Considerations about the inhibition of monophenolase and diphenolase activities of tyrosinase. Characterization of the inhibitor concentration which generates 50 % of inhibition, type and inhibition constants. A review.

Tyrosinase is a copper oxidase enzyme which catalyzes the first two steps in the melanogenesis pathway, L-tyrosine to L-dopa conversion and, then, to o-dopaquinone and dopachrome. Hypopigmentation and, above all, hyperpigmentation issues can be originated depending on their activity. This enzyme also promotes the browning of fruits and vegetables. Therefore, control of their activity by regulators is research topic of great relevance. In this work, we consider the use of inhibitors of monophenolase and diphenolase activities of the enzyme in order to accomplish such control. An experimental design and data analysis which allow the accurate calculation of the degree of inhibition of monophenolase activity (iM) and diphenolase activity (iD) are proposed. The IC50 values (amount of inhibitor that causes 50 % inhibition at a fixed substrate concentration) can be calculated for the two activities and from the values of IC50M (monophenolase) and IC50D(diphenolase). Additionally, the strength and type of inhibition can be deduced from these values. The data analysis from these IC50D values allows to obtain the values of [Formula: see text] or [Formula: see text] , or and [Formula: see text] from the values of IC50M. In all cases, the values of the different must satisfy their relationship with IC50M and IC50D.

Brassica oleracea L. var. italica Aquaporin Reconstituted Proteoliposomes as Nanosystems for Resveratrol Encapsulation.

Aquaporins (AQPs), membrane proteins responsible for facilitating water transport, found in plant membrane vesicles (MV), have been related to the functionality and stability of MV. We focused on AQPs obtained from broccoli, as they show potential for biotechnological applications. To gain further insight into the role of AQPs in MV, we describe the heterologous overexpression of two broccoli AQPs (BoPIP1;2 and BoPIP2;2) in Pichia pastoris, resulting in their purification with high yield (0.14 and 0.99 mg per gram cells for BoPIP1;2 and BoPIP2;2). We reconstituted AQPs in liposomes to study their functionality, and the size of proteoliposomes did not change concerning liposomes. BoPIP2;2 facilitated water transport, which was preserved for seven days at 4 °C and at room temperature but not at 37 °C. BoPIP2;2 was incorporated into liposomes to encapsulate a resveratrol extract, resulting in increased entrapment efficiency (EE) compared to conventional liposomes. Molecular docking was utilized to identify binding sites in PIP2s for resveratrol, highlighting the role of aquaporins in the improved EE. Moreover, interactions between plant AQP and human integrin were shown, which may increase internalization by the human target cells. Our results suggest AQP-based alternative encapsulation systems can be used in specifically targeted biotechnological applications.

Considerations for intensity modulated total body or total marrow and lymphoid irradiation.

We compiled a sampling of the treatment techniques of intensity-modulated total body irradiation, total marrow irradiation and total marrow and lymphoid irradiation utilized by several centers across North America and Europe. This manuscript does not serve as a consensus guideline, but rather is meant to serve as a convenient reference for centers that are considering starting an intensity-modulated program.

Serum phosphate is associated with increased risk of bone fragility fractures in hemodialysis patients.

BACKGROUND: Bone fragility fractures are associated with high morbidity and mortality. This study analysed the association between the current biochemical parameters of CKD-MBD and bone fragility fractures in the COSMOS project. METHODS: COSMOS is a 3-year, multicentre, open cohort, prospective, observational study carried out in 6797 hemodialysis patients (227 centres from 20 European countries). The association of bone fragility fractures (outcome) with serum calcium, phosphate and PTH (exposure), was assessed using Standard Cox proportional hazards regression and Cox proportional hazards regression for recurrent events. Additional analyses were performed considering all-cause mortality as a competitive event for bone fragility fracture occurrence. Multivariable models were used in all strategies, with the fully adjusted model including a total of 24 variables. RESULTS: During a median follow-up of 24 months 252 (4%) patients experienced at least one bone fragility fracture (incident bone fragility fracture rate 28.5 per 1000 patient-years). In the fractured and non-fractured patients, the percentage of men was 43.7% and 61.4%, mean age 68.1 and 63.8 years and a haemodialysis vintage of 55.9 and 38.3 months respectively. Baseline serum phosphate > 6.1 mg/dL (reference value 4.3-6.1 mg/dL) was significantly associated with a higher bone fragility fracture risk in both regression models (HR: 1.53[95%CI: 1.10-2.13] and HR: 1.44[95%CI: 1.02-2.05]. The significant association persisted after competitive risk analysis (subHR: 1.42[95%CI: 1.02-1.98]) but the finding was not confirmed when serum phosphate was considered as a continuous variable. Baseline serum calcium showed no association with bone fragility fracture risk in any regression model. Baseline serum PTH > 800 pg/mL was significantly associated with a higher bone fragility fracture risk in both regression models, but the association disappeared after a competitive risk analysis. CONCLUSIONS: Hyperphosphatemia was independently and consistently associated with an increased bone fracture risk, suggesting serum phosphate could be a novel risk factor for bone fractures in hemodialysis patients.

Comprehensive Cardiovascular and Renal Protection in Patients with Type 2 Diabetes.

Type 2 diabetes (T2DM) is one of the main public health care problems worldwide. It is associated with a marked increased risk of developing atherosclerotic vascular disease, heart failure, chronic kidney disease and death. It is essential to act during the early phases of the disease, through the intensification of lifestyle changes and the prescription of those drugs that have been shown to reduce these complications, with the aim not only of achieving an adequate metabolic control, but also a comprehensive vascular risk control. In this consensus document, developed by the different specialists that treat these patients (endocrinologists, primary care physicians, internists, nephrologists and cardiologists), a more appropriate approach in the management of patients with T2DM or its complications is provided. A particular focus is given to the global control of cardiovascular risk factors, the inclusion of weight within the therapeutic objectives, the education of patients, the deprescription of those drugs without cardiovascular benefit, and the inclusion of GLP-1 receptor agonists and SGLT2 inhibitors as cardiovascular protective drugs, at the same level as statins, acetylsalicylic acid, or renin angiotensin system inhibitors.

Mineral and bone metabolism markers and mortality in diabetic patients on haemodialysis.

BACKGROUND: Diabetic patients on haemodialysis have a higher risk of mortality than non-diabetic patients. The aim of this COSMOS (Current management of secondary hyperparathyroidism: a multicentre observational study) analysis was to assess whether bone and mineral laboratory values [calcium, phosphorus and parathyroid hormone (PTH)] contribute to this risk. METHODS: COSMOS is a multicentre, open-cohort, 3-year prospective study, which includes 6797 patients from 227 randomly selected dialysis centres in 20 European countries. The association between mortality and calcium, phosphate or PTH was assessed using Cox proportional hazard regression models using both penalized splines smoothing and categorization according to KDIGO guidelines. The effect modification of the association between the relative risk of mortality and serum calcium, phosphate or PTH by diabetes was assessed. RESULTS: There was a statistically significant effect modification of the association between the relative risk of mortality and serum PTH by diabetes (P = .011). The slope of the curve of the association between increasing values of PTH and relative risk of mortality was steeper for diabetic compared with non-diabetic patients, mainly for high levels of PTH. In addition, high serum PTH (>9 times the normal values) was significantly associated with a higher relative risk of mortality in diabetic patients but not in non-diabetic patients [1.53 (95% confidence interval 1.07-2.19) and 1.17 (95% confidence interval 0.91-1.52)]. No significant effect modification of the association between the relative risk of mortality and serum calcium or phosphate by diabetes was found (P = .2 and P = .059, respectively). CONCLUSION: The results show a different association of PTH with the relative risk of mortality in diabetic and non-diabetic patients. These findings could have relevant implications for the diagnosis and treatment of chronic kidney disease-mineral and bone disorders.

Risk of Radiation Dermatitis in Patients With Skin of Color Who Undergo Radiation to the Breast or Chest Wall With and Without Regional Nodal Irradiation.

PURPOSE: Acute radiation dermatitis (ARD) is common after radiation therapy for breast cancer, with data indicating that ARD may disproportionately affect Black or African American (AA) patients. We evaluated the effect of skin of color (SOC) on physician-reported ARD in patients treated with radiation therapy. METHODS AND MATERIALS: We identified patients treated with whole breast or chest wall ± regional nodal irradiation or high tangents using 50 Gy in 25 fractions from 2015 to 2018. Baseline skin pigmentation was assessed using the Fitzpatrick scale (I = light/pale white to VI = black/very dark brown) with SOC defined as Fitzpatrick scale IV to VI. We evaluated associations among SOC, physician-reported ARD, late hyperpigmentation, and use of oral and topical treatments for RD using multivariable models. RESULTS: A total of 325 patients met eligibility, of which 40% had SOC (n = 129). On multivariable analysis, Black/AA race and chest wall irradiation had a lower odds of physician-reported grade 2 or 3 ARD (odds ratio [OR], 0.110; 95% confidence interval [CI], 0.030-0.397; P = .001; OR, 0.377; 95% CI, 0.161-0.883; P = .025), whereas skin bolus (OR, 8.029; 95% CI, 3.655-17.635; P = 0) and planning target volume D0.03cc (OR, 1.001; 95% CI, 1.000-1.001; P = .028) were associated with increased odds. On multivariable analysis, SOC (OR, 3.658; 95% CI, 1.236-10.830; P = .019) and skin bolus (OR, 26.786; 95% CI, 4.235-169.432; P = 0) were associated with increased odds of physician-reported late grade 2 or 3 hyperpigmentation. There was less frequent use of topical steroids to treat ARD and more frequent use of oral analgesics in SOC versus non-SOC patients (43% vs 63%, P < .001; 50% vs 38%, P = .05, respectively). CONCLUSIONS: Black/AA patients exhibited lower odds of physician-reported ARD. However, we found higher odds of late hyperpigmentation in SOC patients, independent of self-reported race. These findings suggest that ARD may be underdiagnosed in SOC when using the physician-rated scale despite this late evidence of radiation-induced skin toxicity.

On the Mechanism of Membrane Permeabilization by Tamoxifen and 4-Hydroxytamoxifen.

Tamoxifen (TMX), commonly used in complementary therapy for breast cancer, also displays known effects on the structure and function of biological membranes. This work presents an experimental and simulation study on the permeabilization of model phospholipid membranes by TMX and its derivative 4-hydroxytamoxifen (HTMX). TMX induces rapid and extensive vesicle contents leakage in phosphatidylcholine (PC) liposomes, with the effect of HTMX being much weaker. Fitting of the leakage curves for TMX, yields two rate constants, corresponding to a fast and a slow process, whereas in the case of HTMX, only the slow process takes place. Interestingly, incorporation of phosphatidylglycerol (PG) or phosphatidylethanolamine (PE) protects PC membranes from TMXinduced permeabilization. Fourier-transform infrared spectroscopy (FTIR) shows that, in the presence of TMX there is a shift in the νCH2 band frequency, corresponding to an increase in gauche conformers, and a shift in the νC=O band frequency, indicating a dehydration of the polar region. A preferential association of TMX with PC, in mixed PC/PE systems, is observed by differential scanning calorimetry. Molecular dynamics (MD) simulations support the experimental results, and provide feasible explanations to the protecting effect of PG and PE. These findings add new information to explain the various mechanisms of the anticancer actions of TMX, not related to the estrogen receptor, and potential side effects of this drug.

Effects of a Semisynthetic Catechin on Phosphatidylglycerol Membranes: A Mixed Experimental and Simulation Study.

Catechins have been shown to display a great variety of biological activities, prominent among them are their chemo preventive and chemotherapeutic properties against several types of cancer. The amphiphilic nature of catechins points to the membrane as a potential target for their actions. 3,4,5-Trimethoxybenzoate of catechin (TMBC) is a modified structural analog of catechin that shows significant antiproliferative activity against melanoma and breast cancer cells. Phosphatidylglycerol is an anionic membrane phospholipid with important physical and biochemical characteristics that make it biologically relevant. In addition, phosphatidylglycerol is a preeminent component of bacterial membranes. Using biomimetic membranes, we examined the effects of TMBC on the structural and dynamic properties of phosphatidylglycerol bilayers by means of biophysical techniques such as differential scanning calorimetry, X-ray diffraction and infrared spectroscopy, together with an analysis through molecular dynamics simulation. We found that TMBC perturbs the thermotropic gel to liquid-crystalline phase transition and promotes immiscibility in both phospholipid phases. The modified catechin decreases the thickness of the bilayer and is able to form hydrogen bonds with the carbonyl groups of the phospholipid. Experimental data support the simulated data that locate TMBC as mostly forming clusters in the middle region of each monolayer approaching the carbonyl moiety of the phospholipid. The presence of TMBC modifies the structural and dynamic properties of the phosphatidylglycerol bilayer. The decrease in membrane thickness and the change of the hydrogen bonding pattern in the interfacial region of the bilayer elicited by the catechin might contribute to the alteration of the events taking place in the membrane and might help to understand the mechanism of action of the diverse effects displayed by catechins.

Definitive Radiation With Nodal Boost for Patients With Locally Advanced Breast Cancer.

PURPOSE: The optimal local therapy of patients with nodal disease in supraclavicular (SCV), internal mammary nodes (IMN) and level III axilla is not well studied. We aimed to evaluate the outcomes of patients with breast cancer and advanced nodal disease that received a nodal boost. METHODS AND MATERIALS: This retrospective study included 79 patients with advanced nodal disease who underwent adjuvant radiation with a nodal boost to the SCV, IMNs, and/or axilla. All patients had radiographic changes after systemic therapy concerning for gross nodal disease. Overall survival, disease-free survival (DFS), and local recurrence-free survival were estimated using the Kaplan-Meier method. RESULTS: All patients received an initial 50 Gy to the breast/chest wall and regional nodes, of whom 46.8% received an IMN boost, 38.0% axillary (ax)/SCV boost, and 15.2% both IMN and ax/SCV boost (IMN + ax/SCV). Most patients had hormone receptor positive (74.7%) and human epidermal growth factor receptor 2 negative disease (83.5%). In addition, 12.7% of patients had clinical (c) N2 disease, 21.5% cN3A disease, 51.9% cN3B disease, and 5.1% cN3C disease. Most patients received chemotherapy (97.5%). The median nodal boost dose was 10 Gy (range, 10-20 Gy), with 21.6% of IMN, 16.7% of ax/SCV, and 16.7% of IMN + ax/SCV receiving 14 to 20 Gy. With a median follow up of 30 months, the 3-year local recurrence-free survival, DFS, and overall survival rates were 94.5%, 86.3%, and 93.8%, respectively. Crude rates of failure were 13.9% (10.1% distant failure [DF] alone; 3.8% DF + locoregional failure [LRF]). Rates of failure by boost group were 13.3% for ax/SCV (10.0% DF alone; 3.3% DF + LRF), 5.4% for IMN (2.7% DF alone, 2.7% DF + LRF), and 41.7% for IMN + ax/SCV (33.3% DF, 8.3% DF + LRF). There were no LRFs without DFs. The median time to failure was 22.8 months (interquartile range, 18-34 months). Clinical tumor size and IMN + ax/SCV versus IMN or ax/SCV alone was associated with worse DFS (hazard ratio [HR]: 9.78; 95% confidence interval [CI], 2.07-46.2; P = .004 and HR: 9.49; 95% CI, 2.67-33.7; P = .001, respectively). On multivariate analysis, IMN + ax/SCV versus IMN or ax/SCV alone retained significance (HR: 4.80; 95% CI, 1.27-18.13; P = .02). CONCLUSIONS: In this population of patients with locally advanced breast cancer, the majority of failures were distant with no isolated LRFs. Failures were the highest in the IMN + ax/SCV group (∼40%). Further treatment escalation is necessary for these patients.

Anticancer drugs tamoxifen and 4hydroxytamoxifen as effectors of phosphatidylethanolamine lipid polymorphism.

The interaction of tamoxifen (TMX) and its metabolite 4-hydroxytamoxifen (HTMX) with a biomimetic membrane model system composed of 1,2-dielaidoylphosphatidylethanolamine (DEPE) has been studied using a biophysical approach. Incorporation of TMX into DEPE bilayers gives rise to a progressive broadening of the Lß/Lα phase transition and a downward temperature shift. The Lß/Lα phase transition presents multiple endotherms, indicating a lateral segregation of TMX/DEPE domains within the plane of the bilayer. TMX and HTMX also widen and shift the Lα to hexagonal-HII transition toward lower values, the phase diagrams showing that both compounds facilitate formation of the HII phase. TMX increases motional disorder of DEPE acyl chains in the Lß, Lα and HII phases, whereas the effect of HTMX is clearly different. In addition, neither TMX nor HTMX significantly perturb the hydration state of the polar headgroup region of DEPE. Molecular dynamics (MD) simulations indicate that these drugs do not affect membrane thickness, area per lipid, or the conformation of DEPE molecules. As a general rule, the interaction of HTMX with DEPE is qualitatively similar to TMX but less intense. However, a significant difference shown by MD is that HTMX is mainly placed around the center of each monolayer while TMX is located mainly at the center of the membrane, also having a greater tendency to cluster formation. These results are discussed to understand the modulation of phosphatidylethanolamine lipid polymorphism carried out by these drugs, which could be of relevance to explain their effects on enzyme activity or membrane permeabilization.

3,4,5-Trimethoxybenzoate of Catechin, an Anticarcinogenic Semisynthetic Catechin, Modulates the Physical Properties of Anionic Phospholipid Membranes.

3,4,5-Trimethoxybenzoate of catechin (TMBC) is a semisynthetic catechin which shows strong antiproliferative activity against malignant melanoma cells. The amphiphilic nature of the molecule suggests that the membrane could be a potential site of action, hence the study of its interaction with lipid bilayers is mandatory in order to gain information on the effect of the catechin on the membrane properties and dynamics. Anionic phospholipids, though being minor components of the membrane, possess singular physical and biochemical properties that make them physiologically essential. Utilizing phosphatidylserine biomimetic membranes, we study the interaction between the catechin and anionic bilayers, bringing together a variety of experimental techniques and molecular dynamics simulation. The experimental data suggest that the molecule is embedded into the phosphatidylserine bilayers, where it perturbs the thermotropic gel to liquid crystalline phase transition. In the gel phase, the catechin promotes the formation of interdigitation, and in the liquid crystalline phase, it decreases the bilayer thickness and increases the hydrogen bonding pattern of the interfacial region of the bilayer. The simulation data agree with the experimental ones and indicate that the molecule is located in the interior of the anionic bilayer as monomer and small clusters reaching the carbonyl region of the phospholipid, where it also disturbs the intermolecular hydrogen bonding between neighboring lipids. Our observations suggest that the catechin incorporates well into phosphatidylserine bilayers, where it produces structural changes that could affect the functioning of the membrane.

Pulmonary Toxic Effects After Myeloablative Conditioning With Total Body Irradiation Delivered via Volumetric Modulated Arc Therapy With Fludarabine.

We present the case of a 56-year-old female with a diagnosis of acute T-cell lymphoblastic leukemia who received myeloablative conditioning for bone marrow transplant with total body irradiation (TBI) using volumetric modulated arc therapy (VMAT) to the upper body and anterior-posterior/posterior-anterior (AP/PA) open fields to the lower body followed by hematopoietic stem cell transplant. Her clinical course was complicated by high-grade pulmonary toxic effects 55 days after treatment that resulted in death. We discuss the case, planning considerations by radiation oncologists and radiation physicists, and the multidisciplinary medical management of this patient.

Interaction of Docetaxel with Phosphatidylcholine Membranes: A Combined Experimental and Computational Study.

The antineoplastic drug Docetaxel is a second generation taxane which is used against a great variety of cancers. The drug is highly lipophilic and produces a great array of severe toxic effects that limit its therapeutic effectiveness. The study of the interaction between Docetaxel and membranes is very scarce, however, it is required in order to get clues in relation with its function, mechanism of toxicity and possibilities of new formulations. Using phosphatidylcholine biomimetic membranes, we examine the interaction of Docetaxel with the phospholipid bilayer combining an experimental study, employing a series of biophysical techniques like Differential Scanning Calorimetry, X-Ray Diffraction and Infrared Spectroscopy, and a Molecular Dynamics simulation. Our experimental results indicated that Docetaxel incorporated into DPPC bilayer perturbing the gel to liquid crystalline phase transition and giving rise to immiscibility when the amount of the drug is increased. The drug promotes the gel ripple phase, increasing the bilayer thickness in the fluid phase, and is also able to alter the hydrogen-bonding interactions in the interfacial region of the bilayer producing a dehydration effect. The results from computational simulation agree with the experimental ones and located the Docetaxel molecule forming small clusters in the region of the carbon 8 of the acyl chain palisade overlapping with the carbonyl region of the phospholipid. Our results support the idea that the anticancer drug is embedded into the phospholipid bilayer to a limited amount and produces structural perturbations which might affect the function of the membrane.

Membrane Vesicles for Nanoencapsulated Sulforaphane Increased Their Anti-Inflammatory Role on an In Vitro Human Macrophage Model.

At present, there is a growing interest in finding new non-toxic anti-inflammatory drugs to treat inflammation, which is a key pathology in the development of several diseases with considerable mortality. Sulforaphane (SFN), a bioactive compound derived from Brassica plants, was shown to be promising due to its anti-inflammatory properties and great potential, though its actual clinical use is limited due to its poor stability and bioavailability. In this sense, the use of nanocarriers could solve stability-related problems. In the current study, sulforaphane loaded into membrane vesicles derived from broccoli plants was studied to determine the anti-inflammatory potential in a human-macrophage-like in vitro cell model under both normal and inflammatory conditions. On the one hand, the release of SFN from membrane vesicles was modeled in vitro, and two release phases were stabilized, one faster and the other slower due to the interaction between SFN and membrane proteins, such as aquaporins. Furthermore, the anti-inflammatory action of sulforaphane-loaded membrane vesicles was demonstrated, as a decrease in interleukins crucial for the development of inflammation, such as TNF-α, IL-1ß and IL-6, was observed. Furthermore, these results also showed that membrane vesicles by themselves had anti-inflammatory properties, opening the possibility of new lines of research to study these vesicles, not only as carriers but also as active compounds.

Dissimilar action of tamoxifen and 4-hydroxytamoxifen on phosphatidylcholine model membranes.

The anticancer drug tamoxifen and its primary metabolite 4-hydroxytamoxifen tend to accumulate in membranes due to its strong hydrophobic character. Thus, in this work we have carried out a systematic study to investigate their effects on model phosphatidylcholine membranes. Tamoxifen and 4-hydroxytamoxifen affect the phase behaviour of phosphatidylcholine model membranes, giving rise to formation of drug/dipalmitoylphosphatidylcholine domains, which is more evident in the case of 4-hydroxytamoxifen. These drugs have differential effects on the polar and apolar regions of the phospholipid supporting a different location of both compounds within the bilayer. Both compounds induce contents leakage in fluid phosphatidylcholine unilamellar liposomes, the effect of 4-hydroxytamoxifen being negligible as compared to that of tamoxifen. Molecular dynamics confirmed the tendency of both drugs to form clusters, tamoxifen locating all along the bilayer, whereas 4-hydroxytamoxifen mostly locates near the lipid/water interface, which can explain the different effects of both drugs in fluid phosphatidylcholine membranes.

Clotrimazole Fluidizes Phospholipid Membranes and Localizes at the Hydrophobic Part near the Polar Part of the Membrane.

Clotrimazole (1-[(2-chlorophenyl)-diphenylmethyl]-imidazole) is an azole antifungal drug belonging to the imidazole subclass that is widely used in pharmacology and that can be incorporated in membranes. We studied its interaction with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) phospholipid vesicles by using differential scanning calorimetry and found that the transition temperature decreases progressively as the concentration of clotrimazole increases. However, the temperature of completion of the transition remained constant despite the increase of clotrimazole concentration, suggesting the formation of fluid immiscibility. 1H-NMR and 1H NOESY MAS-NMR were employed to investigate the location of clotrimazole in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) phospholipid membranes. In the presence of clotrimazole, all the resonances originating from POPC were shifted upfield, but mainly those corresponding to C2 and C3 of the fatty acyl, chains suggesting that clotrimazole aromatic rings preferentially locate near these carbons. In the same way, 2D-NOESY measurements showed that the highest cross-relaxation rates between protons of clotrimazole and POPC were with those bound to the C2 and C3 carbons of the fatty acyl chains. Molecular dynamics simulations indicated that clotrimazole is located near the top of the hydrocarbon-chain phase, with the nitrogen atoms of the imidazole ring of clotrimazole being closest to the polar group of the carbonyl moiety. These results are in close agreement with the NMR and the conclusion is that clotrimazole is located near the water-lipid interface and in the upper part of the hydrophobic bilayer.