The Enigma of NTH2 Gene in Yeasts.

The enzymatic hydrolysis of the non-reducing disaccharide trehalose in yeasts is carried out by trehalase, a highly specific α-glucosidase. Two types of such trehalase activity are present in yeasts, and are referred to as neutral and acid enzymes. They are encoded by distinct genes (NTH1 and ATH1, respectively) and exhibit strong differences in their biochemical and physiological properties as well as different subcellular location and regulatory mechanisms. Whereas a single gene ATH1 codes for acid trehalase, the genome of some yeasts appears to predict the existence of a second redundant neutral trehalase, encoded by the NTH2 gene, a paralog of NTH1. In S. cerevisiae the corresponding two proteins share 77% amino acid identity, leading to the suggestion that NTH2 codes for a functional trehalase activity. However, Nth2p lacks any measurable neutral trehalase activity and disruption of NTH2 gene has no effect on this activity compared to a parental strain. Likewise, single nth1Δ and double nth1Δ/nth2Δ null mutants display no detectable neutral activity. Furthermore, disruption of NTH2 does not cause any apparent phenotype apart from a slight involvement in thermotolerance. To date, no evidence of a duplicated NTH gene has been recorded in other archetypical yeasts, like C. albicans or C. parapsilosis, and a possible regulatory mechanism of Nth2p remains unknown. Therefore, although genomic analysis points to the existence, in some yeasts, of two distinct genes encoding trehalase activities, the large body of biochemical and physiological evidence gathered from NTH2 gene does not support this proposal. Indeed, much more experimental evidence would be necessary to firmly validate this hypothesis.

Natural Substances as Valuable Alternative for Improving Conventional Antifungal Chemotherapy: Lights and Shadows.

Fungi are eukaryotic organisms with relatively few pathogenic members dangerous for humans, usually acting as opportunistic infections. In the last decades, several life-threatening fungal infections have risen mostly associated with the worldwide extension of chronic diseases and immunosuppression. The available antifungal therapies cannot combat this challenge because the arsenal of compounds is scarce and displays low selective action, significant adverse effects, and increasing resistance. A growing isolation of outbreaks triggered by fungal species formerly considered innocuous is being recorded. From ancient times, natural substances harvested from plants have been applied to folk medicine and some of them recently emerged as promising antifungals. The most used are briefly revised herein. Combinations of chemotherapeutic drugs with natural products to obtain more efficient and gentle treatments are also revised. Nevertheless, considerable research work is still necessary before their clinical use can be generally accepted. Many natural products have a highly complex chemical composition, with the active principles still partially unknown. Here, we survey the field underlying lights and shadows of both groups. More studies involving clinical strains are necessary, but we illustrate this matter by discussing the potential clinical applications of combined carnosic acid plus propolis formulations.

Hyperglycemia and Oxidative Stress: An Integral, Updated and Critical Overview of Their Metabolic Interconnections.

This review focuses on the multiple and reciprocal relationships that exist between oxidative stress, hyperglycemia and diabetes and related metabolic disorders. Human metabolism uses most of the consumed glucose under aerobic conditions. Oxygen is needed in the mitochondria to obtain energy, as well as for the action of microsomal oxidases and cytosolic pro-oxidant enzymes. This relentlessly generates a certain amount of reactive oxygen species (ROS). Although ROS are intracellular signals necessary for some physiological processes, their accumulation leads to oxidative stress, hyperglycemia, and progressive resistance to insulin. A cellular pro-oxidant versus an antioxidant equilibrium would regulate ROS levels, but oxidative stress, hyperglycemia, and pro-inflammatory conditions feed back to each other and the relevance of the interconnections tends to increase those conditions. Hyperglycemia promotes collateral glucose metabolism through protein kinase C, polyols and hexosamine routes. In addition, it also facilitates spontaneous glucose auto-oxidation and the formation of advanced glycation end products (AGEs), which in turn interact with their receptors (RAGE). The mentioned processes undermine cellular structures, finally giving place to a progressively greater degree of oxidative stress with further hyperglycemia, metabolic alterations, and diabetes complications. NFκB is the major transcription factor involved in the expression of most of the pro-oxidant mediators, while Nrf2 is the major transcription factor regulating the antioxidant response. FoxO is also involved in the equilibrium, but its role is controversial. This review summarizes the key factors linking the diverse glucose metabolic routes enhanced in hyperglycemia with ROS formation and vice versa, emphasizing the role of the major transcription factors involved in the desirable balance between pro-oxidant and antioxidant proteins.

Whole Sequencing and Detailed Analysis of SARS-CoV-2 Genomes in Southeast Spain: Identification of Recurrent Mutations in the 20E (EU1) Variant with Some Clinical Implications.

During the COVID-19 pandemic caused by SARS-CoV-2, new waves have been associated with new variants and have the potential to escape vaccinations. Therefore, it is useful to conduct retrospective genomic surveillance research. Herein, we present a detailed analysis of 88 SARS-CoV-2 genomes belonging to samples taken from COVID-19 patients from October 2020 to April 2021 at the "Reina Sofía" Hospital (Murcia, Spain) focused to variant appeared later. The results at the mentioned stage show the turning point since the 20E (EU1) variant was still prevalent (71.6%), but Alpha was bursting to 14.8%. Concern mutations have been found in 5 genomes classified as 20E (EU1), which were not characteristic of this still little evolved variant. Most of those mutations are found in the spike protein, namely Δ69-70, E484K, Q675H and P681H. However, a relevant deletion in ORF1a at positions 3675-3677 was also identified. These mutations have been reported in many later SARS-CoV-2 lineages, including Omicron. Taken together, our data suggest that preferential emergence mutations could already be present in the early converging evolution. Aside from this, the molecular information has been contrasted with clinical data. Statistical analyses suggest that the correlation between age and severity criteria is significantly higher in the viral samples with more accumulated changes.

Insight into the Antifungal Effects of Propolis and Carnosic Acid-Extension to the Pathogenic Yeast Candida glabrata: New Propolis Fractionation and Potential Synergistic Applications.

Fungi have traditionally been considered opportunistic pathogens in primary infections caused by virulent bacteria, protozoan, or viruses. Consequently, antimycotic chemotherapy is clearly less developed in comparison to its bacterial counterpart. Currently, the three main families of antifungals (polyenes, echinocandins, and azoles) are not sufficient to control the enormous increase in life-threatening fungal infections recorded in recent decades. Natural substances harvested from plants have traditionally been utilized as a successful alternative. After a wide screening of natural agents, we have recently obtained promising results with distinct formulations of carnosic acid and propolis on the prevalent fungal pathogens Candida albicans and Cryptococcus neoformans. Here, we extended their use to the treatment against the emerging pathogenic yeast Candida glabrata, which displayed lower susceptibility in comparison to the fungi mentioned above. Taking into account the moderate antifungal activity of both natural agents, the antifungal value of these combinations has been improved through the obtention of the hydroethanolic fractions of propolis. In addition, we have demonstrated the potential clinical application of new therapeutical designs based on sequential pre-treatments with carnosic/propolis mixtures, followed by exposure to amphotericin B. This approach increased the toxic effect induced by this polyene.

The antifungal effect induced by itraconazole in Candida parapsilosis largely depends on the oxidative stress generated at the mitochondria.

In Candida parapsilosis, homozygous disruption of the two genes encoding trehalase activity increased the susceptibility to Itraconazole compared with the isogenic parental strain. The fungicidal effect of this azole can largely be counteracted by preincubating growing cells with rotenone and the protonophore 2,4-Dinitrophenol. In turn, measurement of endogenous reactive oxygen species formation by flow cytometry confirmed that Itraconazole clearly induced an internal oxidative stress, which can be significantly abolished in rotenone-exposed cells. Analysis of the antioxidant enzymatic activities of catalase and superoxide dismutase pointed to a moderate decrease of catalase in trehalase-deficient mutant cells compared to the wild type, with an additional increase upon addition of rotenone. These enzymatic changes were imperceptible in the case of superoxide dismutase. Alternative assays with Voriconazole led to a similar profile in the results regarding cell growth and antioxidant activities. Collectively, our data suggest that the antifungal action of Itraconazole on C. parapsilosis is dependent on a functional mitochondrial activity. They also suggest that the central metabolic pathways in pathogenic fungi should be considered as preferential antifungal targets in new research.

Lack of Functional Trehalase Activity in Candida parapsilosis Increases Susceptibility to Itraconazole.

Central metabolic pathways may play a major role in the virulence of pathogenic fungi. Here, we have investigated the susceptibility of a Candida parapsilosis mutant deficient in trehalase activity (atc1Δ/ntc1Δ strain) to the azolic compounds fluconazole and itraconazole. A time-course exposure to itraconazole but not fluconazole induced a significant degree of cell killing in mutant cells compared to the parental strain. Flow cytometry determinations indicated that itraconazole was able to induce a marked production of endogenous ROS together with a simultaneous increase in membrane potential, these effects being irrelevant after fluconazole addition. Furthermore, only itraconazole induced a significant synthesis of endogenous trehalose. The recorded impaired capacity of mutant cells to produce structured biofilms was further increased in the presence of both azoles, with itraconazole being more effective than fluconazole. Our results in the opportunistic pathogen yeast C. parapsilosis reinforce the study of trehalose metabolism as an attractive therapeutic target and allow extending the hypothesis that the generation of internal oxidative stress may be a component of the antifungal action exerted by the compounds currently available in medical practice.

Unraveling the Metabolic Hallmarks for the Optimization of Protein Intake in Pre-Dialysis Chronic Kidney Disease Patients.

The daily amount and quality of protein that should be administered by enteral nutrition in pre-dialysis chronic kidney disease (CKD) patients is a widely studied but still controversial issue. This is due to a compromise between the protein necessary to maintain muscular proteostasis avoiding sarcopenia, and the minimal amount required to prevent uremia and the accumulation of nitrogenous toxic substances in blood because of the renal function limitations. This review underlines some intracellular and extracellular features that should be considered to reconcile those two opposite factors. On one hand, the physiological conditions and usual side effects associated with CKD, mTOR and other proteins and nutrients involved in the regulation of protein synthesis in the muscular tissue are discussed. On the other hand, the main digestive features of the most common proteins used for enteral nutrition formulation (i.e., whey, casein and soy protein) are highlighted, due to the importance of supplying key amino acids to serum and tissues to maintain their concentration above the anabolic threshold needed for active protein synthesis, thereby minimizing the catabolic pathways leading to urea formation.

Renal Tissue Expression of BAFF and BAFF Receptors Is Associated with Proliferative Lupus Nephritis.

Background: The B-cell activating factor (BAFF) controls the maturation and survival of B cells. An imbalance in this cytokine has been associated with systemic autoimmunity in SLE and lupus nephritis (LN). However, few investigations have evaluated the tissular expression of BAFF in LN. This study aimed to associate BAFF system expression at the tissular level with the proliferative LN classes. Methods: The analysis included eighteen kidney tissues, with sixteen LN (class III = 5, class IV = 6, class III/IV+V = 4, and class V = 1), and two controls. The tissular expression was evaluated with an immunochemistry assay. A Cytation5 imaging reader and ImageJ software were used to analyze the quantitative expression. A p-value < 0.05 was considered significant. Results: The expressions of BAFF, A proliferation-inducing ligand (APRIL), and their receptors were observed in glomerular, tubular, and interstitial zones, with BAFF being the most strongly expressed in the overall analysis. BAFF-Receptor (BR3), transmembrane activator and CALM interactor (TACI), and B-Cell maturation antigen (BCMA) displayed higher expressions in LN class IV in all zones analyzed (p < 0.05). Additionally, a positive correlation was found between APRIL, TACI, and BCMA at the glomerular level; BCMA and APRIL in the interstitial zone; and BR3, TACI, and BCMA in the tubule (p < 0.05). Conclusions: The expression of BAFF and BAFF receptors is mainly associated with LN class IV, emphasizing the participation of these receptors as an essential pathogenic factor in kidney involvement in SLE patients.

A Specific Mixture of Propolis and Carnosic Acid Triggers a Strong Fungicidal Action against Cryptococcus neoformans.

Current antifungal chemotherapy against the prevalent basidiomycete Cryptococcus neoformans displays some drawbacks. This pathogenic fungus is refractory to echinocandins, whereas conventional treatment with amphotericin B plus 5-fluorocytosine has a limited efficacy. In this study, we explored the potential cryptococcal activity of some natural agents. After conducting a screening test with a set of propolis from different geographical areas, we selected an extract from China, which displayed a certain cytotoxic activity against C. neoformans, due to this extract being cheap and easily available in large amounts. The combination of this kind of propolis with carnosic acid in a 1:4 ratio induced a stronger fungicidal effect, which occurred following a synergistic pattern, without visible alterations in external cell morphology. Furthermore, several carnosic acid-propolis formulations applied onto preformed biofilms decreased the metabolic activity of the sessile cells forming biofilms. These data support the potential application of mixtures containing these two natural extracts in the design of new antifungal strategies in order to combat opportunistic infections caused by prevalent pathogenic fungi.

Novel Bi-Factorial Strategy against Candida albicans Viability Using Carnosic Acid and Propolis: Synergistic Antifungal Action.

The potential fungicidal action of the natural extracts, carnosic acid (obtained from rosemary) and propolis (from honeybees' panels) against the highly prevalent yeast Candida albicans, used herein as an archetype of pathogenic fungi, was tested. The separate addition of carnosic acid and propolis on exponential cultures of the standard SC5314 C. albicans strain caused a moderate degree of cell death at relatively high concentrations. However, the combination of both extracts, especially in a 1:4 ratio, induced a potent synergistic pattern, leading to a drastic reduction in cell survival even at much lower concentrations. The result of a mathematical analysis by isobologram was consistent with synergistic action of the combined extracts rather than a merely additive effect. In turn, the capacity of SC5314 cells to form in vitro biofilms was also impaired by the simultaneous presence of both agents, supporting the potential application of carnosic acid and propolis mixtures in the prevention and treatment of clinical infections as an alternative to antibiotics and other antifungal agents endowed with reduced toxic side effects.

Photoprotection and Skin Pigmentation: Melanin-Related Molecules and Some Other New Agents Obtained from Natural Sources.

Direct sun exposure is one of the most aggressive factors for human skin. Sun radiation contains a range of the electromagnetic spectrum including UV light. In addition to the stratospheric ozone layer filtering the most harmful UVC, human skin contains a photoprotective pigment called melanin to protect from UVB, UVA, and blue visible light. This pigment is a redox UV-absorbing agent and functions as a shield to prevent direct UV action on the DNA of epidermal cells. In addition, melanin indirectly scavenges reactive oxygenated species (ROS) formed during the UV-inducing oxidative stress on the skin. The amounts of melanin in the skin depend on the phototype. In most phenotypes, endogenous melanin is not enough for full protection, especially in the summertime. Thus, photoprotective molecules should be added to commercial sunscreens. These molecules should show UV-absorbing capacity to complement the intrinsic photoprotection of the cutaneous natural pigment. This review deals with (a) the use of exogenous melanin or melanin-related compounds to mimic endogenous melanin and (b) the use of a number of natural compounds from plants and marine organisms that can act as UV filters and ROS scavengers. These agents have antioxidant properties, but this feature usually is associated to skin-lightening action. In contrast, good photoprotectors would be able to enhance natural cutaneous pigmentation. This review examines flavonoids, one of the main groups of these agents, as well as new promising compounds with other chemical structures recently obtained from marine organisms.

The ant Lasius niger is a new source of bacterial enzymes with biotechnological potential for bleaching dye.

Industrial synthetic dyes cause health and environmental problems. This work describes the isolation of 84 bacterial strains from the midgut of the Lasius niger ant and the evaluation of their potential application in dye bioremediation. Strains were identified and classified as judged by rRNA 16S. The most abundant isolates were found to belong to Actinobacteria (49%) and Firmicutes (47.2%). We analyzed the content in laccase, azoreductase and peroxidase activities and their ability to degrade three known dyes (azo, thiazine and anthraquinone) with different chemical structures. Strain Ln26 (identified as Brevibacterium permense) strongly decolorized the three dyes tested at different conditions. Strain Ln78 (Streptomyces ambofaciens) exhibited a high level of activity in the presence of Toluidine Blue (TB). It was determined that 8.5 was the optimal pH for these two strains, the optimal temperature conditions ranged between 22 and 37 °C, and acidic pHs and temperatures around 50 °C caused enzyme inactivation. Finally, the genome of the most promising candidate (Ln26, approximately 4.2 Mb in size) was sequenced. Genes coding for two DyP-type peroxidases, one laccase and one azoreductase were identified and account for the ability of this strain to effectively oxidize a variety of dyes with different chemical structures.

Unprecedented high catecholamine production causing hair pigmentation after urinary excretion in red deer.

Hormones have not been found in concentrations of orders of magnitude higher than nanograms per milliliter. Here, we report urine concentrations of a catecholamine (norepinephrine) ranging from 0.05 to 0.5 g/l, and concentrations of its metabolite DL-3,4-dihydroxyphenyl glycol (DOPEG) ranging from 1.0 to 44.5 g/l, in wild male red deer Cervus elaphus hispanicus after LC-MS analyses. The dark ventral patch of male red deer, a recently described sexually selected signal, contains high amounts of DOPEG (0.9-266.9 mg/l) stuck in the hairs, while DOPEG is not present in non-darkened hair. The formation of this dark patch is explained by the chemical structure of DOPEG, which is a catecholamine-derived o-diphenol susceptible to be oxidized by air and form allomelanins, nitrogen-free pigments similar to cutaneous melanins; by its high concentration in urine; and by the urine spraying behavior of red deer by which urine is spread through the ventral body area. Accordingly, the size of the dark ventral patch was positively correlated with the concentration of DOPEG in urine, which was in turn correlated with DOPEG absorbed in ventral hair. These findings represent catecholamine concentrations about one million higher than those previously reported for any hormone in an organism. This may have favored the evolution of the dark ventral patch of red deer by transferring information on the fighting capacity to rivals and mates. Physiological limits for hormone production in animals are thus considerably higher than previously thought. These results also unveil a novel mechanism of pigmentation based on the self-application of urine over the fur.

Effectiveness of median nerve neural mobilization versus oral ibuprofen treatment in subjects who suffer from cervicobrachial pain: a randomized clinical trial.

INTRODUCTION: Oral ibuprofen (OI) and median nerve neural mobilization (MNNM) are first line treatments for patients who suffer cervicobrachial pain (CP). OI may produce side effects which are not tolerated by all subjects who suffer CP, whereas MNNM has no known side effects. Therefore, the aim of this study was to assess the effectiveness of both treatments (OI vs. MNNM) in CP. MATERIAL AND METHODS: This investigation was a blinded parallel randomized clinical trial (NCT02593721). Sixty-two participants diagnosed with CP were recruited and randomly assigned to 2 groups (n = 31), which received MNNM or 1200 mg/day OI treatment for 6 weeks. The numeric rating scale for pain intensity was the primary outcome. The cervical rotation range of motion (CROM) and the upper limb function were the secondary outcomes. RESULTS: The results showed that OI treatment (η2 = 0.612-0.755) was clearly superior to MNNM (η2 = 0.816-0.821) in all assessments (p < 0.05) except for the CROM device results, which were equivalent to those of the MNNM group (p > 0.05). Three subjects were discharged because of OI side effects. CONCLUSIONS: Oral ibuprofen may be superior to MNNM for pain reduction and upper limb function increase of subjects with CP. Nevertheless, both treatments were effective. Median nerve neural mobilization may be considered an effective non-pharmaceutical treatment option in subjects with CP. Regarding OI adverse effects, our findings challenge the role of pharmacologic versus manual therapy as possible treatments that may improve pain intensity and upper limb functionality in subjects with CP.

Biocatalytic versatility of engineered and wild-type tyrosinase from R. solanacearum for the synthesis of 4-halocatechols.

We evaluated the kinetic characteristics of wild type (WT) and three engineered variants (RVC10, RV145, and C10_N322S) of tyrosinase from Ralstonia solanacearum and their potential as biocatalysts to produce halogenated catechols. RV145 exhibited a 3.6- to 14.5-fold improvement in catalytic efficiency (kcat/Km) with both reductions in Km and increases in kcat compared to WT, making it the best R. solanacearum tyrosinase variant towards halogenated phenols. RVC10 also exhibited increases in catalytic efficiency with all the tested phenols. A single-mutation variant (C10_N322S) exhibited the greatest improvement in kcat but lowest improvement in catalytic efficiency due to an increase in Km compared to WT. Consistent with kinetic characteristics, biotransformation experiments showed that RV145 was a superior biocatalyst in comparison to WT. To prevent through conversion of the catechol to quinone, ascorbic acid (AA) was added to the biotransformation medium in 1:2 (substrate:AA) ratio resulting in a catechol yield of > 90%. Flask experiments with 10 mM 4-iodophenol and 10 µg/mL of the RV145 enzyme yielded 9.5 mM 4-iodocatechol in the presence of 20 mM AA in 30 min. Similarly, 10 mM 4-fluorophenol was completely consumed by 20 µg/mL of RV145 enzyme and yielded 9.2 mM 4-fluorocatechol in the presence of 20 mM AA in 80 min. The biotransformation of 20 mM 4-fluorphenol was incomplete (93%) and the yield of 4-flurocatechol was 87.5%. The 4-halophenol conversion rates and product yields obtained in this study are the highest reported using tyrosinase or any other enzyme.

Is pharmacologic treatment better than neural mobilization for cervicobrachial pain? A randomized clinical trial.

Purpose: This study aim was to compare the effectiveness of the median nerve neural mobilization (MNNM) and cervical lateral glide (CLG) intervention versus oral ibuprofen (OI) in subjects who suffer cervicobrachial pain (CP). Methods: This investigation was a, multicenter, blinded, randomized controlled clinical trial (NCT02595294; NCT02593721). A number of 105 individuals diagnosed with CP were enrolled in the study and treated in 2 different medical facilities from July to November 2015. Participants were recruited and randomly assigned into 3 groups of 35 subjects. Intervention groups received MNNM or CLG neurodynamic treatments, and the (active treatment) control group received an OI treatment for 6 weeks. Primary outcome was pain intensity reported through the Numeric Rating Scale for Pain (NRSP). Secondary outcomes were physical function involving the affected upper limb using the Quick DASH scale, and ipsilateral cervical rotation (ICR) using a cervical range of motion (CROM) device. Assessments were performed before and 1 hour after treatment for NRSP (baseline, 3 and 6 weeks) and CROM (baseline and 6 weeks), as well as only 1 assessment for Quick DASH (baseline and 6 weeks). Results: Repeated-measures ANOVA intergroup statistically significant differences were shown for CP intensity (F(2,72) = 22.343; P < .001; Eta2 = 0.383) and Quick DASH (F(2,72) = 15.338; P < .001; Eta2 = 0.299), although not for CROM (F(2,72) = 1.434; P = .245; Eta2 = 0.038). Indeed, Bonferroni´s correction showed statistically significant differences for CP intensity (P < .01; 95% CI = 0.22 - 3.26) and Quick DASH reduction (P < .01; 95% CI = 8.48 - 24.67) in favor of the OI treatment at all measurement moments after baseline. Conclusions: OI pharmacologic treatment may reduce pain intensity and disability with respect to neural mobilization (MNNM and CLG) in patients with CP during six weeks. Nevertheless, the non-existence of between-groups ROM differences and possible OI adverse effects should be considered.

Effect of antibiotics and NSAIDs on cyclooxygenase-2 in the enamel mineralization.

The objective of this study was to determine whether the use of the most commonly prescribed antibiotics and non-steroidal anti-inflammatory drugs in childhood could disturb enamel mineralization. Forty-two Swiss mice were divided into seven groups: controls; amoxicillin; amoxicillin/clavulanate; erythromycin; acetaminophen; ibuprofen and celecoxib, to inhibit cyclooxygenase 2 (COX2). SEM-EDX analysis was conducted on all cusps of the third molars. Calcium (Ca), phosphorus (P), aluminum, potassium, sodium, magnesium and chlorine were quantified. The stoichiometric Ca/P molar ratios were calculated. Immunohistochemical quantification of COX2 in incisors was carried out by image analysis using COX2-specific immunostaining. Groups treated with antibiotics showed no significant differences in the content of the chemical elements. Only acetaminophen and celecoxib showed a significant decrease in Ca and P compared with the control samples. Ca/P ratios showed no difference. Groups treated with amoxicillin, amoxicillin/clavulanate, erythromycin and acetaminophen showed significantly lower amounts of immunoreactive COX2 at the enamel organ maturation stage of the mouse incisors. Our results suggest that COX2 is involved in the maturation stage of the enamel organ and that its inhibition would appear to alter amelogenesis, producing hypomineralization.

On the Metal Cofactor in the Tyrosinase Family.

The production of pigment in mammalian melanocytes requires the contribution of at least three melanogenic enzymes, tyrosinase and two other accessory enzymes called the tyrosinase-related proteins (Trp1 and Trp2), which regulate the type and amount of melanin. The last two proteins are paralogues to tyrosinase, and they appeared late in evolution by triplication of the tyrosinase gene. Tyrosinase is a copper-enzyme, and Trp2 is a zinc-enzyme. Trp1 has been more elusive, and the direct identification of its metal cofactor has never been achieved. However, due to its enzymatic activity and similarities with tyrosinase, it has been assumed as a copper-enzyme. Recently, recombinant human tyrosinase and Trp1 have been expressed in enough amounts to achieve for the first time their crystallization. Unexpectedly, it has been found that Trp1 contains a couple of Zn(II) at the active site. This review discusses data about the metal cofactor of tyrosinase and Trps. It points out differences in the studied models, and it proposes some possible points accounting for the apparent discrepancies currently appearing. Moreover, some proposals about the possible flexibility of the tyrosinase family to uptake copper or zinc are discussed.

A Protective Eye Shield Reduces Limbal Strain and Its Variability During Simulated Sleep in Adults With Glaucoma.

PURPOSE: To determine the effect of wearing a protective eye shield (mask) on limbal strain magnitude and variability in glaucoma eyes when sleeping with 1 side of the face down (FD) against a pillow. METHODS: A prospective, randomized, interventional trial was conducted at the Wilmer Eye Institute with 36 glaucoma patients. A contact lens sensor measured limbal strain (output in equivalent millivolts) during intervals of up to 60 minutes in lateral decubitus, FD, and supine positions. Eighteen subjects wore a mask during 1 of 2 FD intervals, with randomized assignment of the interval. Data from additional trials with no mask were included in some analyses. In addition, some facial-feature dimensions from 3D scanned images of 23 subjects were compared with limbal strain data. RESULTS: Wearing a mask trends toward a reduced mean change in contact lens sensor output (limbal strain) on moving to a FD positions [+34.1 mVeq, P=0.01 reduced by -22.3 mVeq, P=0.09 (n=36)]. Mask wearing reduced variability in strain while FD [-22.8 mVeq, P=0.04 (n=18)]. In eyes with past progressive visual field loss, the effect of the mask reduced mean strain change when moving to FD [-44.8 mVeq, P=0.02 (n=31)]. Longer corneal apex to nose-tip and to temple lengths were associated with reduced variability while FD [P=0.02 and 0.04, respectively (n=23)]. Treating both lengths as confounding factors increased statistical significance, particularly for analysis of the no-mask change in strain data moving to and from the FD position [P=0.004 to 0.002 and P=0.03 to 0.01 (n=23)]. CONCLUSION AND RELEVANCE: Wearing a mask reduced limbal strain and variation in limbal strain during simulated FD sleep, particularly in eyes with past field worsening, as did some facial features.