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.

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.

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.

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.

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.

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.

Effects of Median Nerve Neural Mobilization in Treating Cervicobrachial Pain: A Randomized Waiting List-Controlled Clinical Trial.

BACKGROUND: There is a current lack of sufficiently high-quality randomized controlled clinical trials that measure the effectiveness of neural tissue mobilization techniques such as median nerve neural mobilization (MNNM) and their specific effects on cervicobrachial pain (CP). The aim of this study was to compare the effectiveness of MNNM in subjects with CP vs. a waiting list control group (WLCG). METHODS: A single-blinded, parallel, randomized controlled clinical trial was performed (NCT02596815). Subjects were recruited with a medical diagnosis of CP corroborated by magnetic resonance imaging. In total, 156 individuals were screened, 60 subjects were recruited, and 51 completed the trial. Pain intensity reported using the Numeric Rating Scale for Pain (NRSP; primary outcome), cervical range of motion (CROM), and functionality using the Quick Disabilities of the Arm, Shoulder and Hand (QuickDASH) scale were the outcome measurements. Assessments were conducted at baseline and 1 hour after treatment (intervention days 1, 15, and 30). Therefore, MNNM was implemented with 30 days of follow-up. RESULTS: The NRSP values of the MNNM group were significantly (P < 0.0001; 95% confidence interval [CI]) superior to those obtained in the WLCG. Subjects treated with MNNM reported an NRSP decrease of 3.08 points at discharge. CROM and QuickDASH outcome values were significantly (P ˂ 0.0001; 95% CI) improved only in the MNNM group. Hedges' g showed a very large effect of the MNNM intervention. CONCLUSION: MNNM may be superior to no treatment in reducing pain and increasing function in the affected upper limbs of subjects with CP.

Cervical Lateral Glide Neural Mobilization Is Effective in Treating Cervicobrachial Pain: A Randomized Waiting List Controlled Clinical Trial.

BACKGROUND: Cervicobrachial pain (CP) is a high-incidence and prevalent condition. Cervical lateral glide (CLG) is a firstline treatment of CP. There is a current lack of enough high-quality randomized controlled double-blind clinical trials that measure the effectiveness of neural tissue mobilization techniques such as the CLG and its specific effect over CP. OBJECTIVES: The aim of the present study was to assess the effect of CLG neural mobilization in treating subjects who suffer from CP, compared with the complete absence of treatment. STUDY DESIGN: This investigation was a single-center, blinded, parallel randomized controlled clinical trial (RCT). SETTING: One hundred forty-seven individuals were screened in a medical center from July to November 2015. Fifty-eight participants were diagnosed with CP. METHODS: Participants were recruited and randomly assigned into two groups of 29 subjects. The intervention group received CLG treatment, and the control group (CG) was assigned to a six-week waiting list to receive treatment. Randomization was carried out by concealed computer software randomized printed cards. The 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 CROM device. Assessments were made at baseline and one hour after treatment. RESULTS: The CLG group NRSP mean value was significantly (P < 0.0001) superior to those obtained by the CG. Subjects treated with CLG reported an average NRSP decrease of 2.16 points (35%). CROM device and Quick DASH outcome values also reported significant (P < 0.0001) improvements only in the CLG group. Cohen's d showed a very large effect of the CLG intervention at subject discharge. LIMITATIONS: Due to the lack of dipper subgroup analysis and additional reproductions of the applied protocol, the authors considered the generalization of the study results to be impossible. CONCLUSIONS: CLG is superior to the absence of treatment in reducing pain and increasing the affected upper limb function of subjects who suffer from CP. CLG may be considered an effective treatment in specific cases of CP.

Melanin and Melanin-Related Polymers as Materials with Biomedical and Biotechnological Applications-Cuttlefish Ink and Mussel Foot Proteins as Inspired Biomolecules.

The huge development of bioengineering during the last years has boosted the search for new bioinspired materials, with tunable chemical, mechanical, and optoelectronic properties for the design of semiconductors, batteries, biosensors, imaging and therapy probes, adhesive hydrogels, tissue restoration, photoprotectors, etc. These new materials should complement or replace metallic or organic polymers that cause cytotoxicity and some adverse health effects. One of the most interesting biomaterials is melanin and synthetic melanin-related molecules. Melanin has a controversial molecular structure, dependent on the conditions of polymerization, and therefore tunable. It is found in animal hair and skin, although one of the common sources is cuttlefish (Sepia officinalis) ink. On the other hand, mussels synthesize adhesive proteins to anchor these marine animals to wet surfaces. Both melanin and mussel foot proteins contain a high number of catecholic residues, and their properties are related to these groups. Dopamine (DA) can easily polymerize to get polydopamine melanin (PDAM), that somehow shares properties with melanin and mussel proteins. Furthermore, PDAM can easily be conjugated with other components. This review accounts for the main aspects of melanin, as well as DA-based melanin-like materials, related to their biomedical and biotechnological applications.

Bird Integumentary Melanins: Biosynthesis, Forms, Function and Evolution.

Melanins are the ubiquitous pigments distributed in nature. They are one of the main pigments responsible for colors in living cells. Birds are among the most diverse animals regarding melanin-based coloration, especially in the plumage, although they also pigment bare parts of the integument. This review is devoted to the main characteristics of bird melanins, including updated views of the formation and nature of melanin granules, whose interest has been raised in the last years for inferring the color of extinct birds and non-avian theropod dinosaurs using resistant fossil feathers. The molecular structure of the two main types of melanin, eumelanin and pheomelanin, and the environmental and genetic factors that regulate avian melanogenesis are also presented, establishing the main relationship between them. Finally, the special functions of melanin in bird feathers are also discussed, emphasizing the aspects more closely related to these animals, such as honest signaling, and the factors that may drive the evolution of pheomelanin and pheomelanin-based color traits, an issue for which birds have been pioneer study models.

Buthionine sulfoximine diverts the melanogenesis pathway toward the production of more soluble and degradable pigments.

Buthionine sulfoximine (BSO) is a specific inhibitor of γ-glutamylcysteine synthetase, thus blocking the synthesis of glutathione (GSH). It is known that this makes that BSO affects melanin synthesis because of the role of thiols in melanogenesis. However, BSO may also react with the intermediate oxidation products of melanogenesis, a possibility that has not been investigated from the initial steps of the pathway. We created in vitro conditions simulating eumelanogenesis (oxidation of L-DOPA in the absence of GSH) and pheomelanogenesis (oxidation of L-DOPA in the presence of GSH) under presence or absence of BSO. BSO made that eumelanogenesis results in pigments more soluble and less resistant to degradation by hydrogen peroxide than pigments obtained without BSO. A similar but less marked effect was observed for pheomelanogenesis only at subsaturating concentrations of GSH. These results suggest that BSO diverts the melanogenesis pathway toward the production of more soluble and degradable pigments.

LodB is required for the recombinant synthesis of the quinoprotein L-lysine-ε-oxidase from Marinomonas mediterranea.

Marinomonas mediterranea is a marine gamma-proteobacterium that synthesizes LodA, a novel L-lysine-ε-oxidase (E.C. 1.4.3.20). This enzyme oxidizes L-lysine generating 2-aminoadipate 6-semialdehyde, ammonium, and hydrogen peroxide. Unlike other L-amino acid oxidases, LodA is not a flavoprotein but contains a quinone cofactor. LodA is encoded by an operon with two genes, lodA and lodB. In the native system, LodB is required for the synthesis of a functional LodA. In this study, we report the recombinant expression of LodA in Escherichia coli using vectors that allow its expression and accumulation in the cytoplasm. To reveal the L-lysine-ε-oxidase activity using the Amplex Red method for hydrogen peroxide detection, it is necessary to first remove the E. coli cytoplasmic catalases. The flavoprotein LodB is the only M. mediterranea protein required in the recombinant system for the generation of the cofactor of LodA. In the absence of LodB, LodA does not contain the quinone cofactor and remains in an inactive form. The results presented indicate that LodB participates in the posttranslational modification of LodA that generates the quinone cofactor.

The urological referral process from primary health care. Reflections on referral criteria.

OBJECTIVES: The flow of patients between Primary Care (PC) and Specialized care (SC) is a common process. It carries many implications for the patient, physician and health system. In Urology, only benign prostatic hyperplasia (BPH) has referral criteria. Urinary incontinence, prostate cancer (PCa), and urological ultrasound, are in the process. The aim of this paper is to communicate, with critical analysis, the characteristics of the information recorded in the referral visit (clinical reasons / rationale) and the effectiveness for urology consultation. METHODS: Observational, descriptive and quantitative study of the referral visits made between PC/SC (Urology) in the health care area of our hospital (December 2010-September 2012). We studied: Referral Visit Database (RVD), consultation document, HORUS system, and specific referral visit survey questionnaire. RESULTS. Referral visits account for 67.89% (all first consultations), 14.79% of the total number of visits. 78% were male (mean age 53 y.o). 11.84% recorded reason for consultation (98% in referral document) with normal priority (94.67%). 34% of them were for BPH. HORUS is not exploited for the referral visit. 40% start the diagnostic process with insufficient exams. 18.1% are listed as closed process / completed. Patient satisfaction was evaluated (20%). Key points in the improvement are: improve referral visit reason for consultations, to know patient's expectations, and to develop protocols (guidelines, and/or referral criteria). CONCLUSIONS. The referral process is complex. The computer system does not include the referral reason for consultation. Institutional agreement between PC/SC Urology must be reached to ensure uniformity in the implementation and support.

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.

Engineering of a bacterial tyrosinase for improved catalytic efficiency towards D-tyrosine using random and site directed mutagenesis approaches.

The tyrosinase gene from Ralstonia solanacearum (GenBank NP518458) was subjected to random mutagenesis resulting in tyrosinase variants (RVC10 and RV145) with up to 3.2-fold improvement in k(cat), 5.2-fold lower K(m) and 16-fold improvement in catalytic efficiency for D-tyrosine. Based on RVC10 and RV145 mutated sequences, single mutation variants were generated with all variants showing increased k(cat) for D-tyrosine compared to the wild type (WT). All single mutation variants based on RV145 had a higher k(cat) and K(m) value compared to the RV145 and thus the combination of four mutations in RV145 was antagonistic for turnover, but synergistic for affinity of the enzyme for D-tyrosine. Single mutation variant 145_V153A exhibited the highest (6.9-fold) improvement in k(cat) and a 2.4-fold increase in K(m) compared to the WT. Two single mutation variants, C10_N322S and C10_T183I reduced the K(m) up to 2.6-fold for D-tyrosine but one variant 145_V153A increased the K(m) 2.4-fold compared to the WT. Homology based modeling of R. solanacearum tyrosinase showed that mutation V153A disrupts the van der Waals interactions with an α-helix providing one of the conserved histidine residues of the active site. The k(cat) and K(m) values for L-tyrosine decreased for RV145 and RVC10 compared to the WT. RV145 exhibited a 2.1-fold high catalytic efficiency compared to the WT which is a 7.6-fold lower improvement compared to D-tyrosine. RV145 exhibited a threefold higher monophenolase:diphenolase activity ratio for D-tyrosine:D-DOPA and a 1.4-fold higher L-tyrosine:L-DOPA activity ratio compared to the WT.

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.

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.

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.