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Antimicrobial resistance is responsible for an alarming number of deaths, estimated at 5 million per year. To combat priority pathogens, like Helicobacter pylori, the development of novel therapies is of utmost importance. Understanding the molecular alterations induced by medications is critical for the design of multi-targeting treatments capable of eradicating the infection and mitigating its pathogenicity. However, the application of bulk omics approaches for unraveling drug molecular mechanisms of action is limited by their inability to discriminate between target-specific modifications and off-target effects. This study introduces a multi-omics method to overcome the existing limitation. For the first time, the Proteome Integral Solubility Alteration (PISA) assay is utilized in bacteria in the PISA-Express format to link proteome solubility with different and potentially immediate responses to drug treatment, enabling us the resolution to understand target-specific modifications and off-target effects. This study introduces a comprehensive method for understanding drug mechanisms and optimizing the development of multi-targeting antimicrobial therapies.

Abstract The objective of this article is to compare the amount of intraoperative blood loss during laparoscopic myomectomy when performing bilateral transient clamping of the uterine and utero-ovarian arteries versus no intervention. It´s a randomized controlled prospective study carried out in the Department of Obstetrics and Gynecology Ramón y Cajal University Hospital and HM Montepríncipe-Sanchinarro University Hospital, Madrid, Spain, in women with fibroid uterus undergoing laparoscopic myomectomy. Eighty women diagnosed with symptomatic fibroid uterus were randomly assigned to undergo laparoscopic myomectomy without additional intervention (Group A) or temporary clamping of bilateral uterine and utero-ovarian arteries prior to laparoscopic myomectomy (Group B). Estimated blood loss, operating time, length of hospital stay, and postoperative hemoglobin values were compared in both groups. The number of fibroids removed was similar in both groups (p = 0.77). Estimated blood loss was lower in the group of patients with prior occlusion of uterine arteries (p = 0.025) without increasing operating time (p = 0.17) nor length of stay (p = 0.17). No patient had either intra or postoperative complications. Only two patients (2.5%) required blood transfusion after surgery. We conclude that temporary clamping of bilateral uterine arteries prior to laparoscopic myomectomy is a safe intervention that reduces blood loss without increasing operative time.

Helicobacter pylori infection constitutes a silent pandemic of global concern. In the last decades, the alarming increase in multidrug resistance evolved by this pathogen has led to a marked drop in the eradication rates of traditional therapies worldwide. By using a high-throughput screening strategy, in combination with in vitro DNA binding assays and antibacterial activity testing, we identified a battery of novel drug-like HsrA inhibitors with MIC values ranging from 0.031 to 4 mg/L against several antibiotic-resistant strains of H. pylori, and minor effects against both Gram-negative and Gram-positive species of human microbiota. The most potent anti-H. pylori candidate demonstrated a high therapeutic index, an additive effect in combination with metronidazole and clarithromycin as well as a strong antimicrobial action against Campylobacter jejuni, another clinically relevant pathogen of phylum Campylobacterota. Transcriptomic analysis suggests that the in vivo inhibition of HsrA triggers lethal global disturbances in H. pylori physiology including the arrest of protein biosynthesis, malfunction of respiratory chain, detriment in ATP generation, and oxidative stress. The novel drug-like HsrA inhibitors described here constitute valuable candidates to a new family of narrow-spectrum antibiotics that allow overcoming the current resistome, protecting from dysbiosis, and increasing therapeutic options for novel personalized treatments against H. pylori.

Objectives: The rise of antibiotic-resistant Streptococcus pneumoniae (Sp) poses a significant global health threat, urging the quest for novel antimicrobial solutions. We have discovered that the human hormone l-thyroxine has antibacterial properties. In order to explore its drugability we perform here the characterization of a series of l-thyroxine analogues and describe the structural determinants influencing their antibacterial efficacy. Method: We performed a high-throughput screening of a library of compounds approved for use in humans, complemented with ITC assays on purified Sp-flavodoxin, to pinpoint molecules binding to this protein. Antimicrobial in vitro susceptibility assays of the hit compound (l-thyroxine) as well as of 13 l-thyroxine analogues were done against a panel of Gram-positive and Gram-negative bacteria. Toxicity of compounds on HepG2 cells was also assessed. A combined structure-activity and computational docking analysis was carried out to uncover functional groups crucial for the antimicrobial potency of these compounds. Results: Human l-thyroxine binds to Sp-flavodoxin, forming a 1:1 complex of low micromolar Kd. While l-thyroxine specifically inhibited Sp growth, some derivatives displayed activity against other Gram-positive bacteria like Staphylococcus aureus and Enterococcus faecalis, while remaining inactive against Gram-negative pathogens. Neither l-thyroxine nor some selected derivatives exhibited toxicity to HepG2 cells. Conclusions: l-thyroxine derivatives targeting bacterial flavodoxins represent a new and promising class of antimicrobials.

Inherited mutations in the CHEK2 gene have been associated with an increased lifetime risk of developing breast cancer (BC). We aim to identify in the study population the prevalence of mutations in the CHEK2 gene in diagnosed BC patients, evaluate the phenotypic characteristics of the tumor and family history, and predict the deleteriousness of the variants of uncertain significance (VUS). A genetic study was performed, from May 2016 to April 2020, in 396 patients diagnosed with BC at the University Hospital Lozano Blesa of Zaragoza, Spain. Patients with a genetic variant in the CHEK2 gene were selected for the study. We performed a descriptive analysis of the clinical variables, a bibliographic review of the variants, and a cosegregation study when possible. Moreover, an in-depth bioinformatics analysis of CHEK2 VUS was carried out. We identified nine genetic variants in the CHEK2 gene in 10 patients (two pathogenic variants and seven VUS). This supposes a prevalence of 0.75% and 1.77%, respectively. In all cases, there was a family history of BC in first- and/or second-degree relatives. We carried out a cosegregation study in two families, being positive in one of them. The bioinformatics analyses predicted the pathogenicity of six of the VUS. In conclusion, CHEK2 mutations have been associated with an increased risk for BC. This risk is well-established for foundation variants. However, the risk assessment for other variants is unclear. The incorporation of bioinformatics analysis provided supporting evidence of the pathogenicity of VUS.

ABSTRACT Antimicrobial resistance of Helicobacter pylori (Hp) threatens currently available treatment regimens and prompts the development of antimicrobial drugs against new bacterial targets. One such class of drugs is Hp-flavodoxin (Hp-fld) inhibitors, which target an essential metabolic pathway in Hp. The aim of this study was to characterize the effects of these inhibitors against Hp over time when used alone or combined with conventional antibiotics. Four benzoxadiazol-based fld inhibitors were initially tested for their in vitro potency, with compound IV displaying the highest efficacy. This compound was then combined with clarithromycin, metronidazole, amoxicillin, levofloxacin, and rifampicin in further experiments. Checkerboard assays indicated that compound IV exhibited additive activity when combined with these antibiotics. The study further investigated the time course of antibacterial effects by exposing a high inoculum of Hp to compound IV and each antibiotic, alone or in combination. At just four times the minimum inhibitory concentration, compound IV demonstrated bactericidal effects within 6 h. However, the regrowth of bacteria occurred between 24 and 48 h of exposure. Similar patterns of killing followed by regrowth were observed for conventional antibiotics alone. However, the addition of compound IV to the antibiotics clearly limited regrowth over 72 h. These findings suggest that compound IV can effectively eliminate spontaneous mutants that are resistant to conventional antibiotics or prevent new mutations during drug exposure. Hp-fld inhibitors, such as compound IV, hold promise as new drugs to be integrated into Hp infection treatment, potentially reducing the development of resistance and shortening the duration of treatment. IMPORTANCE The antimicrobial resistance of Helicobacter pylori (Hp) currently poses a threat to available treatment regimens. Developing antimicrobial drugs targeting new bacterial targets is crucial, and one such class of drugs includes Hp-flavodoxin (Hp-fld) inhibitors that target an essential metabolic pathway in Hp. Our study demonstrated that combining these new drugs with conventional antibiotics used for Hp infection treatment prevented the regrowth observed with drugs used alone. Hp-fld inhibitors show promise as new drugs to be incorporated into the treatment of Hp infection, potentially reducing the development of resistance and shortening the treatment duration.

This review focuses on the role of small extracellular vesicles in the pathophysiological mechanisms of retinal degenerative diseases. Many of these mechanisms are related to or modulated by the oxidative burden of retinal cells. It has been recently demonstrated that cellular communication in the retina involves extracellular vesicles and that their rate of release and cargo features might be affected by the cellular environment, and in some instances, they might also be mediated by autophagy. The fate of these vesicles is diverse: they could end up in circulation being used as markers, or target neighbor cells modulating gene and protein expression, or eventually, in angiogenesis. Neovascularization in the retina promotes vision loss in diseases such as diabetic retinopathy and age-related macular degeneration. The importance of micro RNAs, either as small extracellular vesicles’ cargo or free circulating, in the regulation of retinal angiogenesis is also discussed.

Exosomes or small extracellular vesicles (sEVs) represent a pivotal component in intercellular communication, carrying a diverse array of biomolecules. Several factors can affect sEVs release dynamics, as occurs in hyperglycemia or inflammation. In fact, sEVs release has been associated with the promotion of physio-pathological processes. Among the sEVs cargo, microRNAs play an essential role in cell-to-cell regulation. More concretely, miR-205-5p is related to angiogenesis and cell proliferation. The aim of this study is to understand the specific role of sEVs containing miR-205-5p under high glucose conditions. ARPE-19 cells were cultured with high glucose (HG) for 5 days. sEVs were isolated and characterized. sEVs from ARPE-19 were used for angiogenesis and cell proliferation. HG increased sEVs release but downregulated miR-205-5p cargo expression compared to the control. sEVs from HG-treated ARPE-19 cells promoted tube formation and migration processes. In contrast, miR-205-5p overexpression (by mimic transfection) decreased angiogenesis and cell migration. Our results demonstrate how ARPE-19 cells respond to HG challenge by increasing sEVs with weak miR-205-5p cargo. The absence of this miRNA in sEVs is enough to promote angiogenesis. In contrast, restoring sEVs-miR-205-5p levels decreased it. These findings open new possibilities in sEVs-based therapies containing miR-205-5p against angiogenesis.

Hexachlorocyclohexane (HCH) waste isomers from lindane production are the largest single POPs legacy, with an estimated 4.8 to 7.4 million tonnes of disposed waste. The largest part of this waste – 1.8 to 3 million tonnes – was disposed in Europe, where most producers were located. This paper provides a short overview of projects supported by the European Union (EU) to address this waste legacy and to implement the Stockholm Convention for this group of POPs with associated protection of soil, ecosystems and human health. We report here particularly on the results of a project financed by the EU called the “HCH in EU project”, which aimed to develop a systematic inventory of sites where HCH was handled and potentially resulted in contamination. The compiled information provide guidance for competent authorities to further assess their national HCH inventory and to further develop a strategy to address this large POP legacy in future. The systematic inventory revealed that there were at least 299 sites where HCH was handled. These sites include 54 former production sites, 76 pesticide processing plants that used lindane, 59 uncontrolled HCH waste isomer deposits, 29 landfills with HCH waste, 34 former or current storage sites for stocks of obsolete pesticides including technical HCH or lindane, and 16 HCH treatment or disposal sites. Additionally, at 31 of the sites lindane/technical HCH was used in applications with significant risk of soil pollution, such as wood treatment. The number of sites in this latter category is likely higher and will need further assessment. In addition to this inventory, the “HCH in EU project” produced detailed country reports, a guidance document for how to find potentially HCH-impacted sites, and a strategy document for implementing the sustainable management of these sites EU-wide, with proposed actions at the EU, country, and site level. Furthermore, the project has facilitated information exchange and – together with other related EU projects – has led to sharing information and best practices among member states and to establishing a network of authorities and other stakeholders working on the lindane/HCH waste legacy. This collaboration will facilitate a more systematic and better coordinated process to further assess, secure, and remediate the large HCH waste legacy and reduce and control lindane/HCH releases in the EU and possibly beyond. Such a coordinated effort and exchange of information for inventorying and managing contaminated sites might also be useful for other POPs such as PFOS/PFOA or dioxins.

Protein stability is a requisite for most biotechnological and medical applications of proteins. As natural proteins tend to suffer from a low conformational stability ex vivo, great efforts have been devoted toward increasing their stability through rational design and engineering of appropriate mutations. Unfortunately, even the best currently used predictors fail to compute the stability of protein variants with sufficient accuracy and their usefulness as tools to guide the rational stabilisation of proteins is limited. We present here Protposer, a protein stabilising tool based on a different approach. Instead of quantifying changes in stability, Protposer uses structure- and sequence-based screening modules to nominate candidate mutations for subsequent evaluation by a logistic regression model, carefully trained to avoid overfitting. Thus, Protposer analyses PDB files in search for stabilization opportunities and provides a ranked list of promising mutations with their estimated success rates (eSR), their probabilities of being stabilising by at least 0.5 kcal/mol. The agreement between eSRs and actual positive predictive values (PPV) on external datasets of mutations is excellent. When Protposer is used with its Optimal kappa selection threshold, its PPV is above 0.7. Even with less stringent thresholds, Protposer largely outperforms FoldX, Rosetta and PoPMusiC. Indicating the PDB file of the protein suffices to obtain a ranked list of mutations, their eSRs and hints on the likely source of the stabilization expected. Protposer is a distinct, straightforward and highly successful tool to design protein stabilising mutations, and it is freely available for academic use at http://webapps.bifi.es/the-protposer.

Abstract Background Higher education is responsible for providing education that meets international benchmarks relevant to the needs of the international community. Due to the increase of digital tools in higher education, the possibility of sharing learning resources across nations has expanded. In the current project, a Norwegian university invited universities in Spain and the United Kingdom to adapt and translate e-learning resources originally developed for Norwegian nursing students for use within their respective Bachelor in Nursing programmes. Aim The aim of the current study was to gain insights into the usability and value for learning of e-compendiums shared and implemented across three European universities. Methods The study adopted a descriptive cross-sectional design and included nursing students from the University of Nottingham, Valencia Catholic University, and the University of Stavanger. Data were collected in Autumn 2017 through a questionnaire adapted from the validated “Centre for Excellence in Teaching and Learning Reusable Learning Object evaluation questionnaire” The questionnaire consisted of 19 items that included two aspects: e-compendiums’ value for learning and e-compendiums’ usability. The different study sites were compared using a binary logistic regression analysis. Subgroups of students were compared based on their gender and age. Results A total of 480 nursing students participated in the study. The e -compendiums were overall positively rated, especially for reinforcing and retaining knowledge. Compared to the students from the University of Stavanger, students from Valencia Catholic University rated the e-compendiums more positively in most aspects of learning. Students from University of Nottingham found the e-compendiums to be more important for learning engagement compared to students at the Norwegian study site, and no differences were found in any other aspects of learning. Younger students rated the interactivity and visual components as more important compared to older students. Conclusions Students from the University of Nottingham and Valencia Catholic University seem to accept the e-compendiums despite the fact that they were originally developed for use in another country. We argue that, when sharing e-learning resources across countries, an adaptation and translation process that includes a multicultural and multidisciplinary perspective should be carried out.

Background: Ultrasound features help to differentiate benign from malignant masses, and some of them are included in the ultrasound (US) scores. The main aim of this work is to describe the ultrasound features of certain adnexal masses of difficult classification and to analyse them according to the most frequently used US scores. Methods: Retrospective studies of adnexal lesions are difficult to classify by US scores in women undergoing surgery. Ultrasound characteristics were analysed, and masses were classified according to the Subjective Assessment of the ultrasonographer (SA) and other US scores (IOTA Simple Rules Risk Assessment-SRRA, ADNEX model with and without CA125 and O-RADS). Results: A total of 133 adnexal masses were studied (benign: 66.2%, n:88; malignant: 33.8%, n:45) in a sample of women with mean age 56.5 ± 7.8 years. Malignant lesions were identified by SA in all cases. Borderline ovarian tumors (n:13) were not always detected by some US scores (SRRA: 76.9%, ADNEX model without and with CA125: 76.9% and 84.6%) nor were serous carcinoma (n:19) (SRRA: 89.5%), clear cell carcinoma (n:9) (SRRA: 66.7%) or endometrioid carcinoma (n:4) (ADNEX model without CA125: 75.0%). While most teratomas and serous cystadenomas have been correctly differentiated, other benign lesions were misclassified because of the presence of solid areas or papillae. Fibromas (n:13) were better identified by SA (23.1% malignancy), but worse with the other US scores (SRRA: 69.2%, ADNEX model without and with CA125: 84.6% and 69.2%, O-RADS: 53.8%). Cystoadenofibromas (n:10) were difficult to distinguish from malignant masses via all scores except SRRA (SA: 70.0%, SRRA: 20.0%, ADNEX model without and with CA125: 60.0% and 50.0%, O-RADS: 90.0%). Mucinous cystadenomas (n:12) were misdiagnosed as malignant in more than 15% of the cases in all US scores (SA: 33.3%, SRRA: 16.7%, ADNEX model without and with CA125: 16.7% and 16.7%, O-RADS:41.7%). Brenner tumors are also difficult to classify using all scores. Conclusion: Some malignant masses (borderline ovarian tumors, serous carcinoma, clear cell carcinoma, endometrioid carcinomas) are not always detected by US scores. Fibromas, cystoadenofibromas, some mucinous cystadenomas and Brenner tumors may present solid components/papillae that may induce confusion with malignant lesions. Most teratomas and serous cystadenomas are usually correctly classified.

Background: Several ultrasound (US) features help ultrasound experts in the classification of benign vs. malignant adnexal masses. US scores serve in this differentiation, but they all have misdiagnoses. The main objective of this study is to evaluate what ultrasound characteristics are associated with malignancy influencing ultrasound scores. Methods: This is a retrospective analysis of ultrasound features of adnexal lesions of women managed surgically. Ultrasound characteristics were analyzed, and masses were classified by subjective assessment of the ultrasonographer (SA) and other ultrasound scores (IOTA Simple Rules Risk Assessment SRRA, ADNEX model, and O-RADS). Results: Of a total of 187 adnexal masses studied, 134 were benign (71.7%) and 53 were malignant (28.3%). SA, IOTA SRRA, ADNEX model with or without CA125 and O-RADS had high levels of sensitivity (93.9%, 81.1%, 94.3%, 88.7%, 98.1%) but lower specificity (80.2%, 82.1%, 82.8%, 77.6%, 73.1%) with similar AUC (0.87, 0.87, 0.92, 0.90, 0.86). Ultrasound features significantly related with malignancy were the presence of irregular contour, absence of acoustic shadowing, vascularized solid areas, ≥1 papillae, vascularized septum, and moderate-severe ascites. Conclusion: IOTA SRRA, ADNEX model, and O-RADS can help in the classification of benign and malignant masses. Certain ultrasound characteristics studied in ultrasound scores are associated with malignancy.

AimThe functional prognosis of patients after coma following either cardiac arrest (CA) or acute structural brain injury (ABI) is often uncertain. These patients are associated with high mortality and disability. N20 and N70 somatosensory evoked potentials (SSEP) are used to predict prognosis. We evaluated the utility of SSEP (N20–N70) as an early indicator of long-term prognosis in these patients.MethodsThis was a retrospective cohort study of patients (n = 120) admitted to the intensive care unit (ICU) with a diagnosis of coma after CA (n = 60) or ABI (n = 60). An SSEP study was performed, including N20 and N70 at 24–72 h, after coma onset. Functional recovery was assessed 6–12 months later using the modified Glasgow scale (mGS). The study was approved by our local research ethics committee.ResultsIn the CA and ABI groups, the absence of N20 (36% of CA patients and 41% of ABI patients; specificity = 100%) or N70 (68% of CA patients and 78% of ABI patients) was a strong indicator of poor outcome. Conversely, the presence of N70 was an indicator of a good outcome (AC: specificity = 84.2%, sensitivity = 92.7%; ABI: specificity = 64.2% sensitivity = 91.3%).ConclusionSomatosensory evoked potentials are useful early prognostic markers with high specificity (N20) and sensitivity (N70). Moreover, N70 has additional potential value for improving the prediction of good long-term functional outcomes.Clinical Trial Registration:[https://clinicaltrials.gov/], identifier [2018/01/001].

Subjective ultrasound assessment by an expert examiner is meant to be the best option for the differentiation between benign and malignant adnexal masses. Different ultrasound scores can help in the classification, but whether one of them is significantly better than others is still a matter of debate. The main aim of this work is to compare the diagnostic performance of some of these scores in the evaluation of adnexal masses in the same set of patients. This is a retrospective study of a consecutive series of women diagnosed as having a persistent adnexal mass and managed surgically. Ultrasound characteristics were analyzed according to IOTA criteria. Masses were classified according to the subjective impression of the sonographer and other ultrasound scores (IOTA simple rules -SR-, IOTA simple rules risk assessment -SRRA-, O-RADS classification, and ADNEX model -with and without CA125 value-). A total of 122 women were included. Sixty-two women were postmenopausal (50.8%). Eighty-one women had a benign mass (66.4%), and 41 (33.6%) had a malignant tumor. The sensitivity of subjective assessment, IOTA SR, IOTA SRRA, and ADNEX model with or without CA125 and O-RADS was 87.8%, 66.7%, 78.1%, 95.1%, 87.8%, and 90.2%, respectively. The specificity for these approaches was 69.1%, 89.2%, 72.8%, 74.1%, 67.9%, and 60.5%, respectively. All methods with similar AUC (0.81, 0.78, 0.80, 0.88, 0.84, and 0.75, respectively). We concluded that IOTA SR, IOTA SRRA, and ADNEX models with or without CA125 and O-RADS can help in the differentiation of benign and malignant masses, and their performance is similar to the subjective assessment of an experienced sonographer.

Background: The use of transvaginal ultrasound guided biopsy and puncture of pelvic lesions is a minimally invasive technique that allows for accurate diagnosis. It has many advantages compared to other more invasive (lower complication rate) or non-invasive techniques (accurate diagnosis). Furthermore, it offers greater availability, it does not radiate, enables the study of pelvic masses accessible vaginally with ultrasound control in real time, and it is possible to use the colour Doppler avoiding puncturing large vessels among others. The main aim of the work is to describe a standardized ambulatory technique and to determine its usefulness. Methods: This is a retrospective study of ultrasound transvaginal punctures (core needle biopsies and cytologies) and drainages of pelvic lesions performed on an outpatient basis during the last two years. The punctures were made with local anesthesia, under transvaginal ultrasound guidance with an automatic or semi-automatic 18G biopsy needle with a length of 20–25 cm and a penetration depth of 12 or 22 mm. The material obtained was sent for anatomopathological, cytological and/or microbiological study if necessary. Results: A total of 42 women were recruited in two centers. Fifty procedures (nine punctures, seven drains, and 34 biopsies) were performed. In five cases the punction and drain provided clinical relief in benign pelvic masses. Regarding material of the biopsies performed, 15 were vaginal in women previously histerectomized, finding 10 carcinomas, eight were ovarian tumours in advanced stages or peritoneal carcinomatosis obtaining the appropriate histology in each case, seven were suspicious cervical biopsies finding carcinomas in five of them, three were myometrial biopsies including one breast carcinoma metastasis in the miometrium and a benign placental nodule, and a periurethral biopsy was performed on a woman with a history of endometrial cancer confirming recurrence. The pathological diagnosis was satisfactory in all cases, confirming the nature of the lesion (25 malignant—ten vaginal recurrences of previous gynaecological cancers, eight cases of primary ovarian/peritoneal carcinoma, four new diagnosis of cervical malignant masses, one cervical metastasis of lymphoma, one periurethral recurrence of endometrial carcinoma and one recurrence of breast cancer in the myometrium—and 23 benign). The tolerance was excellent and no complications were detected. Conclusion: The ambulatory ultrasound transvaginal puncture and drainage technique is useful for obtaining a sample for pathological and microbiological diagnosis with excellent tolerance that can be used to rule out the recurrence of malignant lesions or progression of the disease, diagnose masses not accessible to gynecological exploration (vaginal vault, myometrium or cervix) and for early histologic diagnosis in cases of advanced peritoneal carcinomatosis or ovarian carcinoma as well as drainage and cytological study of cystic pelvic masses.

Background: Tubal patency testing constitutes an essential part of infertility work-up. Hysterosalpingo-foam-sonography (HyFoSy) is currently one of the best tests for assessing tubal patency. The objective of our study was to evaluate the post-procedure rate of spontaneous pregnancy among infertile women submitted for an HyFoSy exam with ExEm® foam and the factors associated with this. Methods: Multicenter, prospective, observational study performed at six Spanish centers for gynecologic sonography and human reproduction. From December 2015 to June 2021, 799 infertile women underwent HyFoSy registration consecutively. The patients’ information was collected from their medical records. Multivariable regression analyses were performed, controlling for age, etiology, and time of sterility. The main outcome was to measure post-procedure spontaneous pregnancy rates and the factors associated with the achievement of pregnancy. Results: 201 (26.5%) women got spontaneous conception (SC group), whereas 557 (73.5%) women did not get pregnant (non-spontaneous conception group, NSC). The median time for reaching SC after HyFoSy was 4 months (CI 95% 3.1–4.9), 18.9% of them occurring the same month of the procedure. Couples with less than 18 months of infertility were 93% more likely to get pregnant after HyFoSy (OR 1.93, 95% CI 1.34–2.81; p < 0.001); SC were two times more frequent in women under 35 years with unexplained infertility (OR 2.22, 95% CI 1.07–4.65; P0.033). Conclusion: After HyFoSy, one in four patients got pregnant within the next twelve months. Couples with shorter infertility time, unexplained infertility, and women under 35 years are more likely to achieve SC after HyFoSy.

Yes, we are made of proteins, and yes, we can profit from them [...]

Extracellular vesicles are released from cells under diverse conditions. Widely studied in cancer, they are associated with different diseases playing major roles. Recent reports indicate that oxidative damage promotes the release of small extracellular vesicle (sEVs) from the retinal pigment epithelium (RPE), with an angiogenic outcome and changes in micro-RNA (miRNA) levels. The aim of this study was to determine the role of the miRNA miR-302a-3p, included within RPE-released sEVs, as an angiogenic regulator in cultures of endothelial cells (HUVEC). ARPE-19 cell cultures, treated with H2O2 to cause an oxidative insult, were transfected with a miR-302a-3p mimic. Later, sEVs from the medium were isolated and added into HUVEC or ARPE-19 cultures. sEVs from ARPE-19 cells under oxidative damage presented a decrease of miR-302a-3p levels and exhibited proangiogenic properties. In contrast, sEVs from miR-302a-3p-mimic transfected cells resulted in control angiogenic levels. The results herein indicate that miR-302a-3p contained in sEVs can modify VEGFA mRNA expression levels as part of its antiangiogenic features.

Phenylketonuria (PKU) is a rare metabolic disease caused by variations in a human gene, PAH, encoding phenylalanine hydroxylase (PAH), and the enzyme converting the essential amino acid phenylalanine into tyrosine. Many PKU-causing variations compromise the conformational stability of the encoded enzyme, decreasing or abolishing its catalytic activity, and leading to an elevated concentration of phenylalanine in the blood, which is neurotoxic. Several therapeutic approaches have been developed to treat the more severe manifestations of the disorder, but they are either not entirely effective or difficult to adhere to throughout life. In a search for novel pharmacological chaperones to treat PKU, a lead compound was discovered (compound IV) that exhibited promising in vitro and in vivo chaperoning activity on PAH. The structure of the PAH-IV complex has been reported. Here, using alchemical free energy calculations (AFEC) on the structure of the PAH-IV complex, we design a new generation of compound IV-analogues with a higher affinity for the enzyme. Seventeen novel analogues were synthesized, and thermal shift and isothermal titration calorimetry (ITC) assays were performed to experimentally evaluate their stabilizing effect and their affinity for the enzyme. Most of the new derivatives bind to PAH tighter than lead compound IV and induce a greater thermostabilization of the enzyme upon binding. Importantly, the correspondence between the calculated alchemical binding free energies and the experimentally determined ΔΔGb values is excellent, which supports the use of AFEC to design pharmacological chaperones to treat PKU using the X-ray structure of their complexes with the target PAH enzyme.

Synucleinopathies are a group of disorders characterized by the accumulation of α-Synuclein amyloid inclusions in the brain. Preventing α-Synuclein aggregation is challenging because of the disordered nature of the protein and the stochastic nature of fibrillogenesis, but, at the same time, it is a promising approach for therapeutic intervention in these pathologies. A high-throughput screening initiative allowed us to discover ZPDm, the smallest active molecule in a library of more than 14.000 compounds. Although the ZPDm structure is highly related to that of the previously described ZPD-2 aggregation inhibitor, we show here that their mechanisms of action are entirely different. ZPDm inhibits the aggregation of wild-type, A30P, and H50Q α-Synuclein variants in vitro and interferes with α-Synuclein seeded aggregation in protein misfolding cyclic amplification assays. However, ZPDm distinctive feature is its strong potency to dismantle preformed α-Synuclein amyloid fibrils. Studies in a Caenorhabditis elegans model of Parkinson’s Disease, prove that these in vitro properties are translated into a significant reduction in the accumulation of α-Synuclein inclusions in ZPDm treated animals. Together with previous data, the present work illustrates how different chemical groups on top of a common molecular scaffold can result in divergent but complementary anti-amyloid activities.

La obesidad es un problema de salud pública, que viene en aumento a nivel mundial. Está relacionada con numerosas enfermedades como son la hipertensión arterial, diabetes mellitus tipo 2, dislipemia y apnea obstructiva del sueño. El objetivo del estudio fue determinar la prevalencia de sobrepeso y obesidad en pacientes internados en el Hospital de Clínicas y su asociación con comorbilidades asociados a las mismas. Realizamos un estudio observacional, transversal en 117 pacientes adultos internados. Se realizó un breve cuestionario de comorbilidades. Las variables medidas fueron peso, talla y perímetro abdominal. Se calculó el índice de masa corporal y se definió sobrepeso (25-29.9 kg/m2) y obesidad (≥30-kg/m2). Se encontró una prevalencia de obesidad de 30,8% y de sobrepeso de 33,5%, sin diferencias significativas entre los sexos. Asimismo, encontramos un alto porcentaje de pacientes con sedentarismo. Se halló una relación positiva entre índice de masa corporal alto y presencia de comorbilidades: hipertensión arterial (p

α-Synuclein (α-Syn) forms toxic intracellular protein inclusions and transmissible amyloid structures in Parkinson’s disease (PD). Preventing α-Syn self-assembly has become one of the most promising approaches in the search for disease-modifying treatments for this neurodegenerative disorder. Here, we describe the capacity of a small molecule (ZPD-2), identified after a high-throughput screening, to inhibit α-Syn aggregation. ZPD-2 inhibits the aggregation of wild-type α-Syn and the A30P and H50Q familial variants in vitro at substoichiometric compound:protein ratios. In addition, the molecule prevents the spreading of α-Syn seeds in protein misfolding cyclic amplification assays. ZPD-2 is active against different α-Syn strains and blocks their seeded polymerization. Treating with ZPD-2 two different PD Caenorhabditis elegans models that express α-Syn either in muscle or in dopaminergic (DA) neurons substantially reduces the number of α-Syn inclusions and decreases synuclein-induced DA neurons degeneration. Overall, ZPD-2 is a hit compound worth to be explored in order to develop lead molecules for therapeutic intervention in PD.

Antimicrobial resistant (AMR) bacteria constitute a global health concern. Helicobacter pylori is a Gram-negative bacterium that infects about half of the human population and is a major cause of peptic ulcer disease and gastric cancer. Increasing resistance to triple and quadruple H. pylori eradication therapies poses great challenges and urges the development of novel, ideally narrow spectrum, antimicrobials targeting H. pylori. Here, we describe the antimicrobial spectrum of a family of nitrobenzoxadiazol-based antimicrobials initially discovered as inhibitors of flavodoxin: an essential H. pylori protein. Two groups of inhibitors are described. One group is formed by narrow-spectrum compounds, highly specific for H. pylori, but ineffective against enterohepatic Helicobacter species and other Gram-negative or Gram-positive bacteria. The second group includes extended-spectrum antimicrobials additionally targeting Gram-positive bacteria, the Gram-negative Campylobacter jejuni, and most Helicobacter species, but not affecting other Gram-negative pathogens. To identify the binding site of the inhibitors in the flavodoxin structure, several H. pylori-flavodoxin variants have been engineered and tested using isothermal titration calorimetry. An initial study of the inhibitors capacity to generate resistances and of their synergism with antimicrobials commonly used in H. pylori eradication therapies is described. The narrow-spectrum inhibitors, which are expected to affect the microbiota less dramatically than current antimicrobial drugs, offer an opportunity to develop new and specific H. pylori eradication combinations to deal with AMR in H. pylori. On the other hand, the extended-spectrum inhibitors constitute a new family of promising antimicrobials, with a potential use against AMR Gram-positive bacterial pathogens.

Human phenylalanine hydroxylase (PAH) is a metabolic enzyme involved in the catabolism of L-Phe in liver. Loss of conformational stability and decreased enzymatic activity in PAH variants result in the autosomal recessive disorder phenylketonuria (PKU), characterized by developmental and psychological problems if not treated early. One current therapeutic approach to treat PKU is based on pharmacological chaperones (PCs), small molecules that can displace the folding equilibrium of unstable PAH variants toward the native state, thereby rescuing the physiological function of the enzyme. Understanding the PAH folding equilibrium is essential to develop new PCs for different forms of the disease. We investigate here the urea and the thermal-induced denaturation of full-length PAH and of a truncated form lacking the regulatory and the tetramerization domains. For either protein construction, two distinct transitions are seen in chemical denaturation followed by fluorescence emission, indicating the accumulation of equilibrium unfolding intermediates where the catalytic domains are partly unfolded and dissociated from each other. According to analytical centrifugation, the chemical denaturation intermediates of either construction are not well-defined species but highly polydisperse ensembles of protein aggregates. On the other hand, each protein construction similarly shows two transitions in thermal denaturation measured by fluorescence or differential scanning calorimetry, also indicating the accumulation of equilibrium unfolding intermediates. The similar temperatures of mid denaturation of the two constructions, together with their apparent lack of response to protein concentration, indicate the catalytic domains are unfolded in the full-length PAH thermal intermediate, where they remain associated. That the catalytic domain unfolds in the first thermal transition is relevant for the choice of PCs identified in high throughput screening of chemical libraries using differential scanning fluorimetry.

Objective: Persistent right umbilical vein (PRUV) is a vascular anomaly where the right umbilical vein remains as the only conduit that returns oxygenated blood to the fetus. It has classically been described as associated with numerous defects. We distinguish the intrahepatic variant (better prognosis) and the extrahepatic variant (associated with worse prognosis). The objective of this study was to compare rates of congenital malformations in fetuses with intrahepatic PRUV (I-PRUV) versus singleton pregnancies without risk factors. Materials and Methods: A multicenter, crossover design, comparative study was performed between 2003 and 2013 on fetuses diagnosed with I-PRUV (n=56), and singleton pregnancies without congenital malformation risk factors (n=4050). Results: Fifty-six cases of I-PRUV were diagnosed (incidence 1:770). A statistically significant association between I-PRUV and the presence of congenital malformations (odds ratio 4.321; 95% confidence interval 2.15–8.69) was found. This positive association was only observed with genitourinary malformations (odds ratio 3.038; 95% confidence interval 1.08–8.56). Conclusion: Our rate of malformations associated with I-PRUV (17.9%) is similar to previously published rates. I-PRUV has shown a significant increase in the rate of associated malformations, although this association has only been found to be statistically significant in the genitourinary system. Noteworthy is the fact that this comparative study has not pointed to a significant increase in the congenital heart malformation rate. Diagnosis of isolated I-PRUV does not carry a worse prognosis.

Passive flow control devices are included in the design of wind turbine blades in order to obtain better performance and reduce loads without consuming any external energy. Vortex Generators are one of the most popular flow control devices, whose main objective is to delay the flow separation and increase the maximum lift coefficient. Computational Fluid Dynamics (CFD) simulations of a Vortex Generator (VG) on a flat plate in negligible streamwise pressure gradient conditions with the fully-resolved mesh model and the cell-set model using Large Eddy Simulation (LES) and Reynolds-Averaged Navier-Stokes (RANS) were carried out, with the objective of evaluating the accuracy of the cell-set model taking the fully-resolved mesh model as benchmark. The implementation of the cell-set model entailed a considerable reduction of the number of cells, which entailed saving simulation time and resources. The coherent structures, vortex path, wall shear stress and size, strength and velocity profiles of the primary vortex have been analyzed. The results show good agreements between the fully-resolved mesh model and the cell-set mode with RANS in all the analyzed parameters. With LES, acceptable results were obtained in terms of coherent structures, vortex path and wall shear stress, but slight differences between models are visible in the size, strength and velocity profiles of the primary vortex. As this is considered the first application of the cell-set model on VGs, further research is proposed, since the implementation of the cell-set model can represent an advantage over the fully-resolved mesh model.

The retinal pigment epithelium (RPE) plays a key role in retinal health, being essential for the protection against reactive oxygen species (ROS). Nevertheless, excessive oxidative stress can induce RPE dysfunction, promoting visual loss. Our aim is to clarify the possible implication of CYP2E1 in ethanol (EtOH)-induced oxidative stress in RPE alterations. Despite the increase in the levels of ROS, measured by fluorescence probes, the RPE cells exposed to the lowest EtOH concentrations were able to maintain cell survival, measured by the Cell Proliferation Kit II (XTT). However, EtOH-induced oxidative stress modified inflammation and angiogenesis biomarkers, analyzed by proteome array, ELISA, qPCR and Western blot. The highest EtOH concentration used stimulated a large increase in ROS levels, upregulating the cytochrome P450-2E1 (CYP2E1) and promoting cell death. The use of antioxidants such as N-acetylcysteine (NAC) and diallyl sulfide (DAS), which is also a CYP2E1 inhibitor, reverted cell death and oxidative stress, modulating also the upstream angiogenesis and inflammation regulators. Because oxidative stress plays a central role in most frequent ocular diseases, the results herein support the proposal that CYP2E1 upregulation could aggravate retinal degeneration, especially in those patients with high baseline oxidative stress levels due to their ocular pathology and should be considered as a risk factor.

Helicobacter pylori is considered the most prevalent bacterial pathogen in humans. The increasing antibiotic resistance evolved by this microorganism has raised alarm bells worldwide due to the significant reduction in the eradication rates of traditional standard therapies. A major challenge in this antibiotic resistance crisis is the identification of novel microbial targets whose inhibitors can overcome the currently circulating resistome. In the present study, we have validated the use of the essential response regulator ArsR as a novel and promising therapeutic target against H. pylori infections. A high-throughput screening of a repurposing chemical library using a fluorescence-based thermal shift assay identified several ArsR binders. At least four of these low-molecular weight compounds noticeably inhibited the DNA binding activity of ArsR and showed bactericidal effects against antibiotic-resistant strains of H. pylori. Among the ArsR inhibitors, a human secondary bile acid, lithocholic acid, quickly destroyed H. pylori cells and exhibited partial synergistic action in combination with clarithromycin or levofloxacin, while the antimicrobial effect of this compound against representative members of the normal human microbiota such as Escherichia coli and Staphylococcus epidermidis appeared irrelevant. Our results enhance the battery of novel therapeutic tools against refractory infections caused by multidrug-resistant H. pylori strains.

Flavodoxins are small soluble electron transfer proteins widely present in bacteria and absent in vertebrates. Flavodoxins participate in different metabolic pathways and, in some bacteria, they have been shown to be essential proteins representing promising therapeutic targets to fight bacterial infections. Using purified flavodoxin and chemical libraries, leads can be identified that block flavodoxin function and act as bactericidal molecules, as it has been demonstrated for Helicobacter pylori (Hp), the most prevalent human gastric pathogen. Increasing antimicrobial resistance by this bacterium has led current therapies to lose effectiveness, so alternative treatments are urgently required. Here, we summarize, with a focus on flavodoxin, opportunities for pharmacological intervention offered by the potential protein targets described for this bacterium and provide information on other gastrointestinal pathogens and also on bacteria from the gut microbiota that contain flavodoxin. The process of discovery and development of novel antimicrobials specific for Hp flavodoxin that is being carried out in our group is explained, as it can be extrapolated to the discovery of inhibitors specific for other gastric pathogens. The high specificity for Hp of the antimicrobials developed may be of help to reduce damage to the gut microbiota and to slow down the development of resistant Hp mutants.

Este artículo pretende mostrar una visión global de la educación en ingeniería. En concreto se centra en las posibilidades que ofrecen las llamadas metodologías activas. Hoy más que nunca los profesionales de la ingeniería deben tratan con ciertos asuntos como incertidumbres, especificaciones incompletas y solicitudes que en ocasiones pueden entrar en conflicto con los clientes, gobiernos, organizaciones medioambientalistas y el público en general. Ello requiere del profesional, no sólo unas sólidas competencias técnicas, sino también ciertas habilidades en el ámbito de las relaciones humanas. Además, los futuros profesionales deben estar preparados para adaptarse a cambios continuos debidos a razones tecnológicas u organizacionales. También, deben satisfacer las realidades comerciales de la práctica industrial en el mundo moderno, así como conocer las consecuencias legales de cualquier decisión que tomen. Como consecuencia de estos requisitos, los estudiantes deberían recibir una educación multidisciplinar que proporcione una amplia perspectiva de los temas que conciernen a su trabajo: medioambientales, sociales y económicos. Pero además de una sólida formación técnica, los futuros ingenieros necesitan grandes habilidades de comunicación y de trabajo en equipo. A pesar de todo ello, continúa mayoritariamente presente en ingeniería el modelo de enseñanza tradicional, basado en la clase magistral. Es por ello necesaria la introducción de un nuevo paradigma, basado en el empleo de las denominadas metodologías activas, en los planes de estudio de los grados en ingeniería.

Vortex generators (VG) are passive flow control devices used for avoiding or delaying the separation of the boundary layer by bringing momentum from the higher layers of the fluid towards the surface. The Vortex generator usually has the same height as the local boundary layer thickness, and these Vortex generators can produce overload drag in some cases. The aim of the present study was to analyze the characteristics and path of the primary vortex produced by a single rectangular vortex generator on a flat plate for the incident angles of β = 10 ∘ , 15 ∘ , 18 ∘ and 20 ∘ . A parametric study of the induced vortex was performed for six VG heights using Reynolds average Navier–Stokes equations at Reynodls number R e = 27,000 based on the local boundary layer thickness, using computational fluid dynamics techniques with OpenFOAM open-source code. In order to determine the vortex size, the so-called half-life radius was computed and compared with experimental data. The results showed a similar trend for all the studied vortex generator heights and incident angles with small variations for the vertical and the lateral paths. Additionally, 0.4H and 0.6H VG heights at incident angles of β = 18 ∘ and β = 20 ∘ showed the best performance in terms of vortex strength and generation of wall shear stress.

The potential of non-invasive ventilation procedures and new minimally invasive techniques has resulted in the research of alternative approaches as the aerosolization for the treatment of respiratory distress syndrome (RDS). The aim of this work was to design two nebulizer prototypes and to evaluate them studying the particle size distribution of the inhaled droplets generated with distilled water and two perfluorocarbons (PFCs). Different experiments were performed with driving pressures of 1–3 bar for each compound. An Aerodynamic Particle Sizer was used to measure the aerodynamic diameter (Da), the mass median aerodynamic diameter (MMAD) and the geometric standard deviation (GSD). The results showed that both prototypes produced heterodisperse aerosols with Da mean values in all cases below 5 µm. The initial experiments with distilled water showed MMAD values lower than 9 µm and up to 15 µm with prototype 1 and prototype 2, respectively. Regarding the PFCs, relatively uniform MMAD values close to 12 µm were achieved. The air delivery with outer lumens of prototype 1 presented more suitable mass distribution for the generation and delivery of a uniform aerosol than the two half-circular ring geometry proposed in the prototype 2.

Streptococcus pneumoniae (Sp) strain TIGR4 is a virulent, encapsulated serotype that causes bacteremia, otitis media, meningitis and pneumonia. Increased bacterial resistance and limited efficacy of the available vaccine to some serotypes complicate the treatment of diseases associated to this microorganism. Flavodoxins are bacterial proteins involved in several important metabolic pathways. The Sp flavodoxin (Spfld) gene was recently reported to be essential for the establishment of meningitis in a rat model, which makes SpFld a potential drug target. To facilitate future pharmacological studies, we have cloned and expressed SpFld in E. coli and we have performed an extensive structural and biochemical characterization of both the apo form and its active complex with the FMN cofactor. SpFld is a short-chain flavodoxin containing 146 residues. Unlike the well-characterized long-chain apoflavodoxins, the Sp apoprotein displays a simple two-state thermal unfolding equilibrium and binds FMN with moderate affinity. The X-ray structures of the apo and holo forms of SpFld differ at the FMN binding site, where substantial rearrangement of residues at the 91-100 loop occurs to permit cofactor binding. This work will set up the basis for future studies aiming at discovering new potential drugs to treat S. pneumoniae diseases through the inhibition of SpFld.

There exist two isoforms of cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) in pig populations that differ in a single amino acid (Met139Leu). The isoenzymes have different kinetic properties, affecting more strongly the Km and Vmax of nucleotides. They are associated to different phenotypes modifying traits of considerable economic interest. In this work we use inhibitors of phosphoenolpyruvate carboxykinase activity to search for further differences between these isoenzymes. On the one hand we have used the well-known inhibitor 3-mercaptopicolinic acid. Its inhibition patterns were the same for both isoenzymes: a three-fold decrease of the Ki values for GTP in 139Met and 139Leu (273 and 873 μM, respectively). On the other hand, through screening of a chemical library we have found two novel compounds with inhibitory effects of a similar magnitude to that of 3-mercaptopicolinic acid but with less solubility and specificity. One of these novel compounds, (N'1-({5-[1-methyl-5-(trifluoromethyl)-1H-pyrazol-3-yl]-2-thienyl}methylidene)-2,4-dichlorobenzene-1-carbohydrazide), exhibited significantly different inhibitory effects on either isoenzyme: it enhanced threefold the apparent Km value for GTP in 139Met, whereas in 139Leu, it reduced it from 99 to 69 μM. The finding of those significant differences in the binding of GTP reinforces the hypothesis that the Met139Leu substitution affects strongly the nucleotide binding site of PEPCK-C.

The increasing capability of Wind Turbine (WT) based power generation systems has derived in an increment of the WT rotor diameter, i.e., longer rotor blades. This results in an increase of the electrical power generated but also in instabilities in the operation of the WT, especially due to the mechanical fatigue loads generated in its elements. In this context, flow control has appeared as a solution to improve the aerodynamic performance of the blades. These devices not only increase lift coefficient but also reduce mechanical fatigue loads. This paper presents a detailed numerical analysis of the effects of placing a passive flow control element, a Gurney Flap (GF), in a DU91W250 airfoil. Moreover, a numerical study of the influence of the GF length on the aerodynamic performance of the blade has been carried out. This study is considered as a basis for the development of an optimization technique of the GF length for long WT blades.

Several flow control devices have been studied in recent years. Majority of them were designed firstly for aeronautical purposes. At present many research is aimed to introduce these devices in wind turbines (WTs) in order to optimize their aerodynamic performance. The main goal of the present work is to analyze the influence of passive flow control devices, Vortex Generators and Gurney Flaps, on the Annual Energy Production (AEP) of a large Horizontal Axis Wind Turbine (HAWT). Consequently, BEM based calculations were performed in order to study their effect on the NREL offshore 5 MW Baseline Wind Turbine. Obtained results show an increment in the maximum value of the power coefficient, Cp_max, and a considerable improvement of the AEP.

El objeto de este trabajo se centra en analizar el papel que están jugando y pueden llegar a jugar los documentales científicos que se difunden ampliamente a través de la TV, venta en quioscos, etc., en la formación de una ciudadanía consciente de los problemas a los que se enfrenta hoy la humanidad y preparada para participar en la adopción de las medidas necesarias.

Over the past few years, it has been established that vibration energy harvesters with intentionally designed components can be used for frequency bandwidth enhancement under excitation for sufficiently high vibration amplitudes. Pipelines are often necessary means of transporting important resources such as water, gas, and oil. A self-powered wireless sensor network could be a sustainable alternative for in-pipe monitoring applications. A new control algorithm has been developed and implemented into an underwater energy harvester. Firstly, a computational study of a piezoelectric energy harvester for underwater applications has been studied for using the kinetic energy of water flow at four different Reynolds numbers Re = 3000, 6000, 9000, and 12,000. The device consists of a piezoelectric beam assembled to an oscillating cylinder inside the water of pipes from 2 to 5 inches in diameter. Therefore, unsteady simulations have been performed to study the dynamic forces under different water speeds. Secondly, a new control law strategy based on the computational results has been developed to extract as much energy as possible from the energy harvester. The results show that the harvester can efficiently extract the power from the kinetic energy of the fluid. The maximum power output is 996.25 µW and corresponds to the case with Re = 12,000.

Ferredoxin-NADP(H) reductases (FNRs) deliver NADPH or low potential one-electron donors to redox-based metabolism in plastids and bacteria. Xanthomonas citri subsp. citri (Xcc) is a Gram-negative bacterium responsible for citrus canker disease that affects commercial citrus crops worldwide. The Xcc fpr gene encodes a bacterial type FNR (XccFPR) that contributes to the bacterial response to oxidative stress conditions, usually found during plant colonization. Therefore, XccFPR is relevant for the pathogen survival and its inhibition might represent a strategy to treat citrus canker. Because of mechanistic and structural differences from plastidic FNRs, XccFPR is also a potential antibacterial target. We have optimized an activity-based high-throughput screening (HTS) assay that identifies XccFPR inhibitors. We selected 43 hits from a chemical library and narrowed them down to the four most promising inhibitors. The antimicrobial effect of these compounds was evaluated on Xcc cultures, finding one with antimicrobial properties. Based on the functional groups of this compound and their geometric arrangement, we identified another three XccFPR inhibitors. Inhibition mechanisms and constants were determined for these four XccFPR inhibitors. Their specificity was also evaluated by studying their effect on the plastidic Anabaena PCC 7119 FNR, finding differences that can become interesting tools to discover Xcc antimicrobials.

Este número especial Ikastorratza.e-Revista de Didáctica recoge varias acciones de experiencia formativa docente interna en metodologías activas denominadas “píldoras” del y para el profesorado de la Escuela Universitaria de Ingeniería de Vitoria-Gasteiz (UPV/EHU) en el ámbito de la ingeniería.

Diabetes and alcohol misuse are two of the major challenges in health systems worldwide. These two diseases finally affect several organs and systems including the central nervous system. Hippocampus is one of the most relevant structures due to neurogenesis and memory-related processing among other functions. The present review focuses on the common profile of diabetes and ethanol exposure in terms of oxidative stress and proinflammatory and prosurvival recruiting transcription factors affecting hippocampal neurogenesis. Some aspects around antioxidant strategies are also included. As a global conclusion, the present review points out some common hits on both diseases giving support to the relations between alcohol intake and diabetes.

The LDL receptor internalizes circulating LDL and VLDL particles for degradation. Its extracellular binding domain contains ten (seven LA and three EGF) cysteine-rich modules, each bearing three disulfide bonds. Despite the enormous number of disulfide combinations possible, LDLR oxidative folding leads to a single native species with 30 unique intradomain disulfides. Previous folding studies of the LDLR have shown that non native disulfides are initially formed that lead to compact species. Accordingly, the folding of the LDLR has been described as a "coordinated nonvectorial" reaction, and it has been proposed that early compaction funnels the reaction toward the native structure. Here we analyze the oxidative folding of LA4 and LA5, the modules critical for ApoE binding, isolated and in the LA45 tandem. Compared to LA5, LA4 folding is slow and inefficient, resembling that of LA5 disease-linked mutants. Without Ca++, it leads to a mixture of many two-disulfide scrambled species and, with Ca++, to the native form plus two three-disulfide intermediates. The folding of the LA45 tandem seems to recapitulate that of the individual repeats. Importantly, although the folding of the LA45 tandem takes place through formation of scrambled isomers, no interdomain disulfides are detected, i.e. the two adjacent modules fold independently without the assistance of interdomain covalent interactions. Reduction of incredibly large disulfide combinatorial spaces, such as that in the LDLR, by intradomain confinement of disulfide bond formation might be also essential for the efficient folding of other homologous disulfide-rich receptors.

Proteins perform many useful molecular tasks, and their biotechnological use continues to increase. As protein activity requires a stable native conformation, protein stabilisation is a major scientific and practical issue. Towards that end, many successful protein stabilisation strategies have been devised in recent years. In most cases, model proteins with a two-state folding equilibrium have been used to study and demonstrate protein stabilisation. Many proteins, however, display more complex folding equilibria where stable intermediates accumulate. Stabilising these proteins requires specifically stabilising the native state relative to the intermediates, as these are expected to lack activity. Here we discuss how to investigate the ‘relevant’ stability of proteins with equilibrium intermediates and propose a way to dissect the contribution of side chain interactions to the overall stability into the ‘relevant’ and ‘nonrelevant’ terms. Examples of this analysis performed on apoflavodoxin and in a single-chain mini antibody are presented.

An increasing number of neurodegenerative diseases are being found to be associated with the abnormal accumulation of aggregated proteins in the brain. In Parkinson’s disease, this process involves the aggregation of alpha-synuclein (α-syn) into intraneuronal inclusions. Thus, compounds that inhibit α-syn aggregation represent a promising therapeutic strategy as disease-modifying agents for neurodegeneration. The formation of α-syn amyloid aggregates can be reproduced in vitro by incubation of the recombinant protein. However, the in vitro aggregation of α-syn is exceedingly slow and highly irreproducible, therefore precluding fast high throughput anti-aggregation drug screening. Here, we present a simple and easy-to-implement in-plate method for screening large chemical libraries in the search for α-syn aggregation modulators. It allows us to monitor aggregation kinetics with high reproducibility, while being faster and requiring lower protein amounts than conventional aggregation assays. We illustrate how the approach enables the identification of strong aggregation inhibitors in a library of more than 14,000 compounds.

The nonstructural protein 3 (NS3) from the hepatitis C virus processes the non-structural region of the viral precursor polyprotein in infected hepatic cells. The NS3 protease activity has been considered a target for drug development since its identification two decades ago. Although specific inhibitors have been approved for clinical therapy very recently, resistance-associated mutations have already been reported for those drugs, compromising their long-term efficacy. Therefore, there is an urgent need for new anti-HCV agents with low susceptibility to resistance-associated mutations. Regarding NS3 protease, two strategies have been followed: competitive inhibitors blocking the active site and allosteric inhibitors blocking the binding of the accessory viral protein NS4A. In this work we exploit the intrinsic Zn(+2)-regulated plasticity of the protease to identify a new type of allosteric inhibitors. In the absence of Zn(+2), the NS3 protease adopts a partially-folded inactive conformation. We found ligands binding to the Zn(+2)-free NS3 protease, trap the inactive protein, and block the viral life cycle. The efficacy of these compounds has been confirmed in replicon cell assays. Importantly, direct calorimetric assays reveal a low impact of known resistance-associated mutations, and enzymatic assays provide a direct evidence of their inhibitory activity. They constitute new low molecular-weight scaffolds for further optimization and provide several advantages: 1) new inhibition mechanism simultaneously blocking substrate and cofactor interactions in a non-competitive fashion, appropriate for combination therapy; 2) low impact of known resistance-associated mutations; 3) inhibition of NS4A binding, thus blocking its several effects on NS3 protease.

Human Amylin, or islet amyloid polypeptide (hIAPP), is a small hormone secreted by pancreatic β-cells that forms aggregates under insulin deficiency metabolic conditions, and it constitutes a pathological hallmark of type II diabetes mellitus. In type II diabetes patients, amylin is abnormally increased, self-assembled into amyloid aggregates, and ultimately contributes to the apoptotic death of β-cells by mechanisms that are not completely understood. We have screened a library of approved drugs in order to identify inhibitors of amylin aggregation that could be used as tools to investigate the role of amylin aggregation in type II diabetes or as therapeutics in order to reduce β-cell damage. Interestingly, three of the compounds analyzed—benzbromarone, quercetin, and folic acid—are able to slow down amylin fiber formation according to Thioflavin T binding, turbidimetry, and Transmission Electron Microscopy assays. In addition to the in vitro assays, we have tested the effect of these compounds in an amyloid toxicity cell culture model and we have found that one of them, quercetin, has the ability to partly protect cultured pancreatic insulinoma cells from the cytotoxic effect of amylin. Our data suggests that quercetin can contribute to reduce oxidative damage in pancreatic insulinoma β cells by modulating the aggregation propensity of amylin.

The early stages of the thermal unfolding of apoflavodoxin have been determined by using atomistic multi microsecond-scale molecular dynamics (MD) simulations complemented with a variety of experimental techniques. Results strongly suggest that the intermediate is reached very early in the thermal unfolding process and that it has the properties of an "activated" form of the native state, where thermal fluctuations in the loops break loop-loop contacts. The unrestrained loops gain then kinetic energy corrupting short secondary structure elements without corrupting the core of the protein. The MD-derived ensembles agree with experimental observables and draw a picture of the intermediate state inconsistent with a well-defined structure and characteristic of a typical partially disordered protein. Our results allow us to speculate that proteins with a well packed core connected by long loops might behave as partially disordered proteins under native conditions, or alternatively behave as three state folders. Small details in the sequence, easily tunable by evolution, can yield to one or the other type of proteins.