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The emerging field of precision medicine is rapidly growing, fostered by the advances in genome mapping and molecular diagnosis. In general, the translation of these advances into precision treatments relies on the use of biological macromolecules, whose structure offers a high specificity and potency. Unfortunately, due to their complex structure and limited ability to overcome biological barriers, these macromolecules need to be administered via injection. The scientific community has devoted significant effort to making the oral administration of macromolecules plausible thanks to the implementation of drug delivery technologies. Here, an overview of the current situation and future prospects in the field of oral delivery of biologics is provided. Technologies in clinical trials, as well as recent and disruptive delivery systems proposed in the literature for local and systemic delivery of biologics including peptides, antibodies, and nucleic acids, are described. Strategies for the specific targeting of gastrointestinal regions-stomach, small bowel, and colon-cell populations, and internalization pathways, are analyzed. Finally, challenges associated with the clinical translation, future prospects, and identified opportunities for advancement in this field are also discussed., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Enzymatic glycosylation of polyphenols is a tool to improve their physicochemical properties and bioavailability. On the other hand, glycosidic enzymes can be inhibited by phenolic compounds. In this work, we studied the specificity of various phenolics (hydroquinone, hydroxytyrosol, epigallocatechin gallate, catechol and p-nitrophenol) as fructosyl acceptors or inhibitors of the β-fructofuranosidase from Xanthophyllomyces dendrorhous (pXd-INV). Only hydroquinone and hydroxytyrosol gave rise to the formation of glycosylated products. For the rest, an inhibitory effect on both the hydrolytic (H) and transglycosylation (T) activity of pXd-INV, as well as an increase in the H/T ratio, was observed. To disclose the binding mode of each compound and elucidate the molecular features determining its acceptor or inhibitor behaviour, ternary complexes of the inactive mutant pXd-INV-D80A with fructose and the different polyphenols were analyzed by X-ray crystallography. All the compounds bind by stacking against Trp105 and locate one of their phenolic hydroxyls making a polar linkage to the fructose O2 at 3.6-3.8 Å from the C2, which could enable the ulterior nucleophilic attack leading to transfructosylation. Binding of hydroquinone was further investigated by soaking in absence of fructose, showing a flexible site that likely allows productive motion of the intermediates. Therefore, the acceptor capacity of the different polyphenols seems mediated by their ability to make flexible polar links with the protein, this flexibility being essential for the transfructosylation reaction to proceed. Finally, the binding affinity of the phenolic compounds was explained based on the two sites previously reported for pXd-INV.

Metschnikowia reukaufii is a widespread yeast able to grow in the plants' floral nectaries, an environment of extreme conditions with sucrose concentrations exceeding 400 g l -1 , which led us into the search for enzymatic activities involved in this sugar use/transformation. New oligosaccharides were produced by transglucosylation processes employing M. reukaufii cell extracts in overload-sucrose reactions. These products were purified and structurally characterized by MS-ESI and NMR techniques. The reaction mixture included new sugars showing a great variety of glycosidic bonds including α-(1→1), α-(1→3) and α-(1→6) linkages. The main product synthesized was the trisaccharide isomelezitose, whose maximum concentration reached 81 g l -1 , the highest amount reported for any unmodified enzyme or microbial extract. In addition, 51 g l -1 of the disaccharide trehalulose was also produced. Both sugars show potential nutraceutical and prebiotic properties. Interestingly, the sugar mixture obtained in the biosynthetic reactions also contained oligosaccharides such as esculose, a rare trisaccharide with no previous NMR structure elucidation, as well as erlose, melezitose and theanderose. All the sugars produced are naturally found in honey. These compounds are of biotechnological interest due to their potential food, cosmeceutical and pharmaceutical applications., (© 2019 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

A dose of proanthocyanidins with satiating properties proved to be able to limit body weight increase several weeks after administration under exposure to a cafeteria diet. Here we describe some of the molecular targets and the duration of the effects. We treated rats with 500 mg grape seed proanthocyanidin extract (GSPE)/kg BW for ten days. Seven or seventeen weeks after the last GSPE dose, while animals were on a cafeteria diet, we used reverse transcriptase-polymerase chain reaction (RT-PCR) to measure the mRNA of the key energy metabolism enzymes from the liver, adipose depots and muscle. We found that a reduction in the expression of adipose Lpl might explain the lower amount of adipose tissue in rats seven weeks after the last GSPE dose. The liver showed increased expression of Cpt1a and Hmgs2 together with a reduction in Fasn and Dgat2 . In addition, muscle showed a higher fatty oxidation ( Oxct 1 and Cpt1b mRNA). However, after seventeen weeks, there was a completely different gene expression pattern. At the conclusion of the study, seven weeks after the last GSPE administration there was a limitation in adipose accrual that might be mediated by an inhibition of the gene expression of the adipose tissue Lpl . Concomitantly there was an increase in fatty acid oxidation in liver and muscle., Competing Interests: The authors declare no conflict of interest.

A case of a femoropopliteal recanalization procedure using a new bailout technical maneuver to aid reentry into the true lumen is described. After a failed standard reentry attempt, at the level of the vessel reconstitution in the subintimal space, the guidewire was exchanged for a GooseNeck Snare Kit. A needle was inserted across the target artery, piercing both arterial walls and passing through the snare loop. Once the needle had exited the artery, a wire was inserted and the needle withdrawn. The snare was closed, withdrawn, and the wire externalized through the femoral access. Finally, a catheter was advanced from the antegrade sheath up to the arterial reconstitution. Selective injection at the site of reentry confirmed the intraluminal position, and the procedure was successfully completed from the antegrade sheath., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Harvesting of broccoli at several moments of the day affects the rate of senescence during storage. In this work, broccoli heads were harvested at several moments and then kept at 20°C in order to analyze protein metabolism and antioxidant compounds. Almost no differences were detected in the contents of total and soluble proteins, and free amino acids. Only an increment in free amino acids was detected by day 3 in samples obtained at 8:00 hr. With reference to antioxidants, the contents of ascorbic acid, carotenoids and xanthophylls, phenols, and flavonoids were similar in samples harvested at different moments. However, an increment was detected in carotenoids, phenols, and flavonoids during storage of samples collected at 18:00 hr on day 3 and samples collected at 13:00 hr on day 5. The combination of delay of senescence and increment in antioxidants suggest harvesting at 12:00 or 18:00 hr. PRACTICAL APPLICATION: Broccoli is a vegetable with an important level of nutrients. However, it is also highly perishable and suffers a high rate of senescence and loss of quality during postharvest. In this work, it is demonstrated that the simple practice of harvests in different moments of the day can affect the postharvest behavior of broccoli, and it is suggested to carry out the harvest toward the end of the day., (© 2019 Wiley Periodicals, Inc.)

Purpose: To evaluate the efficacy of a new guidewire threading instrument (GTI) in reducing guidewire threading times compared with the traditional freehand method., Materials and Methods: This prospective, controlled, single-center study recruited 100 subjects (mean age 45.2±11.3 years; 43 men) and divided them into 2 groups depending on their professional background: 50 experienced medical personnel (performed or assisted in >50 catheter-based procedures) and 50 medical personnel with no experience in endovascular techniques (inexperienced group). The threading time of both groups was recorded using the conventional freehand method and with the GTI for both 0.035- and 0.014-inch platforms. Users of eyeglasses for presbyopia were tested with and without glasses. Median values are reported with the interquartile range (IQR; Q1, Q3) in parentheses., Results: The mean insertion times with both the 0.035-inch and 0.014-inch guidewires in the overall study group were significantly better with the use of the GTI compared with the traditional freehand method (p<0.001). Both the experienced and the inexperienced participants improved their insertion times using the GTI with both guidewire platforms (p<0.001). The threading time with the new device was also significantly reduced (p<0.001) for both participants with presbyopia and those without. When comparing the median absolute time improvement (difference between freehand/GTI insertion times) for the 0.014-inch platform, the inexperienced group showed a greater improvement in their performance [3.52 seconds (IQR 2.76, 5.12)] compared with the experienced group [1.87 seconds (IQR 1.37, 2.66), p<0.001]. The median "absolute time improvement" was also significantly greater for the presbyopic group [5.75 seconds (IQR 3.14, 8.20)] vs the group without age-related visual impairment [2.64 seconds (IQR 1.65, 3.36), p<0.001]., Conclusion: This simple and inexpensive homemade device facilitates wire threading of low-profile catheters and seems to be especially helpful for trainees with no experience and presbyopic operators.

Background: The golimumab safety awareness study commenced in 2010 to measure, periodically, the awareness of golimumab prescriber healthcare professionals (HCPs) of specific risks associated with golimumab as well as awareness of the requirement to provide a patient alert card to each patient treated with golimumab, as described in the European golimumab educational program. The aim of this study was to measure the awareness of HCPs who prescribe or who intend to prescribe SIMPONI ® (golimumab) of the risks potentially related to golimumab and of the requirements for distributing the patient alert card, as described in the golimumab educational program., Methods: A structured, quantitative Web-based survey was conducted in 2010, 2012, 2014, and 2016 in eight European countries among HCPs who were at that time current or future prescribers of golimumab for patients with rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, nonradiographic axial spondyloarthritis, or ulcerative colitis., Results: The overall golimumab risk awareness was high for golimumab prescriber HCPs across the risk statement categories (median awareness in 2016 across categories: 91%). The awareness of the golimumab risks was generally slightly higher among rheumatologists (75-98%) and gastroenterologists (73-97%) than among dermatologists (67-94%). Overall, the awareness of the requirements for handing out the patient alert card to golimumab-treated patients remained steady or increased slightly in 2016 relative to the other surveys., Conclusions: The results of this study show that the awareness of risks associated with golimumab by golimumab prescriber HCPs is high. The information made available to golimumab prescriber HCPs appears to have been sufficient with respect to golimumab risk awareness education., Competing Interests: Conflict of interest statement: L. Felo is an employee of Merck Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Kenilworth, NJ, USA and holds stock/stock options in the company. M. Otero-Lobato and A. Geldhof are employees of Janssen Biologics BV, Leiden, The Netherlands, and W. Noël is an employee of Janssen Cilag, Belgium; all three of these employees hold stock/stock options in Johnson & Johnson. All of the authors are responsible for the work described in this manuscript. All authors were involved in at least one of the following: (1) conception, design, acquisition, analysis, statistical analysis, or interpretation of data and (2) drafting the manuscript or revising it for important intellectual content. All authors provided final approval of the version to be published.

Purpose: To describe a maneuver to facilitate percutaneous arteriovenous fistula creation during venous arterialization procedures in patients with no-option critical limb ischemia., Technique: Following a failed arterial recanalization attempt, a balloon catheter is passed up to the tip of the guidewire. Venous access is gained distally, a 4-F sheath is antegradely passed, and a 4-mm GooseNeck snare is advanced through it. A fluoroscopic view that overlaps the snare and the inflated balloon is obtained. If the vein remains anterior with respect to the artery, a needle is inserted across the vein, passing through the snare loop and puncturing the intra-arterial balloon. A wire is inserted and placed inside the punctured balloon. The balloon is retrieved and the wire externalized through the femoral access. A catheter is advanced antegradely over this wire from the artery into the vein. If the vein remains posterior to the artery, a needle is inserted, puncturing the balloon and thereafter the vein (crossing through the snare). A wire is inserted, captured by the snare, and externalized through the vein sheath. A catheter is finally advanced over this wire from the vein into the artery., Conclusion: This maneuver is a simple alternative to create an arteriovenous fistula during venous arterialization procedures in patients with no-option critical limb ischemia.

Xanthophyllomyces dendrorhous is a basidiomycete yeast that synthesizes carotenoids, mainly astaxanthin, which are of great commercial interest. Currently, there are many unknown aspects related to regulatory mechanisms on the synthesis of carotenoids in this yeast. Our recent studies showed that changes in sterol levels and composition resulted in upregulation of genes in the mevalonate pathway required for the synthesis of carotenoid precursors, leading to increased production of these pigments. Sterol Regulatory Element-Binding Proteins (SREBP), called Sre1 in yeast, are conserved transcriptional regulators of sterol homeostasis and other cellular processes. Given the results linking sterols and carotenoids, we investigated the role of SREBP in sterol and carotenoid synthesis in X. dendrorhous. In this study, we present the identification and functional characterization of the X. dendrorhous SRE1 gene, which encodes the transcription factor Sre1. The deduced protein has the characteristic features of SREBP/Sre1 and binds to consensus DNA sequences in vitro. RNA-seq analysis and chromatin-immunoprecipitation experiments showed that genes of the mevalonate pathway and ergosterol biosynthesis are directly regulated by Sre1. The sre1 - mutation reduced sterol and carotenoid production in X. dendrorhous , and expression of the Sre1 N-terminal domain (Sre1N) increased carotenoid production more than twofold compared to wild-type. Overall, our results indicate that in X. dendrorhous transcriptional regulation of genes in the mevalonate pathway control production of the isoprenoid derivatives, carotenoids and sterol. Our results provide new insights into the conserved regulatory functions of SREBP/Sre1 and identify pointing to the SREBP pathway as a potential target to enhance carotenoid production in X. dendrorhous .

The β-fructofuranosidase Ffase from the yeast Schwanniomyces occidentalis produces potential prebiotic fructooligosaccharides with health-promoting properties, making it of biotechnological interest. Ffase is one of the highest and more selective known producers of 6-kestose by transfructosylation of sucrose. In this work, production of 6-kestose was simplified by directly using cultures of S. occidentalis and Saccharomyces cerevisiae expressing both the wild-type enzyme and a mutated Ffase variant including the Ser196Leu substitution (Ffase-Leu196). Best results were obtained using yeast cultures supplemented with sucrose and expressing the Ffase-Leu196, which after only 4 h produced ~ 116 g/L of 6-kestose, twice the amount obtained with the corresponding purified enzyme. 6-Kestose represented ~ 70% of the products synthesized. In addition, a small amount of 1-kestose and the neofructoligosaccharides neokestose and blastose were also produced. The Ser196Leu substitution skewed production of 6-kestose and neofructooligosaccharides resulting in an increase of ~ 2.2- and 1.5-fold, respectively, without affecting production of 1-kestose. Supplementing yeast cultures with glucose clearly showed that blastose originates from direct fructosylation of glucose, a property that has not been described for other similar proteins from yeasts. Modeling neokestose and blastose into the Ffase-active site revealed the molecular basis explaining the peculiar specificity of this enzyme.

Purpose: To compare white matter bundles and fiber tract changes in seizure-free and non-seizure-free idiopathic generalized epilepsy (IGE) patients., Method: Forty adult patients with IGE underwent a 3 T brain MRI with DTI sequences. According to seizure control status, eighteen patients were classified as refractory (R) if they had presented at least one incapacitating seizure in the previous six months, while on appropriate antiepileptic drug treatment. Twenty two seizure-free patients with adequate seizure control were considered non-refractory (NR). We compared fractional anisotropy (FA) and mean diffusivity (MD) values in sixteen white matter tracts in the R and NR groups, and in twenty healthy subjects., Results: R and NR groups did not differ in gender, age and education. We found decreased FA in two tracts in the R group (forceps major and right uncinate fasciculus) and approaching statistical significance in two tracts in the NR group (right cingulate gyrus and right uncinate fasciculus) group, as well as increased MD in six tracts in the R group (forceps minor, left thalamic anterior radiation, right inferior longitudinal fasciculus, right longitudinal superior parietal and temporal fasciculi, and right cingulate gyrus) and in five tracts in the NR group (forceps minor, left thalamic anterior radiation, right inferior longitudinal fasciculus, right longitudinal superior parietal and temporal fasciculi), compared to controls. No differences were noted comparing FA and MD values between R and NR groups., Conclusions: In our patient population, refractory IGE patients on adequate antiepileptic drug treatment did not present more severe white matter tract involvement compared to non-refractory patients., (Copyright © 2018 British Epilepsy Association. Published by Elsevier Ltd. All rights reserved.)

Objectives: To gain greater understanding of the development of emergency medicine in Spain by analyzing the presentations at conferences of the Spanish Society of Emergency Medicine (SEMES) over the past 30 years (1988-2017)., Material and Methods: We examined the programs of all SEMES conferences and described the characteristics of both presentations and presenters. We also analyzed changes occurring between 1988 and 2017 of some of the characteristics observed. The Web of Science was searched to evaluate the scientific productivity of the most frequent presenters and to calculate h-indexes for those presenters to assesstheir scientific relevance., Results: SEMES did not hold a conference in 1992. The total of 29 conferences included 2182 presentations (112 listing presenters from abroad) given by 1410 presenters (89 from abroad). The presenters' affiliations named 616 centers. The number of presentations and presenters increased linearly during the first period and then leveled off. The number increased exponentially in the final phase. Men gave 79.6% of the presentations; 70.6% of the presenters were physicians, 11.9% were nurses and 4.0% were ambulance staff. Specialists in emergency medicine accounted for 60.8% of the presenters who were physicians. Presenters from the Spanish autonomous community organizing the conference gave 29.8% of the presentations. The contributions of presenters from the local organizing community were nearly always more numerous than the average number of contributions from that community in all 29 conferences overall. Conference contributions from some autonomous communities (Extremadura, Andalusia, and Catalonia) were considerably fewer than would be expected given the scientific productivity of those communities. However, communities (Murcia, Balearic Islands, Asturias, Castile-Leon, Madrid), gave many more presentations than their productivity metrics would predict. Analysis of the 59 most frequent presenters (at 5 conferences or more) showed that 64.4% of them had published at least 20 articles and that 71.2% had an h-index of 5 or higher. The number of women on the program increased significantly between 1988 and 2017. Likewise, geographic diversity increased significantly (presentations from centers outside the local organizing area) as did the participation of hospitalbased emergency medicine specialists., Conclusion: SEMES conference programs have attracted significantly more presentations and presenters over the years. We also detected changes in descriptive characteristics. The analysis of those characteristics can help future SEMES conference planners to plan ways to correct aspects such as scarce geographic diversity, low international participation, and few women among presenters.

Purpose: To describe a simple maneuver to facilitate the retrograde puncture of challenging tibial vessels in the lower leg., Technique: Because of the depth of the tibial vessels at the mid to upper calf, especially in obese or muscular patients, a 7- or 9-cm, 21-G micropuncture needle is often needed to reach the artery. However, the low profile of the needle contributes to its flexibility and therefore the tendency to bend as it progresses through the deep tissues, hindering access to the target vessel. To overcome this obstacle, a 4- to 5-cm, 18-G needle is initially placed pointing to the artery. The 21-G needle is advanced through it to the target site. This simple maneuver increases the support to the 21-G needle, preventing its bending and facilitating arterial puncture in the presence of calcified vessels., Conclusion: The telescoping needle technique may be considered as a bailout procedure in the retrograde tibial approach after failed attempts secondary to needle bending or calcified vessels.

Introduction: West syndrome (WS) is an age-dependent epileptic encephalopathy in which the prognosis varies according to the, not always identified, underlying origin., Objectives: To define the profile of cryptogenic (a least studied isolated sub-group) WS, in Spain. To study its outcome, response to different treatments, and to establish prognostic factors., Patients and Methods: The study included a review of the medical records of 16 patients diagnosed with cryptogenic WS during the period, 2000-2015. The mean follow-up time was 6.6 years, with a minimum of 2 years., Results: The large majority (11/16) were male. The mean age at onset was 6 months, and 6/16 had a family history of idiopathic epilepsy. The first line treatment with vigabatrin had an electrical-clinical response in 5/16 patients, with the remaining cases responding to adrenocorticotropic hormone (ACTH). Almost half (44%) of the patients progressed to other types of epilepsy, with no difference between those treated with vigabatrin or ACTH. A greater number of adverse effects were obtained with ACTH, with no retinal involvement being observed with vigabatrin. The aetiological cause was found in 2/16. Being female, late onset, and early control of the hypsarrhythmia, were factors of a good prognosis., Conclusions: The overall prognosis of cryptogenic WS was more serious than expected. Although the incidence of Lennox-Gastaut syndrome was low, the progression to focal epilepsy was the most common, with it appearing within the first 2 years of the diagnosis. The initial response to vigabatrin was lower than expected, but the long-term result was comparable to ACTH., (Copyright © 2017 Asociación Española de Pediatría. Publicado por Elsevier España, S.L.U. All rights reserved.)

The synthesis of a novel α-glucosylated derivative of pterostilbene was performed by a transglycosylation reaction using starch as glucosyl donor, catalyzed by cyclodextrin glucanotransferase (CGTase) from Thermoanaerobacter sp. The reaction was carried out in a buffer containing 20% ( v / v ) DMSO to enhance the solubility of pterostilbene. Due to the formation of several polyglucosylated products with CGTase, the yield of monoglucoside was increased by the treatment with a recombinant amyloglucosidase (STA1) from Saccharomyces cerevisiae ( var. diastaticus) . This enzyme was not able to hydrolyze the linkage between the glucose and pterostilbene. The monoglucoside was isolated and characterized by combining ESI-MS and 2D-NMR methods. Pterostilbene α-d-glucopyranoside is a novel compound. The α-glucosylation of pterostilbene enhanced its solubility in water to approximately 0.1 g/L. The α-glucosylation caused a slight loss of antioxidant activity towards ABTS˙⁺ radicals. Pterostilbene α-d-glucopyranoside was less toxic than pterostilbene for human SH-S5Y5 neurons, MRC5 fibroblasts and HT-29 colon cancer cells, and similar for RAW 264.7 macrophages.

The objective of this work was the development of a new drug nanocarrier intended to overcome the barriers associated to the oral modality of administration and to assess its value for the systemic or local delivery of peptides. The nanocarrier was rationally designed taking into account the nature of the intestinal barriers and was loaded with insulin, which was selected as a model peptide. The nanocarrier consisted of a complex between insulin and a hydrophobically-modified cell penetrating peptide (CPP), enveloped by a protecting polymer. The selected CPP was octaarginine (r8), chemically conjugated with cholesterol (Chol) or lauric acid (C12), whereas the protecting polymer was poly (glutamic acid)-poly (ethylene glycol) (PGA-PEG). This enveloping material was intended to preserve the stability of the nanocomplex in the intestinal medium and facilitate its diffusion across the intestinal mucus. The enveloped nanocomplexes (ENCPs) exhibited a number of key features, namely (i) a unimodal size distribution with a mean size of 200 nm and a neutral zeta potential, (ii) the capacity to associate insulin (~100% association efficiency) and protect it from degradation in simulated intestinal fluids, (iii) the ability to diffuse through intestinal mucus and, most importantly, (iv) the capacity to interact with the Caco-2 model epithelium, resulting in a massive insulin cell uptake (47.59 ± 5.79%). This enhanced accumulation of insulin at the epithelial level was not translated into an enhanced insulin transport. In fact, only 2% of insulin was transported across the monolayer, and this was correlated with a moderate response of insulin following oral administration to healthy rats. Despite of this, the accumulation of the insulin-loaded nanocarriers in the intestinal mucosa could be verified in vivo upon their labeling with 99m Tc. Overall, these data underline the capacity of the nanocarriers to overcome substantial barriers associated to the oral modality of administration and to facilitate the accumulation of the associated peptide at the intestinal level., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Chitinases are ubiquitous enzymes that have gained a recent biotechnological attention due to their ability to transform biological waste from chitin into valued chito-oligomers with wide agricultural, industrial or medical applications. The biological activity of these molecules is related to their size and acetylation degree. Chitinase Chit42 from Trichoderma harzianum hydrolyses chitin oligomers with a minimal of three N-acetyl-D-glucosamine (GlcNAc) units. Gene chit42 was previously characterized, and according to its sequence, the encoded protein included in the structural Glycoside Hydrolase family GH18., Results: Chit42 was expressed in Pichia pastoris using fed-batch fermentation to about 3 g/L. Protein heterologously expressed showed similar biochemical properties to those expressed by the natural producer (42 kDa, optima pH 5.5-6.5 and 30-40 °C). In addition to hydrolyse colloidal chitin, this enzyme released reducing sugars from commercial chitosan of different sizes and acetylation degrees. Chit42 hydrolysed colloidal chitin at least 10-times more efficiently (defined by the k cat /K m ratio) than any of the assayed chitosan. Production of partially acetylated chitooligosaccharides was confirmed in reaction mixtures using HPAEC-PAD chromatography and mass spectrometry. Masses corresponding to (D-glucosamine) 1-8 -GlcNAc were identified from the hydrolysis of different substrates. Crystals from Chit42 were grown and the 3D structure determined at 1.8 Å resolution, showing the expected folding described for other GH18 chitinases, and a characteristic groove shaped substrate-binding site, able to accommodate at least six sugar units. Detailed structural analysis allows depicting the features of the Chit42 specificity, and explains the chemical nature of the partially acetylated molecules obtained from analysed substrates., Conclusions: Chitinase Chit42 was expressed in a heterologous system to levels never before achieved. The enzyme produced small partially acetylated chitooligosaccharides, which have enormous biotechnological potential in medicine and food. Chit42 3D structure was characterized and analysed. Production and understanding of how the enzymes generating bioactive chito-oligomers work is essential for their biotechnological application, and paves the way for future work to take advantage of chitinolytic activities.

Double-stranded RNA (dsRNA) molecules are widely found in yeasts and filamentous fungi. It has been suggested that these molecules may play an important role in the evolution of eukaryote genomes and could be a valuable tool in yeast typing. The characterization of these extrachromosomal genetic elements is usually a laborious process, especially when trying to analyze a large number of samples. In this chapter, we describe a simple method to isolate dsRNA elements from yeasts using low amounts of starting material and their application to different Xanthophyllomyces dendrorhous strains and other psychrotolerant carotenogenic yeasts. Furthermore, the methodologies for enzymatic and hybridization characterizations and quantification of relative dsRNA abundance are detailed.

Purpose: To describe a technical trick to overcome a prior failed retrograde puncture of the tibial vessels secondary to arterial calcification., Technique: Following a prior failed retrograde puncture attempt, the needle is left in place next to the artery. The tip of the needle is then moved toward the artery as a lever so that the artery is fixed between the needle, the skin, and the surrounding tissues. With the artery fixed, a standard puncture procedure is performed. Alternatively, if additional support is needed, 2 micropuncture needles are inserted one on either side of the artery, crossing under the target vessel and hence trapping it between them, providing additional support and therefore enhancing the chance of successful arterial access., Conclusion: The buddy needle technique may be considered as an alternative procedure in a retrograde pedal/tibial approach after prior failed standard attempts in patients with heavily calcified vessels.

Antibiotic A201A produced by Saccharothrix mutabilis subsp. capreolus NRRL3817 contains an aminonucleoside (N 6 , N 6 -dimethyl-3'-amino-3'-deoxyadenosyl), a polyketide (α-methyl-p-coumaric acid) and a disaccharide moiety. The heterologous expression in Streptomyces lividans and Streptomyces coelicolor of a S. mutabilis genomic region of ~34 kb results in the production of A201A, which was identified by microbiological, biochemical and physicochemical approaches, and indicating that this region may contain the entire A201A biosynthetic gene cluster (ata). The analysis of the nucleotide sequence of the fragment reveals the presence of 32 putative open reading frames (ORF), 28 of which according to boundary gene inactivation experiments are likely to be sufficient for A201A biosynthesis. Most of these ORFs could be assigned to the biosynthesis of the antibiotic three structural moieties. Indeed, five ORFs had been previously implicated in the biosynthesis of the aminonucleoside moiety, at least nine were related to the biosynthesis of the polyketide (ata-PKS1-ataPKS4, ata18, ata19, ata2, ata4 and ata7) and six were associated with the synthesis of the disaccharide (ata12, ata13, ata16, ata17, ata5 and ata10) moieties. In addition to AtaP5, three putative methyltransferase genes are also found in the ata cluster (Ata6, Ata8 and Ata11), and no regulatory genes were found.

Urinary biomarkers can predict the progression of chronic kidney disease (CKD). In this study, kidney injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL), and N-acetyl-β-D-glucosaminidase (NAG) were correlated with the stages of CKD, and the association of these biomarkers with CKD progression and adverse outcomes was determined. A total of 250 patients, including 111 on hemodialysis, were studied. Urinary KIM-1, NGAL, and NAG were measured at baseline. Patients not on dialysis at baseline who progressed to a worse CKD stage were compared with those who did not progress. The association of each biomarker and selected covariates with progression to more advanced stages of CKD, end-stage kidney disease, or death was evaluated by Poisson regression. NGAL was moderately correlated (rs=0.467, P<0.001) with the five stages of CKD; KIM-1 and NAG were also correlated, but weakly. Sixty-four patients (46%) progressed to a more advanced stage of CKD. Compared to non-progressors, those patients exhibited a trend to higher levels of KIM-1 (P=0.064) and NGAL (P=0.065). In patients not on dialysis at baseline, NGAL was independently associated with progression of CKD, ESKD, or death (RR=1.022 for 300 ng/mL intervals; CI=1.007-1.037, P=0.004). In patients on dialysis, for each 300-ng/mL increase in urinary NGAL, there was a 1.3% increase in the risk of death (P=0.039). In conclusion, urinary NGAL was associated with adverse renal outcomes and increased risk of death in this cohort. If baseline urinary KIM-1 and NGAL predict progression to worse stages of CKD is something yet to be explored.

Purpose: The purpose of the study is to examine (1) whether family and peer marijuana use are independently related to adolescent marijuana use in Chile, (2) whether family and peer marijuana use are associated with adolescent marijuana dependence in adolescents using marijuana, and (3) whether the adolescent's age moderates the association between family or peer use and adolescent marijuana use and/or dependence., Method: This study used data from the National Survey on Drug Use in the General Population in Chile (a cross-sectional observational study), which was conducted in 2008 and 2010 in 4413 adolescents aged 12-19. Adolescents answered questions about their past-year marijuana use and dependence (ICD-10 criteria) and the marijuana use of their family and peers. Logistic regressions were performed while controlling for confounders., Results: Adolescents who had a family member who used marijuana were five times more likely to use the drug. Adolescents with a close friend who used marijuana were eight times more likely to use marijuana. When adolescents were using marijuana, they were three times more likely to be dependent if they had a family member who used the drug. However, no significant relationship was found between peer use and dependence. No statistically significant interactions were found between family or peer use and age., Conclusion: Family and peer marijuana use was independently associated with adolescent's past-year marijuana use; however, only family marijuana use was statistically associated with adolescent's marijuana dependence., Competing Interests: Compliance with Ethical Standards Funding This study was conducted in the context of the doctoral studies of the first author, who is supported by a scholarship from the Chilean government. However, the sponsor played no role in any part of this study. No other funding was involved in this research. Conflict of Interest The authors declare that they have no conflict of interest. Human Subject Statement All the authors declare that they conformed to the Declaration of Helsinki concerning human rights and informed consent and that they followed the correct procedures concerning the treatment of humans in research. We used data from the National Survey on Drug Use in the General Population, which was conducted by the Chilean government and is publicly available de-identified secondary data, following the national procedures to use these databases. Informed consent was verbally obtained from all individual participants included in the study.

Background: The yeast Xanthophyllomyces dendrorhous produces carotenoids of commercial interest, including astaxanthin and β-carotene. Although carotenogenesis in this yeast and the expression profiles of the genes controlling this pathway are known, the mechanisms regulating this process remain poorly understood. Several studies have demonstrated that glucose represses carotenogenesis in X. dendrorhous, suggesting that this pathway could be regulated by catabolic repression. Catabolic repression is a highly conserved regulatory mechanism in eukaryotes and has been widely studied in Saccharomyces cerevisiae. Glucose-dependent repression is mainly observed at the transcriptional level and depends on the DNA-binding regulator Mig1, which recruits the co-repressor complex Cyc8-Tup1, which then represses the expression of target genes. In this work, we studied the regulation of carotenogenesis by catabolic repression in X. dendrorhous, focusing on the role of the co-repressor complex Cyc8-Tup1., Results: The X. dendrorhous CYC8 and TUP1 genes were identified, and their functions were demonstrated by heterologous complementation in S. cerevisiae. In addition, cyc8 - and tup1 - mutant strains of X. dendrorhous were obtained, and both mutations were shown to prevent the glucose-dependent repression of carotenogenesis in X. dendrorhous, increasing the carotenoid production in both mutant strains. Furthermore, the effects of glucose on the transcript levels of genes involved in carotenogenesis differed between the mutant strains and wild-type X. dendrorhous, particularly for genes involved in the synthesis of carotenoid precursors, such as HMGR, idi and FPS. Additionally, transcriptomic analyses showed that cyc8 - and tup1 - mutations affected the expression of over 250 genes in X. dendrorhous., Conclusions: The CYC8 and TUP1 genes are functional in X. dendrorhous, and their gene products are involved in catabolic repression and carotenogenesis regulation. This study presents the first report involving the participation of Cyc8 and Tup1 in carotenogenesis regulation in yeast.

The β-fructofuranosidase from the yeast Schwanniomyces occidentalis (Ffase) produces the prebiotic sugars 6-kestose and 1-kestose by transfructosylation of sucrose, which makes it of biotechnological interest. In this study, the hydrolase and transferase activity of this enzyme was kinetically characterized and its potential to synthesize new fructosylated products explored. A total of 40 hydroxylated compounds were used as potential fructosyl-acceptor alternatives to sucrose. Only 17 of them, including some monosaccharides, disaccharides, and oligosaccharides as well as alditols and glycosides were fructosylated. The best alternative acceptors were the alditols. The major transfer product of the reaction including mannitol was purified and characterized as 1-O-β-D-fructofuranosyl-D-mannitol, whose maximum concentration reached 44 g/L, representing about 7.3 % of total compounds in the mixture and 89 % of all products generated by transfructosylation. The reactions including erythritol produced 35 g/L of an isomer mixture comprising 1- and 4-O-β-D-fructofuranosyl-D-erythritol. In addition, Ffase produced 24 g/L of the disaccharide blastose by direct fructosylation of glucose, which makes it the first enzyme characterized from yeast showing this ability. Thus, novel fructosylated compounds with potential applications in food and pharmaceutical industries can be obtained due to the Ffase fructosyl-acceptor promiscuity.

The red yeast X. dendrorhous is one of the few natural sources of astaxanthin, a carotenoid used in aquaculture for salmonid fish pigmentation and in the cosmetic and pharmaceutical industries for its antioxidant properties. Genetic control of carotenogenesis is well characterized in this yeast; however, little is known about the regulation of the carotenogenesis process. Several lines of evidence have suggested that carotenogenesis is regulated by catabolic repression, and the aim of this work was to identify and functionally characterize the X. dendrorhous MIG1 gene encoding the catabolic repressor Mig1, which mediates transcriptional glucose-dependent repression in other yeasts and fungi. The identified gene encodes a protein of 863 amino acids that demonstrates the characteristic conserved features of Mig1 proteins, and binds in vitro to DNA fragments containing Mig1 boxes. Gene functionality was demonstrated by heterologous complementation in a S. cerevisiae mig1- strain; several aspects of catabolic repression were restored by the X. dendrorhous MIG1 gene. Additionally, a X. dendrorhous mig1- mutant was constructed and demonstrated a higher carotenoid content than the wild-type strain. Most important, the mig1- mutation alleviated the glucose-mediated repression of carotenogenesis in X. dendrorhous: the addition of glucose to mig1- and wild-type cultures promoted the growth of both strains, but carotenoid synthesis was observed only in the mutant strain. Transcriptomic and RT-qPCR analyses revealed that several genes were differentially expressed between X. dendrorhous mig1- and the wild-type strain when cultured with glucose as the sole carbon source. The results obtained in this study demonstrate that catabolic repression in X. dendrorhous is an active process in which the identified MIG1 gene product plays a central role in the regulation of several biological processes, including carotenogenesis., Competing Interests: The authors have declared that no competing interests exist.

Basidiomycetous yeast Xanthophyllomyces dendrorhous expresses an α-glucosidase with strong transglycosylation activity producing prebiotic sugars such as panose and an unusual tetrasaccharides mixture including α-(1-6) bonds as major products, which makes it of biotechnological interest. Initial analysis pointed to a homodimeric protein of 60 kDa subunit as responsible for this activity. In this study, the gene Xd-AlphaGlu was characterized. The 4131-bp-long gene is interrupted by 13 short introns and encodes a protein of 990 amino acids (Xd-AlphaGlu). The N-terminal sequence of the previously detected 60 kDa protein resides in this larger protein at residues 583-602. Functionality of the gene was proved in Saccharomyces cerevisiae, which produced a protein of about 130 kDa containing Xd-AlphaGlu sequences. All properties of the heterologously expressed protein, including thermal and pH profiles, activity on different substrates, and ability to produce prebiotic sugars were similar to that of the α-glucosidase produced in X. dendrorhous. No activity was detected in S. cerevisiae containing exclusively the 1256-bp from gene Xd-AlphaGlu that would encode synthesis of the 60 kDa protein previously detected. Data were compatible with an active monomeric α-glucosidase of 990 amino acids and an inactive hydrolysis product of 60 kDa. Protein Xd-AlphaGlu contained most of the elements characteristic of α-glucosidases included in the glycoside hydrolases family GH31 and its structural model based on the homologous human maltase-glucoamylase was obtained. Remarkably, the Xd-AlphaGlu C-terminal domain presents an unusually long 115-residue insertion that could be involved in this enzyme's activity against long-size substrates such as maltoheptaose and soluble starch.

Xanthophyllomyces dendrorhousβ-fructofuranosidase (XdINV)is a highly glycosylated dimeric enzyme that hydrolyzes sucrose and releases fructose from various fructooligosaccharides (FOS) and fructans. It also catalyzes the synthesis of FOS, prebiotics that stimulate the growth of beneficial bacteria in human gut. In contrast to most fructosylating enzymes, XdINV produces neo-FOS, which makes it an interesting biotechnology target. We present here its three-dimensional structure, which shows the expected bimodular arrangement and also a long extension of its C terminus that together with anN-linked glycan mediate the formation of an unusual dimer. The two active sites of the dimer are connected by a long crevice, which might indicate its potential ability to accommodate branched fructans. This arrangement could be representative of a group of GH32 yeast enzymes having the traits observed in XdINV. The inactive D80A mutant was used to obtain complexes with relevant substrates and products, with their crystals structures showing at least four binding subsites at each active site. Moreover, two different positions are observed from subsite +2 depending on the substrate, and thus, a flexible loop (Glu-334-His-343) is essential in binding sucrose and β(2-1)-linked oligosaccharides. Conversely, β(2-6) and neo-type substrates are accommodated mainly by stacking to Trp-105, explaining the production of neokestose and the efficient fructosylating activity of XdINV on α-glucosides. The role of relevant residues has been investigated by mutagenesis and kinetics measurements, and a model for the transfructosylating reaction has been proposed. The plasticity of its active site makes XdINV a valuable and flexible biocatalyst to produce novel bioconjugates., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

This work aims at developing and optimizing a valuable oral delivery carrier for the cannabinoid derivative CB13, which presents a high therapeutic potential in chronic pain states that respond poorly to conventional analgesics, but also shows highly unfavorable physicochemical properties. CB13-loaded lipid nanoparticles (LNP) formulations were developed through solvent-emulsion evaporation and optimized in terms of physicochemical properties, long-term stability, integrity under gastric simulated conditions and in vitro interaction with NIH 3T3, HEK 293T and Caco-2 cells. An optimized formulation of LNP containing CB13 was obtained from a wide range of conditions assayed and analyzed. The selection of the lipid core, production conditions and the inclusion of lecithin proved to be key factors for the final properties of encapsulation, integrity and performance of the carriers. The LNP formulation proposed proved to be a promising carrier for the oral delivery of CB13, a cannabinoid with high therapeutic potential in chronic pain states that currently lack a valid oral treatment.

Lysosomal storage diseases (LSDs) comprise a group of rare inherited chronic syndromes that cause deficiency of specific native enzymes within the lysosomes. The macromolecular compounds that are usually catabolized by lysosomal enzymes are accumulated within these organelles, causing progressive damage to tissues, skeleton and organs and, in several cases, the central nervous system (CNS). The damage caused by substrate accumulation finally results in physical deterioration, functional impairment and potential death. Up to date, the most promising therapy for most LSDs is enzyme-replacement therapy (ERT), which provides patients with the corresponding active enzyme. However, these enzymes do not have enough stability in blood, the treatment must be therefore periodically administrated by i.v. infusion under medical supervision, and immunogenicity issues are frequent. In addition, affected areas within the CNS, where the blood-brain barrier (BBB) is a major obstacle, cannot be reached by the enzymes. Nanotechnology can provide useful carriers to successfully protect and preserve enzymes, and transport them through the BBB towards brain locations. Several strategies based on targeting specific receptors on the BBB have led to nanoparticles that successfully carry sensitive molecules to the brain. Then, the main LSDs are described and a thorough review of nanotechnology strategies for brain delivery studied up to date is presented.

The new cobas® Cdiff and cobas® MRSA/SA tests were compared with conventional methods for the rapid detection of toxigenic Clostridium difficile and methicillin-resistant Staphylococcus aureus. The final concordance between cobas Cdiff Test and GDH/toxin gene screening was 97.62% and between cobas MRSA/SA Test and chromogenic culture, 91.30%, respectively., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Two dsRNAs of estimated lengths of 5 (L1) and 3.7 (L2) kpb are commonly found in strains of the basidiomycetous yeast Xanthophyllomyces dendrorhous, and the presence of virus-like particles (VLPs) have been described in some strains. Recently, two putative totiviruses (XdV-L1A and XdV-L1B) were identified from L1 dsRNA and one (XdV-L2) from L2 dsRNA in the strain UCD 67-385. In some strains, there are smaller dsRNAs (0.9-1.4 kb) that probable are satellite elements. In this work, the VLPs from several strains of X. dendrorhous, which differ in their dsRNAs content, were separated by sucrose gradient and characterized in relation to the dsRNAs and proteins that compose them. It was found that all types of dsRNAs were encapsidated into VLPs, supporting the hypothesis that the smaller dsRNAs are satellite molecules. A main protein of approx. 76 or 37 kDa composed the virions that only have the L1-dsRNA or L2-dsRNA, respectively. In the strain UCD 67-385, these both proteins were identified as viral capsid protein (CP), allow to confirm the gag predicted ORFs in XdV-L1A, XdV-L1B, and XdV-L2, with CPs of 76.6, 76.2, and 38.8 kDa, respectively. Analysis of predicted structures of CPs of XdV-L1A and XdV-L1B, showed high similitudes with the CPs of ScV-L-A and other totiviruses.

The β-fructofuranosidase Xd-INV from the yeast Xanthophyllomyces dendrorhous is the largest microbial enzyme producing neo-fructooligosaccharides (neo-FOS) known to date. It mainly synthesizes neokestose and neonystose, oligosaccharides with potentially improved prebiotic properties. The Xd-INV gene comprises an open reading frame of 1995 bp, which encodes a 665-amino acid protein. Initial N-terminal sequencing of Xd-INV pointed to a majority extracellular protein of 595 amino acids lacking the first 70 residues (potential signal peptide). Functionality of the last 1785 bp of Xd-INV gene was previously proved in Saccharomyces cerevisiae but only weak β-fructofuranosidase activity was quantified. In this study, different strategies to improve this enzyme level in a heterologous system have been used. Curiously, best results were obtained by increasing the protein N-terminus sequence in 39 amino acids, protein of 634 residues. The higher β-fructofuranosidase activity detected in this study, about 15 U/mL, was obtained using Pichia pastoris and represents an improvement of about 1500 times the level previously obtained in a heterologous organism and doubles the best level of activity obtained by the natural producer. Heterologously expressed protein was purified and characterized biochemically and kinetically. Except by its glycosylation degree (10 % lower) and thermal stability (4-5 °C lower in the 60-85 °C range), the properties of the heterologous enzyme, including ability to produce neo-FOS, remained unchanged. Interestingly, besides the neo-FOS referred before blastose was also detected (8-22 g/L) in the reaction mixtures, making Xd-INV the first yeast enzyme producing this non-conventional disaccharide reported to date.

The objective of this work is to develop a nanoplatform that can potentiate the oral administration of Δ9-tetrahidrocannabinol, a highly lipophilic active agent with very promising antiproliferative and antiemetic activities. To that aim, colloidal carriers based on the biodegradable and biocompatible poly(D,L-lactide-co-glycolide) were investigated. Such delivery systems were prepared by nanoprecipitation, and nanoparticle engineering further involved surface modification with a poly(ethylene glycol), chitosan, or poly(ethylene glycol)-chitosan shells to assure the greatest uptake by intestinal cells and to minimize protein adsorption. Characterization of the nanoplatforms included particle geometry (size and shape), electrophoretic properties (surface charge). Δ9-tetrahydrocannabinol vehiculization capabilities (loading and release), blood compatibility, and cellular uptake and cytotoxicity. Results were satisfactorily used to define the optimum engineering conditions to formulate surface modified nanoparticles for the efficient oral administration of Δ9-tetrahydrocannabinol. To the best of our knowledge, this is the first time that biocompatible polymeric nanoparticles have been formulated for Δ9-tetrahydrocannabinoldelivery., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Antegrade puncture is a routinely used technique of obtaining access to the common femoral artery to perform infrainguinal interventions. However, antegrade arterial access can be challenging in the presence of hostile, scarred groins, obesity, or a high common femoral artery bifurcation. A simple method of converting a retrograde femoral access to an antegrade catheterization using an inexpensive and universally available monofilament suture is presented., (Copyright © 2014 Society for Vascular Surgery. Published by Mosby, Inc. All rights reserved.)

Oral drug delivery is a route of choice for vaccine administration because of its noninvasive nature and thus efforts have focused on efficient delivery of vaccine antigens to mucosal sites. An effective oral vaccine delivery system must protect the antigen from degradation upon mucosal delivery, penetrate mucosal barriers, and control the release of the antigen and costimulatory and immunomodulatory agents to specific immune cells (i.e., APCs). In this paper, mannan-modified pH-responsive P(HEMA-co-MAA) nanogels were synthesized and assessed as carriers for oral vaccination. The nanogels showed pH-sensitive properties, entrapping and protecting the loaded cargo at low pH values, and triggered protein release after switching to intestinal pH values. Surface decoration with mannan as carbohydrate moieties resulted in enhanced internalization by macrophages as well as increasing the expression of relevant costimulatory molecules. These findings indicate that mannan-modified P(HEMA-co-MAA) nanogels are a promising approach to a more efficacious oral vaccination regimen.

This article aimed to produce, characterize and evaluate different surface-modified naphthalen-1-yl-(4-pentyloxynaphthalen-1-yl)methanone (CB13) loaded poly(lactic-co-glycolic acid) nanoparticles in order to improve their oral absorption and in vivo biodistribution. Plain and surface-modified PLGA nanoparticles were successfully prepared using a nanoprecipitation method. Chitosan, Eudragit RS, lecithin and vitamin E were used as surface modifying agents. The NPs were evaluated in terms of mean diameter and size distribution, zeta potential, morphology, drug loading, drug release profiles, mucoadhesive properties, in vitro cell viability and uptake and in vivo biodistribution. Mean particle size distributions in the range of 253-344 nm, spherical shape and controlled zeta potential values were observed depending on the additive employed. High values of entrapment efficiency were obtained for all the formulations. Lecithin and vitamin E modified particles showed higher release rates when compared to the rest of formulations. A clear improvement in ex vivo mucoadhesion properties was observed in the case of chitosan- and Eudragit RS-modified nanoparticles. Chitosan-poly(lactic-co-glycolic acid) nanoparticles showed the highest uptake values on Caco-2 cells. Biodistribution assays proved that most of the particles were accumulated in liver and spleen. An important goal has been achieved in this investigation: CB13, a highly lipophilic drug with low water solubility, can reach the interior of cells more efficiently when it is included in these surface-modified polymeric carriers.

Control of energy metabolism is an essential process for life. In insects, egg formation (oogenesis) and embryogenesis is dependent on stored molecules deposited by the mother or transcribed later by the zygote. In oviparous insects the egg becomes an isolated system after egg laying with all energy conversion taking place during embryogenesis. Previous studies in a few vector species showed a strong correlation of key morphogenetic events and changes in glucose metabolism. Here, we investigate glycogen and glucose metabolism in the red flour beetle Tribolium castaneum, an insect amenable to functional genomic studies. To examine the role of the key enzymes on glycogen and glucose regulation we cloned and analyzed the function of glycogen synthase kinase 3 (GSK-3) and hexokinase (HexA) genes during T. castaneum embryogenesis. Expression analysis via in situ hybridization shows that both genes are expressed only in the embryonic tissue, suggesting that embryonic and extra-embryonic cells display different metabolic activities. dsRNA adult female injection (parental RNAi) of both genes lead a reduction in egg laying and to embryonic lethality. Morphological analysis via DAPI stainings indicates that early development is impaired in Tc-GSK-3 and Tc-HexA1 RNAi embryos. Importantly, glycogen levels are upregulated after Tc-GSK-3 RNAi and glucose levels are upregulated after Tc-HexA1 RNAi, indicating that both genes control metabolism during embryogenesis and oogenesis, respectively. Altogether our results show that T. castaneum embryogenesis depends on the proper control of glucose and glycogen.

The wide-range transformation/expression platform, Xplor2, was employed for the assessment of Schwanniomyces occidentalis as a potential producer of the recombinant proteins human IFNα2a (IFNα2a) and S. occidentalis fructofuranosidase (SFfase), and its efficiency was compared to that of Arxula adeninivorans. ADE2 and URA3 genes from both yeast species were isolated, characterized and used as selection markers in combination with the IFNα2a and SFfase expression modules, which used the strong constitutive A. adeninivorans-derived TEF1 promoter. Yeast rDNA integrative expression cassettes and yeast integrative expression cassettes equipped with a selection marker and expression modules were transformed into auxotrophic S. occidentalis and A. adeninivorans strains and a quantitative comparison of the expression efficiency was made. Whilst IFNα2a was mainly accumulated extracellularly (>95 %) in A. adeninivorans, extracellular SFfase (>90 %) was detected in both yeast species. The DNA composition of the selection marker modules and expression modules, especially their open reading frame codon usage, affects auxotrophy recovery as well as protein expression. Auxotrophy recovery was only achieved with selection marker modules of the homologous gene donor yeast. The concentration of recombinant IFNα2a was fivefold higher in A. adeninivorans (1 mg L(-1)), whereas S. occidentalis accumulated 1.5- to 2-fold more SFfase (0.5 Units ml(-1)). These results demonstrate the extension of the use of the wide-range expression platform Xplor2 to another yeast species of biotechnological interest.

This article presents the potential of PLGA nanoparticles for the oral administration of drugs. Different strategies are used to improve oral absorption of these nanoparticles. These strategies are based on modification of nanoparticle surface properties. They can be achieved either by coating the nanoparticle surface with stabilizing hydrophilic bioadhesive polymers or surfactants, or by incorporating biodegradable copolymers containing a hydrophilic moiety. Some substances such as chitosan, vitamin E, methacrylates, lectins, lecithins, bile salts and RGD molecules are employed for this purpose. Of especial interest are nanoparticles production methods and, in order to improve oral bioavailability, the mechanism of each additive.

Setting: Health Department Tuberculosis (TB) Control program, Connecticut, United States, Objective: 1) To assess TB-relatedness of deaths and missed opportunities among Connecticut patients who died with TB, and 2) to identify factors associated with death., Design: The study population consisted of all persons diagnosed with TB and reported to the Connecticut TB Control Program during 2007-2009. TB Control Program records, medical records, autopsy reports and death certificates of decedents were reviewed. A tool was used to categorize TB-relatedness of deaths and identify missed opportunities in diagnosis and medical treatment among TB-related deaths. Surveillance data regarding TB survivors were used for comparison to identify factors associated with death., Results: During 2007-2009, 20/300 (7%) persons with TB died; 14 (70%) decedents had at least one medical comorbidity and 17 (85%) deaths were TB-related. Among patients who had a TB-related death, 16 (94%) had ≥1 missed opportunity identified. Excess alcohol use (risk ratio [RR] 4.4, 95% confidence interval [CI] 1.8-11.0) and age > 64 years (RR 5.7, 95%CI 2.5-13.1) were associated with death., Conclusions: The majority of deaths among Connecticut TB patients were TB-related. Missed opportunities were common. Excess alcohol use and older age might indicate a need for monitoring to prevent death.

A total of 91 wild olive accessions and 31 olive cultivars growing in the Extremadura region of central-western Spain were analyzed using morphological traits and RAPD markers. We focused on three main and 16 minor Spanish olive cultivars that are recognized as native or local to the Extremadura region. The five arbitrary 10-mer primers tested on the olive cultivars gave 67 polymorphic bands, representing 91% of the total amplification products. The number of bands per primer ranged from 9 to 18, whereas the number of polymorphic bands ranged from 8 to 17. All the cultivars could be identified by a combination of three primers (OPF-6, OPA-8, and OPK-16); four cultivar-specific markers were detected. The minor local "Jariego" and "Tempranillo" cultivars showed the most distal similarities. The resulting dendrogram, using the unweighted pair-group method with arithmetic mean clustering algorithm, depicted the pattern of relationships between the local Extremadura cultivars and the cultivars from geographically connected regions. This analysis showed a correlation between most of the minor local cultivars and the geographical origin; there was no apparent clustering according to morphological traits or fruit use of olive cultivars when these parameters were used as analysis criteria.

Objective: To evaluate the prevalence and potential associations of hearing impairment in patients 30 to 50 years old with diabetes diagnosed before age 40 years-early-onset type 2 diabetes mellitus (T2DM)., Methods: The study cohorts consisted of 46 consecutive patients with early-onset T2DM and 47 age-matched control subjects with rheumatoid arthritis. All study subjects completed clinical, serologic, and auditory assessments., Results: The patients with T2DM had a mean age of 42 ± 6 years and a mean disease duration of 11 ± 6 years. Microalbuminuria was present in 26.1%, proliferative retinopathy in 26.1%, and symptomatic peripheral neuropathy in 23.9%. The prevalence of unilateral or bilateral hearing loss was significantly higher in the patients with T2DM than in the patients with rheumatoid arthritis (21.7% versus 6.4%, respectively; P = .01). Most cases of hearing loss were mild and involved high or acute tones. After multivariate analysis with adjustment for age, there was a significant association between hearing loss and hemoglobin A1c (odds ratio, 1.3; 95% confidence interval, 1.02 to 1.81; P = .035). In the patients with T2DM, the lengthening of the brainstem response was not significantly increased; however, the wave morphologic features were abnormal and the reproducibility was poor in both ears in 11 patients (24%)., Conclusion: Patients with early-onset T2DM and poor glycemic control have an increased prevalence of subclinical hearing loss and impaired auditory brainstem responses. Hearing impairment may be an underrecognized complication of diabetes.

Schwanniomyces occidentalis β-fructofuranosidase (Ffase) is a GH32 dimeric enzyme that releases fructose from the nonreducing end of various oligosaccharides and essential storage fructans such as inulin. It also catalyzes the transfer of a fructosyl unit to an acceptor producing 6-kestose and 1-kestose, prebiotics that stimulate the growth of bacteria beneficial for human health. We report here the crystal structure of inactivated Ffase complexed with fructosylnystose and inulin, which shows the intricate net of interactions keeping the substrate tightly bound at the active site. Up to five subsites were observed, the sugar unit located at subsite +3 being recognized by interaction with the β-sandwich domain of the adjacent subunit within the dimer. This explains the high activity observed against long substrates, giving the first experimental evidence of the direct role of a GH32 β-sandwich domain in substrate binding. Crucial residues were mutated and their hydrolase/transferase (H/T) activities were fully characterized, showing the involvement of the Gln-228/Asn-254 pair in modulating the H/T ratio and the type β(2-1)/β(2-6) linkage formation. We generated Ffase mutants with new transferase activity; among them, Q228V gives almost specifically 6-kestose, whereas N254T produces a broader spectrum product including also neokestose. A model for the mechanism of the Ffase transfructosylation reaction is proposed. The results contribute to an understanding of the molecular basis regulating specificity among GH-J clan members, which represent an interesting target for rational design of enzymes, showing redesigned activities to produce tailor-made fructooligosaccharides.