathologic study, immunohistochemical determination of COX-2 protein expression, and automatic quantification of connective tissue by image analysis. RESULTS The protein expression of COX-2, mainly produced by plasma cells and monocytes, was significantly related to the presence of periodontal disease, bleeding index, intensity of inflammatory infiltrate, and loss of connective tissue in the lamina propria of gingival biopsies (P <0.01, Spearman test). COX-2 expression was also directly correlated with attachment loss (P <0.05, Spearman test). CONCLUSIONS COX-2 protein expression is higher in patients with GV and CP than in individuals without periodontal disease and is inversely correlated with the amount of connective tissue in the lamina propria as determined by image analysis. This finding suggests that COX-2 participates in mechanisms and pathway signaling related to the destruction of fibrillar support structures of the periodontium.

Substituent and Solvent Effects on the UV–vis Absorption Spectrum of the Photoactive Yellow Protein Chromophore

Solvent effects on the UV-vis absorption spectra and molecular properties of four models of the photoactive yellow protein (PYP) chromophore have been studied with ASEP/MD, a sequential quantum mechanics/molecular mechanics method. The anionic trans-p-coumaric acid (pCA(-)), thioacid (pCTA(-)), methyl ester (pCMe(-)), and methyl thioester (pCTMe(-)) derivatives have been studied in gas phase and in water solution. We analyze the modifications introduced by the substitution of sulfur by oxygen atoms and hydrogen by methyl in the coumaryl tail. We have found some differences in the absorption spectra of oxy and thio derivatives that could shed light on the different photoisomerization paths followed by these compounds. In solution, the spectrum substantially changes with respect to that obtained in the gas phase. The n → π1* state is destabilized by a polar solvent like water, and it becomes the third excited state in solution displaying an important blue shift. Now, the π → π1* and π → π2* states mix, and we find contributions from both transitions in S1 and S2. The presence of the sulfur atom modulates the solvent effect and the first two excited states become practically degenerate for pCA(-) and pCMe(-) but moderately well-separated for pCTA(-) and pCTMe(-).

An Ex Vivo Model in Human Femoral Heads for Histopathological Study and Resonance Frequency Analysis of Dental Implant Primary Stability

Objective. This study was designed to explore relationships of resonance frequency analysis (RFA)—assessed implant stability (ISQ values) with bone morphometric parameters and bone quality in an ex vivo model of dental implants placed in human femoral heads and to evaluate the usefulness of this model for dental implant studies. Material and Methods. This ex vivo study included femoral heads from 17 patients undergoing surgery for femoral neck fracture due to osteoporosis (OP) (n = 7) or for total prosthesis joint replacement due to severe hip osteoarthrosis (OA) (n = 10). Sixty 4.5 × 13 mm Dentsply Astra implants were placed, followed by RFA. CD44 immunohistochemical analysis for osteocytes was also carried out. Results. As expected, the analysis yielded significant effects of femoral head type (OA versus OA) (P < 0.001), but not of the implants (P = 0.455) or of the interaction of the two factors (P = 0.848). Bonferroni post hoc comparisons showed a lower mean ISQ for implants in decalcified (50.33 ± 2.92) heads than in fresh (66.93 ± 1.10) or fixated (70.77 ± 1.32) heads (both P < 0.001). The ISQ score (fresh) was significantly higher for those in OA (73.52 ± 1.92) versus OP (67.13 ± 1.09) heads. However, mixed linear analysis showed no significant association between ISQ scores and morphologic or histomorphometric results (P > 0.5 in all cases), and no significant differences in ISQ values were found as a function of the length or area of the cortical layer (both P > 0.08). Conclusion. Although RFA-determined ISQ values are not correlated with morphometric parameters, they can discriminate bone quality (OP versus OA). This ex vivo model is useful for dental implant studies.