Controlled manipulation of enzyme specificity through immobilization-induced flexibility constraints

It is thought that during immobilization enzymes, as dynamic biomolecules, may become distorted and this may alter their catalytic properties. However, the effects of different immobilization strategies on enzyme rigidity or flexibility and their consequences in specificity and stereochemistry at large scale has not been yet clearly evaluated and understood. This was here investigated by using as model an ester hydrolase, isolated from a bacterium inhabiting a karstic lake, with broad substrate spectrum (72 esters being converted; 61.5 U mg−1 for glyceryl tripropionate) but initially non-enantiospecific. We found that the enzyme (7 nm × 4.4 nm × 4.2 nm) could be efficiently ionic exchanged inside the pores (9.3 nm under dry conditions) of amino-functionalized ordered mesoporous material (NH2-SBA-15), achieving a protein load of 48 mg g−1, and a specific activity of 4.5 ± 0.1 U mg−1. When the enzyme was site-directed immobilized through His interaction with an immobilized cationon the surface of two types of magnetic micro-particles through hexahistidine-tags, protein loads up to 10.2 μg g−1 and specific activities of up to 29.9 ± 0.3 U mg−1, were obtained. We found that ionically exchanged enzyme inside pores of NH2-SBA-15 drastically narrowed the substrate range (17 esters), to an extent much higher than ionically exchanged enzyme on the surface of magnetic micro-particles (up to 61 esters). This is attributed to differences in surface chemistry, particle size, and substrate accessibility to the active site tunnel. Our results also suggested, for the first time, that immobilization of enzymes in pores of similar size may alter the enzyme structures and produce enzyme active centers with different configuration which promote stereochemical conversions in a manner different to those arising from surface immobilization, where the strength of the ionic exchange also has an influence. This was shown by demonstrating that when the enzyme was introduced inside pores with a diameter (under dry conditions) slightly higher than that of the enzyme crystal structure a biocatalyst enantiospecific for ethyl (R)-4-chloro-3-hydroxybutyrate was produced, a feature not found when using wider pores. By contrast, immobilization on the surface of ferromagnetic microparticles produced selective biocatalysts for methyl (S)-(+)-mandelate or methyl (S)-lactate depending on the functionalization. This study illustrates the benefits of extensive analysis of the substrate spectra to better understand the effects of different immobilization strategies on enzyme flexibility/rigidity, as well as substrate specificity and stereochemistry. Our results will help to design tunable materials and interfaces for a controlled manipulation of specificity and to transform non-enantiospecific enzymes into stereo-chemically substrate promiscuous biocatalysts capable of converting multiple chiral molecules.This project received funding from the European Union’s Horizon 2020 research and innovation program Blue Growth: Unlockingthe potential of Seas and Oceans under grant agreement no. 634486 (project acronym INMARE). This research was also supported by the grants PCIN-2014-107 (within ERA NET IB2 grant nr. ERA-IB-14-030 - MetaCat), PCIN-2017-078 (within the ERA-MarineBiotech grant ProBone), BIO2014-54494-R, MAT2016-77496-R, BIO2017-85522-R, and CTQ2016-79138-R from the Spanish Ministry of Economy, Industry and Competitiveness. A.B. acknowledges the support from the Spanish Ministry of Economy, Industry and Competitiveness (MAT2017-88808-R grant), María de Maeztu Units of Excellence Programme (MDM-2016-0618), and the Diputación de Guipúzcoa for current funding in the frame of Gipuzkoa Fellows program. G.D. thanks the German Federal Ministry of Education and Research (BMBF, Grant No. 031A095C) for funding in the frame of the Molecular Interaction Engineering program (Biotechnologie 2020+). The authors gratefully acknowledge financial support provided by the European Regional Development Fund (ERDF). C.C. thanks the Spanish Ministry of Economy, Industry and Competitiveness for a PhD fellowship (Grant BES-2015-073829).Peer ReviewedPostprint (published version

Prevention of ventilator-associated pneumonia: the multimodal approach of the spanish ICU pneumonia zero program.

Objectives: The “Pneumonia Zero” project is a nationwide multimodal intervention based on the simultaneous implementation of a comprehensive evidence-based bundle measures to prevent ventilator-associated pneumonia in critically ill patients admitted to the ICU. Design: Prospective, interventional, and multicenter study. Setting: A total of 181 ICUs throughout Spain. Patients: All patients admitted for more than 24 hours to the participating ICUs between April 1, 2011, and December 31, 2012. Intervention: Ten ventilator-associated pneumonia prevention measures were implemented (seven were mandatory and three highly recommended). The database of the National ICU-Acquired Infections Surveillance Study (Estudio Nacional de Vigilancia de Infecciones Nosocomiales [ENVIN]) was used for data collection. Ventilator-associated pneumonia rate was expressed as incidence density per 1,000 ventilator days. Ventilator-associated pneumonia rates from the incorporation of the ICUs to the project, every 3 months, were compared with data of the ENVIN registry (April–June 2010) as the baseline period. Ventilator-associated pneumonia rates were adjusted by characteristics of the hospital, including size, type (public or private), and teaching (postgraduate) or university-affiliated (undergraduate) status. Measurements and Main Results: The 181 participating ICUs accounted for 75% of all ICUs in Spain. In a total of 171,237 ICU admissions, an artificial airway was present on 505,802 days (50.0% of days of stay in the ICU). A total of 3,474 ventilator-associated pneumonia episodes were diagnosed in 3,186 patients. The adjusted ventilator-associated pneumonia incidence density rate decreased from 9.83 (95% CI, 8.42–11.48) per 1,000 ventilator days in the baseline period to 4.34 (95% CI, 3.22–5.84) after 19–21 months of participation. Conclusions: Implementation of the bundle measures included in the “Pneumonia Zero” project resulted in a significant reduction of more than 50% of the incidence of ventilator-associated pneumonia in Spanish ICUs. This reduction was sustained 21 months after implementation

Occurrence of persistent and mobile chemicals and other contaminants of emerging concern in Spanish and Portuguese wastewater treatment plants, transnational river basins and coastal water

This work investigated, during one year, the occurrence and fate of 52 contaminants of emerging concern (CECs) in transnational river basins and coastal areas of the North of Portugal and Galicia (NW Spain) and the wastewater treatment plants (WWTPs) discharging on these environments. The different CECs investigated included pharmaceuticals, personal care products, industrial chemicals, among others, of which ca. 90 % would fulfill the persistence, mobility and toxicity criteria proposed by the German Environmental Agency. The results showed the ubiquitous presence of these CECs and an incomplete removal of over 60 % of them with current conventional WWTPs. These findings highlight the requirement of a prominent and coordinated upgrade of WWTP treatments in order to meet the future European Union regulations on urban wastewater treatment and surface water quality. In fact, even some compounds exhibiting high removals, such as caffeine or xylene sulfonate, were frequently detected in river and estuarine waters at the high ng L−1 level. Thus, our preliminary risk assessment study concluded that 18 of the CECs presented a potential risk for the environment, being caffeine, sulpiride, perfluorooctanoic acid (PFOA), diclofenac, fipronil and perfluorobutanoic acid (PFBA) the most concerning ones. Yet, additional toxicity data as well as a more robust information on persistence and mobility of CECs are necessary for better estimating the magnitude of the problem and improve risk assessment. As an example, in the case of the antidiabetic metformin, recent research has revealed toxicity for model fish species at concentration levels below those found in 40 % of the river water samples analyzed in this workThis work was financed by the European Regional Development Fund through the Interreg V-A Spain-Portugal Programme (POCTEP) 2014-2020 (ref. 0725_NOR_WATER_1_P), the Agencia Estatal de Investigación MCIN/AEI/10.13039/501100011033 (ref. PID2020-117686RB-C32) and the Consellería de Cultura, Educación y Universidades da Xunta de Galicia (ref. ED431C 2021/06). The study was also supported by the Portuguese Foundation for Science and Technology (FCT) under the projects [UIDB/04423/2020 and UIDP/04423/2020]. R.M. acknowledges Banco Santander and Universidade de Santiago de Compostela for her outstanding researcher contract, N. A. acknowledges the FCT for his Ph.D. grant DFA/BD/6218/2020 and T.N. acknowledges FCT Individual Call to Scientific Employment Stimulus 2022 (2022.02925.CEECIND). The authors also want to acknowledge Agilent Technologies for providing the equipment described in Section 2.4 and the different entities which have collaborated in the sampling campaign: VIAQUA, Augas de Galicia, Confederación Hidrográfica do Miño-Sil, Águas do Porto, Efacec and Águas do NorteS

Normal ossification of the glenoid mimicking a glenoid fracture in an adolescent patient: a case report

A 13-year-old male was diagnosed with a glenoid fracture following direct shoulder trauma, for which surgical treatment was considered. After referral to a center for pediatric orthopedic care, physical examination, contralateral shoulder X-ray, and detailed computed tomography examination ruled out the presence of fracture; these findings were later confirmed by magnetic resonance imaging. Normal ossification patterns in the adolescent shoulder may simulate a fracture in traumatic settings. To accurately diagnose and manage pediatric shoulder pathology, orthopedic surgeons must be aware of the normal anatomy of the growing shoulder, its secondary ossification centers, and growth plates. Level of evidence V

Spatial dynamics of bovine tuberculosis in the Autonomous Community of Madrid, Spain (2010-2012)

Progress in control of bovine tuberculosis (bTB) is often not uniform, usually due to the effect of one or more sometimes unknown epidemiological factors impairing the success of eradication programs. Use of spatial analysis can help to identify clusters of persistence of disease, leading to the identification of these factors thus allowing the implementation of targeted control measures, and may provide some insights of disease transmission, particularly when combined with molecular typing techniques. Here, the spatial dynamics of bTB in a high prevalence region of Spain were assessed during a three year period (2010-2012) using data from the eradication campaigns to detect clusters of positive bTB herds and of those infected with certain Mycobacterium bovis strains (characterized using spoligotyping and VNTR typing). In addition, the within-herd transmission coefficient (β) was estimated in infected herds and its spatial distribution and association with other potential outbreak and herd variables was evaluated. Significant clustering of positive herds was identified in the three years of the study in the same location ("high risk area"). Three spoligotypes (SB0339, SB0121 and SB1142) accounted for >70% of the outbreaks detected in the three years. VNTR subtyping revealed the presence of few but highly prevalent strains within the high risk area, suggesting maintained transmission in the area. The spatial autocorrelation found in the distribution of the estimated within-herd transmission coefficients in herds located within distances <14 km and the results of the spatial regression analysis, support the hypothesis of shared local factors affecting disease transmission in farms located at a close proximity

Are Peripheral Biomarkers Determinants of Eating Styles in Childhood and Adolescence Obesity? A Cross-Sectional Study

Disturbances in eating behaviors have been widely related to obesity. However, little is known about the role of obesity-related biomarkers in shaping habitual patterns of eating be haviors (i.e., eating styles) in childhood. The objective of the present study was to explore the relationships between several biomarkers crucially involved in obesity (ghrelin, insulin resistance, and leptin/adiponectin ratio) and eating styles in children and adolescents with obesity. Seventy participants aged between 8 and 16 (56.2% men) fulfilled the Spanish version of the Dutch Eating Behavior Questionnaire for Children to measure external, emotional, and restrained eating styles. In addition, concentrations of ghrelin, leptin, adiponectin, insulin, and glucose were obtained through a blood test. Hierarchical multiple regression analyses controlling for age and sex were computed for each eating style. Results indicated that individuals with higher ghrelin concentration levels showed lower scores in restrained eating (β = −0.61, p < 0.001). The total model explained 32% of the variance of the restrained pattern. No other relationships between obesity-related biomarkers and eating behaviors were found. This study highlights that one of the obesity-risk factors, namely lower plasma ghrelin levels, is substantially involved in a well-known maladaptive eating style, restraint eating, in childhood obesity

Utility of clinical-epidemiological profiles in outbreaks of foodborne disease, Catalonia, 2002 through 2006

The objective of this study was to evaluate the use of clinical- epidemiological profiles for classifying non-laboratory-confirmed outbreaks of foodborne disease (FBD) in Catalonia between 2002 and 2006 and for elucidating associations among factors contributing to these outbreaks. A total of 275 nonfamily outbreaks were studied, of which 190 (69.1%) were laboratory confirmed and 85 (30.9%) were not. In 176 (92.6%) of laboratory-confirmed outbreaks and 69 (81.2%) of non-laboratory-confirmed outbreaks, information was obtained on contributing factors (P = 0.009). In 72% of non-laboratory-confirmed outbreaks, the etiology was assigned by using clinical-epidemiological profiles; thus, 93% of outbreaks eventually were associated with an etiology. In laboratory-confirmed outbreaks, poor personal hygiene was positively associated with norovirus (odds ratio [OR], 2.69; 95% confidence interval [CI], 1.47 to 4.89; P = 0,0007) and negatively associated with Salmonella and Campylobacter (OR, 0.54; 95% CI, 0.33 to 0.89; P = 0.01), and an unsafe source was positively associated with Salmonella and Campylobacter (OR, 4.07; 95% CI, 1.72 to 10.09; P = 0.001) and negatively associated with norovirus (OR, 0.14; 95% CI, 0.04 to 0.58; P = 0.001). No differences were found among contributing factors associated with outbreaks with a laboratoryconfirmed etiology and those associated with outbreaks with an etiology assigned according to the clinical-epidemiological profiles. Clinical-epidemiological profiles are useful for determining what prevention and control strategies are appropriate to the agents involved in each community and for designing outbreak investigations. Copyright ©, International Association for Food Protection.This work was partially funded by the Centro de Investigación Biomédica en Red de Epidemiología y Salud Pública (CIBERESP), Spain, and by the Fondo de Investigaciones Sanitarias (project PI 030877).Peer Reviewe

The origin and collapse of rock glaciers during the Bølling-Allerød interstadial: A new study case from the Cantabrian Mountains (Spain)

.During the Late Pleistocene, the main mountain ranges of the Iberian Peninsula were covered by small icefields and cirque and alpine glaciers. The deglaciation triggered paraglacial processes that generated landforms, mostly within the ice-free glacial cirques. In this research we analyse the deglaciation process in the Muxivén Cirque (42°15′N – 6°16′W), in the upper Sil River Basin, which includes some of the largest relict rock glaciers of the Cantabrian Mountains. We addressed this objective by means of accurate geomorphological reconstructions, sedimentological analysis, Schmidt-hammer surface weathering measurements and a dataset of 10 10Be Cosmic-Ray Exposure ages. Results reveal that after ~16 ka, glaciers retreated to the bottom of the cirques at the headwaters of the valley, leaving the walls free of ice and triggering rock avalanches onto the remnants of these glaciers. This paraglacial process supplied debris to a small glacier within Muxivén Cirque, which transformed in two rock glaciers. These debris isolated the ice inside the rock glaciers only for a very short period of time and ended up melting completely before the Younger Dryas. The lower sector of the largest one stabilized at 14.5 ± 1.5 ka, while the upper sector remained active until 13.5 ± 0.8 ka. Previous to the stabilization of the lower sector of the northern rock glacier, at its margin a high-energy debris avalanche occurred at ~14.0 ± 0.9 ka. These data agree with previous research, corroborating the paraglacial origin of most Iberian rock glaciers during the Bølling-Allerød interstadial.S

A DNA damage repair gene-associated signature predicts responses of patients with advanced soft-tissue sarcoma to treatment with trabectedin

Predictive biomarkers of trabectedin represent an unmet need in advanced soft-tissue sarcomas (STS). DNA damage repair (DDR) genes, involved in homologous recombination or nucleotide excision repair, had been previously described as biomarkers of trabectedin resistance or sensitivity, respectively. The majority of these studies only focused on specific factors (ERCC1, ERCC5, and BRCA1) and did not evaluate several other DDR-related genes that could have a relevant role for trabectedin efficacy. In this retrospective translational study, 118 genes involved in DDR were evaluated to determine, by transcriptomics, a predictive gene signature of trabectedin efficacy. A six-gene predictive signature of trabectedin efficacy was built in a series of 139 tumor samples from patients with advanced STS. Patients in the high-risk gene signature group showed a significantly worse progression-free survival compared with patients in the low-risk group (2.1 vs 6.0 months, respectively). Differential gene expression analysis defined new potential predictive biomarkers of trabectedin sensitivity (PARP3 and CCNH) or resistance (DNAJB11 and PARP1). Our study identified a new gene signature that significantly predicts patients with higher probability to respond to treatment with trabectedin. Targeting some genes of this signature emerges as a potential strategy to enhance trabectedin efficacy.This study was funded by the Spanish Group for Research on Sarcoma (GEIS) and partially by PharmaMar. The authors would like to thank the GEIS data center for data management. The authors also thank the donors and the Hospital Universitario Virgen del Rocío—Instituto de Biomedicina de Sevilla Biobank (Andalusian Public Health System Biobank and ISCIII-Red de Biobancos PT17/0015/0041) for part of the human specimens used in this study. David S. Moura is recipient of a Sara Borrell postdoctoral fellowship funded by the National Institute of Health Carlos III (ISCIII) (CD20/00155)