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The first step in cheesemaking is the milk clotting process, in which κ-caseinolytic enzymes contribute to micelle precipitation. The best enzyme for this purpose is chymosin because of its high degree of specificity toward κ-casein. Although recombinant bovine chymosin is the most frequently used chymosin in the industry, new sources of recombinant chymosin, such as goat, camel, or buffalo, are now available. The present work represents a comparative study of 4 different recombinant chymosins (goat and buffalo chymosins expressed in Pichia pastoris, and bovine and camel chymosin expressed in Aspergillus niger). Recombinant goat chymosin exhibited the best catalytic efficiency compared with the buffalo, bovine, or camel recombinant enzymes. Moreover, recombinant goat chymosin exhibited the best specific proteolytic activity, a wider pH range of action, and a lower glycosylation degree than the other 3 enzymes. In conclusion, we propose that recombinant goat chymosin represents a serious alternative to recombinant bovine chymosin for use in the cheesemaking industry. [ABSTRACT FROM AUTHOR]

Polygalacturonases are enzymes involved in plant cell wall growth and reorganization. Transgenic Arabidopsis thaliana plants with a Saccharomyces cerevisiae endopolygalacturonase gene ( PGU1) were obtained. The yeast gene was properly expressed in the plants as it has been shown by RT-PCR as well as by the increase in the endopolygalacturonase activity. The transgenic plants showed conspicuous malformations in early stages of development probably due to a weak cell adhesion. On the other hand, adult plants exhibited almost no phenotypic differences as compared to the wild type plants, this suggesting the appearance of some mechanisms on the plant side to counteract the effect of the overexpressed polygalacturonase. [ABSTRACT FROM AUTHOR]

Milk-clotting proteases, which are widely used in the cheese-making industry, are enzymes that use soluble caseins as their preferential substrates. Here, we propose a modification to a method previously described for the specific determination of milk-clotting proteases by using K-casein labeled with fluorescein isothiocyanate as substrate. Validation of the modified method was confirmed using natural bacterial, fungal, plant, and animal milk-clotting proteases, as well as a milk-clotting enzyme of recombinant origin. The new modified method described here allowed specific quantification of the activity of milk-clotting proteases in a very sensitive way and permitted determination of the appropriate kinetic parameters of all the enzymes tested, consistent with their origin and degree of purity. [ABSTRACT FROM AUTHOR]

Acinetobacter baumanniiis a hospital-acquired pathogen that shows an extraordinary capacity to stay in the hospital environment. Adherence of the bacteria to eukaryotic cells or to abiotic surfaces is the first step for establishing an infection. TheA. baumanniistrain AbH12O-A2 showed an exceptional ability to adhere to A549 epithelial cells. The AbFhaB/FhaC 2-partner secretion (TPS) system involved in adhesion was discovered after the screening of the recently determinedA. baumanniiAbH12O-A2 strain genome (CP009534.1). The AbFhaB is a large exoprotein which transport to the bacterial surface is mediated by the AbFhaC protein. In the present study, the role of this TPS system in the AbH12O-A2 adherence phenotype was investigated. The functional inactivation of this 2-partner secretion system was addressed by analyzing the outer membrane vesicles (OMV) proteomic profile from the wild-type strain and its derivative mutant AbH12O-A2ΔfhaC demonstrating that AbFhaB is no longer detected in the absence of AbFhaC. Scanning electron microscopy (SEM) and adhesion experiments demonstrated that inactivation of the AbFhaB/FhaC system significantly decreases bacterial attachment to A549 alveolar epithelial cells. Moreover, it has been demonstrated that this 2-partner secretion system is involved in fibronectin-mediated adherence of theA. baumanniiAbH12O-A2 isolate. Finally, we report that the AbFhaB/FhaC system is involved in virulence when tested using invertebrate and vertebrate hosts. These data suggest the potential role that this AbFhaB/FhaC secretion system could play in the pathobiology ofA. baumannii. [ABSTRACT FROM AUTHOR]

Objectives The objectives of this study were to investigate the acceptability of rapid HIV testing among general practitioners in Spain and to identify perceived barriers and needs in order to implement rapid testing in primary care settings. Methods An anonymous questionnaire was distributed online to all members of the two largest Spanish scientific medical societies for family and community medicine. The study took place between 15th June and 31st October 2010. Results Completed questionnaires were returned by 1308 participants. The majority (90.8%) of respondents were General Practitioners (GP). Among all respondents, 70.4% were aware of the existence of rapid tests for the diagnosis of HIV but they did not know how to use them. Nearly 80% of participants would be willing to offer rapid HIV testing in their practices and 74.7% would be confident of the results obtained by these tests. The barriers most commonly identified by respondents were a lack of time and a need for training, both in the use of rapid tests (44.3% and 56.4%, respectively) and required pre- and post-test counselling (59.2% and 34.5%, respectively). Conclusions This study reveals a high level of acceptance and willingness on the part of GPs to offer rapid HIV testing in their practices. Nevertheless, the implementation of rapid HIV testing in primary care will not be possible without moving from comprehensive pre-test counselling towards brief pre-test information and improving training in the use of rapid tests. [ABSTRACT FROM AUTHOR]

Aim: The study of a milk-clotting protease secreted by Bacillus licheniformis strain USC13. Methods and Results: Growth of B. licheniformis USC13 in LB medium resulted in the production of a serine protease with a molecular weight of 62 kDa processed to its mature form of 34 kDa, both forms were found in the extracellular medium. The enzyme exhibited typical milk-clotting kinetics. Conclusions: The capacity of this protease to produce milk curds could make it useful as a new source of milk coagulants. Significance and Impact of the Study: Cheese-making industry seeks for novel enzyme sources, and microbial coagulants have several advantages over animal and plant counterparts. The protease from B. licheniformis has the ability to produce milk curds although more studies about quality of both the enzyme and the milk curds formed should be carried out in the future to confirm its usefulness in the dairy industry. [ABSTRACT FROM AUTHOR]

Aims: Gordonia jacobaea is a recently isolated bacterial species with potential industrial application on account of its ability to store large quantities of trans-canthaxanthin. Its genetic manipulation is, however, difficult and cumbersome owing to the presence of mycolic acids in the cell wall and, especially, because of current lack of knowledge about its basic genetics. The present work describes a method for the genetic transformation of G. jacobaea. Methods and Results: Gordonia jacobaea was grown in media supplemented with different glycine, penicillin G and isoniazid concentrations. The temperature, carbon source, growth phase and ultrasounds were analyzed for improving the method efficiency. The cells were finally transformed by electroporation. Finally, the method was applied to Brevibacterium lactofermentum and Gordonia bronchialis. Conclusions: The growth of G. jacobaea in the presence of glycine and isoniazid is essential for obtaining electrocompetents cells. The temperature, growth phase and ultrasounds appeared as the main factors for increasing the transformation efficiency. The use of shuttle plasmids became necessary. The method described can be used with other Corynebacteria species. Significance and Impact of the Study: Because of the importance of the CNM group ( Corynebacteria, Nocardia and Mycobacteria genera) in different areas such as industry, bioremediation improve the knowledge of their molecular mechanisms are becoming essential. The method described here improves the genetic manipulation of this group of bacteria. [ABSTRACT FROM AUTHOR]

Abstract: The existence of a diverse trypsin gene family with a main role in the proteolytic digestion process has been proved in vertebrate and invertebrate organisms. In lepidopteran insects, a diversity of trypsin-like genes expressed in midgut has also been identified. Genomic DNA and cDNA trypsin-like sequences expressed in the Mediterranean corn Borer (MCB), Sesamia nonagrioides, midgut are reported in this paper. A phylogenetic analysis revealed that at least three types of trypsin-like enzymes putatively involved in digestion are conserved in MCB and other lepidopteran species. As expected, a diversity of sequences has been found, including four type-I (two subtypes), four type-II (two subtypes) and one type-III. In parallel, four different trypsins have been purified from midgut lumen of late instar MCB larvae. N-terminal sequencing and mass spectrometric analyses of purified trypsins have been performed in order to identify cDNAs coding for major trypsins among the diversity of trypsin-like sequences obtained. Thus, it is revealed that the four purified trypsins in MCB belong to the three well-defined phylogenetic groups of trypsin-like sequences detected in Lepidoptera. Major active trypsins present in late instar MCB lumen guts are trypsin-I (type-I), trypsin-IIA and trypsin-IIB (type-II), and trypsin-III (type-III). Trypsin-I, trypsin-IIA and trypsin-III showed preference for Arg over Lys, but responded differently to proteinaceous or synthetic inhibitors. As full-length cDNA clones coding for the purified trypsins were available, three-dimensional protein models were built in order to study the implication of specific residues on their response to inhibitors. Thus, it is predicted that Arg73, conserved in type-I lepidopteran trypsins, may favour reversible inhibition by the E-64. Indeed, the substitution of Val213Cys, unique for type-II lepidopteran trypsins, may be responsible for their specific inhibition by HgCl2. The implication of these results on the optimisation of the use of protease inhibitors for pest control, and on the identification of endoprotease-mediated resistance to Bacillus thuringiensis Cry-toxins is discussed. [Copyright &y& Elsevier]

The degeneration of nigral dopaminergic neurons in Parkinson’s disease is believed to be associated with a glial reaction and inflammatory changes. In turn, local factors may induce changes in vascularization and contribute to neuronal vulnerability. Among these factors, Vascular Endothelial Growth Factor (VEGF) is released in adults under pathological conditions and is thought to induce angiogenesis. In order to determine whether changes in brain vasculature are observed in the affected brain regions in parkinsonism, we quantitatively analysed the VEGF-expressing cells and blood vessels in the substantia nigra of monkeys rendered parkinsonian by MPTP injection and compared the results with those obtained in control monkeys. Using stereological methods, we observed an increase in the number of VEGF-expressing neurons and an increase of the number of blood vessels and their volume occupying the substantia nigra pars compacta of monkeys rendered parkinsonian by chronic MPTP intoxication. These changes in vascularization may therefore modify the neuronal availability of blood nutrients, blood cells or toxic substances and neuronal susceptibility to parkinsonism. [ABSTRACT FROM AUTHOR]

The parkinsonian symptoms of primates after MPTP exposure can be measured by several visual methods (classical motor scores). However, these methods have a subjective bias, especially as regards the evaluation of the motor activity. Computerized monitoring systems represent an unbiased method for measuring the motor disability of monkeys after MPTP administration. In this work the motor activity of monkeys before and after MPTP administration is measured and compared with the activity of a control intact group by means of a telemetry system. A pronounced decrease in motor activity was observed after MPTP administration. These results suggest the monitoring method used is suited for characterizing the motor incapacity and possible improvements following treatments to test different therapies to control Parkinson’s disease in MPTP models involving primates. [Copyright &y& Elsevier]

Treated sewage contains a large diversity of pathogens that can be transmitted to the environment and, directly or indirectly, infect humans through water use (i.e., consumption, bathing, or irrigation). In urban environments, wastewater normally flows into wastewater treatment plants (WWTPs), where it is subjected to different processes in order to eliminate the greatest amount of waste. However, there are inequalities among European countries concerning wastewater management. In this context, we evaluate the potential of freshwater mussels to improve water quality (i.e., reduce bacterial abundance) in rivers receiving primary, secondary, or tertiary sewage-treated effluents. Additionally, because freshwater mussels are declining at a global scale and empty niches are progressively occupied by non-native counterparts, we evaluate if depauperate communities and the Asian clams, Corbicula genus, can provide equivalent ecosystem services (i.e., water quality improvement by biofiltration) formerly provided by diverse native communities. For this, an analysis of the bacterial biodiversity of the samples filtered by the different bivalve communities was carried out. The experimental approach was performed by metabarcoding the 16S rRNA gene using Illumina technologies. According to the results obtained, secondary treatment processes were effective in reducing the bacterial diversity. Furthermore, the waters filtered by the bivalves presented a lower bacterial abundance for certain genera. Biofiltration differs, however, among species, with Corbicula reducing a large number of taxa much more efficiently than native freshwater mussels in both diverse and depauperated communities. These results are likely related to Corbicula being a generalist species in front of native mussels, which may be more selective. Considering it is not possible to eradicate Corbicula from European rivers, its filtering capacity should be considered when managing freshwater ecosystems., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Noé Ferreira-Rodriguez reports financial support was provided by Government of Galicia, Department of Culture Education and Universities. Margarita Poza reports financial support was provided by Carlos III Health Institute. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

The dynamics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission are influenced by a variety of factors, including social restrictions and the emergence of distinct variants. In this study, we delve into the origins and dissemination of the Alpha, Delta, and Omicron-BA.1 variants of concern in Galicia, northwest Spain. For this, we leveraged genomic data collected by the EPICOVIGAL Consortium and from the GISAID database, along with mobility information from other Spanish regions and foreign countries. Our analysis indicates that initial introductions during the Alpha phase were predominantly from other Spanish regions and France. However, as the pandemic progressed, introductions from Portugal and the United States became increasingly significant. The number of detected introductions varied from 96 and 101 for Alpha and Delta to 39 for Omicron-BA.1. Most of these introductions left a low number of descendants (<10), suggesting a limited impact on the evolution of the pandemic in Galicia. Notably, Galicia's major coastal cities emerged as critical hubs for viral transmission, highlighting their role in sustaining and spreading the virus. This research emphasizes the critical role of regional connectivity in the spread of SARS-CoV-2 and offers essential insights for enhancing public health strategies and surveillance measures., (© 2024 The Author(s). Journal of Medical Virology published by Wiley Periodicals LLC.)

Background: The aim of our study is to analyse the trends in the diagnosis of sexually transmitted infections (STIs) during the COVID-19 pandemic., Methods: We conducted an observational retrospective population-based study using data from primary care electronic health records spanning from January 2016 to December 2022 (involving 5.1 million people older than 14 years). We described the daily number of new STI diagnoses from 2016 to 2022; as well as the monthly accumulation of new STI diagnoses for each year. We compared the monthly averages of new diagnoses in 2019, 2020, 2021 and 2022 using the T-test. Finally, we performed a segmented regression analysis of the daily number of STI diagnoses., Results: We analysed 200,676 new STI diagnoses. The number of diagnoses abruptly decreased coinciding with the lockdown. Overall in 2020, we observed a reduction of 15%, with higher reductions for specific STIs such as gonorrhoea (-21%), chlamydia (-24%), and HIV (-31%) compared to 2019. Following this drastic drop, which was temporarily associated with the lockdown, we observed a rapid rebound. In 2021, the number of STI diagnoses was similar to that of 2019. Notably, we found a considerable increase in 2022, particularly for non-specific STI, which lack laboratory confirmation (67% increase). HIV was the only STI with a reduction of up to -38% in diagnoses at the end of 2022 compared to 2019., Conclusions: After a significant reduction in 2020, the number of STIs recorded in primary care rapidly rebounded, and the current trend is similar to that of 2019, except for HIV. These findings underscore the dynamic impact of the COVID-19 pandemic on STI diagnoses and highlight the importance of ongoing monitoring and public health interventions in the post-pandemic period., (© 2024. The Author(s).)

The incidence of colorectal cancer (CRC) has increased worldwide, and early diagnosis is crucial to reduce mortality rates. Therefore, new noninvasive biomarkers for CRC are required. Recent studies have revealed an imbalance in the oral and gut microbiomes of patients with CRC, as well as impaired gut vascular barrier function. In the present study, the microbiomes of saliva, crevicular fluid, feces, and non-neoplastic and tumor intestinal tissue samples of 93 CRC patients and 30 healthy individuals without digestive disorders (non-CRC) were analyzed by 16S rRNA metabarcoding procedures. The data revealed that Parvimonas, Fusobacterium, and Bacteroides fragilis were significantly over-represented in stool samples of CRC patients, whereas Faecalibacterium and Blautia were significantly over-abundant in the non-CRC group. Moreover, the tumor samples were enriched in well-known periodontal anaerobes, including Fusobacterium, Parvimonas, Peptostreptococcus, Porphyromonas, and Prevotella. Co-occurrence patterns of these oral microorganisms were observed in the subgingival pocket and in the tumor tissues of CRC patients, where they also correlated with other gut microbes, such as Hungatella. This study provides new evidence that oral pathobionts, normally located in subgingival pockets, can migrate to the colon and probably aggregate with aerobic bacteria, forming synergistic consortia. Furthermore, we suggest that the group composed of Fusobacterium, Parvimonas, Bacteroides, and Faecalibacterium could be used to design an excellent noninvasive fecal test for the early diagnosis of CRC. The combination of these four genera would significantly improve the reliability of a discriminatory test with respect to others that use a single species as a unique CRC biomarker., (© 2024 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Oral and intestinal samples from a cohort of 93 colorectal cancer (CRC) patients and 30 healthy controls (non-CRC) were collected for microbiome analysis. Saliva (28 non-CRC and 94 CRC), feces (30 non-CRC and 97 CRC), subgingival fluid (20 CRC), and tumor tissue samples (20 CRC) were used for 16S metabarcoding and/or RNA sequencing (RNAseq) approaches. A differential analysis of the abundance, performed with the ANCOM-BC package, adjusting the P-values by the Holm-Bonferroni method, revealed that Parvimonas was significantly over-represented in feces from CRC patients (P-value < 0.001) compared to healthy controls. A total of 11 Parvimonas micra isolates were obtained from the oral cavity and adenocarcinoma of CRC patients. Genome analysis identified a pair of isolates from the same patient that shared 99.2% identity, demonstrating that P. micra can translocate from the subgingival cavity to the gut. The data suggest that P. micra could migrate in a synergistic consortium with other periodontal bacteria. Metatranscriptomics confirmed that oral bacteria were more active in tumor than in non-neoplastic tissues. We suggest that P. micra could be considered as a CRC biomarker detected in non-invasive samples such as feces., (© 2023 The Authors. Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Competing Interests: The authors declare no conflict of interest.

Wastewater treatment plants (WWTPs) are recognized as important niches of antibiotic-resistant bacteria that can be easily spread to the environment. In this study, we collected wastewater samples from the WWTP of A Coruña (NW Spain) from April 2020 to February 2022 to evaluate the presence of Gram-negative bacteria harboring carbapenemase genes. Bacteria isolated from wastewater were classified and their antimicrobial profiles were determined. In total, 252 Gram-negative bacteria carrying various carbapenemase genes were described. Whole-genome sequencing was conducted on 55 selected carbapenemase producing isolates using Oxford Nanopore technology. This study revealed the presence of a significant population of bacteria carrying carbapenemase genes in WWTP, which constitutes a public health problem due to their risk of dissemination to the environment. This emphasizes the usefulness of WWTP monitoring for combating antibiotic resistance. Data revealed the presence of different types of sequences harboring carbapenemase genes, such as bla KPC-2 , bla GES-5 , bla GES-6 , bla IMP-11 , bla IMP-28 , bla OXA-24 , bla OXA-48 , bla OXA-58 , bla OXA-217 , and bla VIM-2 . Importantly, the presence of the bla KPC-2 gene in wastewater, several months before any clinical case was detected in University Hospital of A Coruña, suggests that wastewater-based epidemiology can be used as an early warning system for the surveillance of antibiotic-resistant bacteria.

Attention deficit hyperactivity disorder (ADHD) is a highly prevalent developmental disorder in children. However, accurately identifying ADHD in early childhood remains a crucial challenge. Electronic health (e-health) systems offer promising possibilities to enhance the diagnostic process for ADHD, particularly concerning the executive functions (EFs) that play a direct role. This study aims to validate an evidence-based tool for screening ADHD through EFs in the school environment. The tool, named Sendero Gris, is designed for tablet devices and is based on a previously validated test with the same name. To ensure its validity, a comparison was made between the results obtained from the tool to be validated and the original format of the test. The analysis revealed no statistically significant differences between the two approaches at a 90% confidence level ( p -value = 0.49). Moreover, a user experience study focusing on usability was conducted to assess the children's inclination to use the developed tool, yielding highly positive results. The implementation of Sendero Gris on a tablet device, with its objective and versatile nature, seems to maintain the potential of the original format as a screening tool for ADHD.

Purpose: To assess the potential value of LiqBio as a complementary tool for diagnosis and surveillance of BCL., Methods: This prospective multi-center study included 78 patients (25 follicular lymphomas (FL) and 53 large B-cell lymphomas (LBCL)). We performed next-generation sequencing (NGS) of cfDNA LiqBio and paired gDNA tissue biopsies at diagnosis and compared the mutational statuses. Also, through NGS of LiqBio, we identified MRD biomarkers and compared this novel LiqBio-MRD method with PET/CT in detecting MRD at follow-up., Results: We identified mutations in 71% of LiqBio and 95% of tissue biopsies, and found a correlation between variant allele frequency of somatic mutations. Additionally, we identified mutations in 73% of LiqBio from patients with no available tissue samples or no mutations in them. Regarding the utility of LiqBio-MRD as a dynamic monitoring tool, when compared with the PET/CT method, a lower sensitivity was observed for LiqBio-MRD at 92.3% (vs. 100% for PET/CT), but a higher specificity of 91.3% (vs. 86.9% for PET/CT)., Conclusion: Genetic profiling of tumor cfDNA in plasma LiqBio is a complementary tool for BCL diagnosis and MRD surveillance.

Wastewater-based epidemiology has been widely used as a cost-effective method for tracking the COVID-19 pandemic at the community level. Here we describe COVIDBENS, a wastewater surveillance program running from June 2020 to March 2022 in the wastewater treatment plant of Bens in A Coruña (Spain). The main goal of this work was to provide an effective early warning tool based in wastewater epidemiology to help in decision-making at both the social and public health levels. RT-qPCR procedures and Illumina sequencing were used to weekly monitor the viral load and to detect SARS-CoV-2 mutations in wastewater, respectively. In addition, own statistical models were applied to estimate the real number of infected people and the frequency of each emerging variant circulating in the community, which considerable improved the surveillance strategy. Our analysis detected 6 viral load waves in A Coruña with concentrations between 10 3 and 10 6 SARS-CoV-2 RNA copies/L. Our system was able to anticipate community outbreaks during the pandemic with 8-36 days in advance with respect to clinical reports and, to detect the emergence of new SARS-CoV-2 variants in A Coruña such as Alpha (B.1.1.7), Delta (B.1.617.2), and Omicron (B.1.1.529 and BA.2) in wastewater with 42, 30, and 27 days, respectively, before the health system did. Data generated here helped local authorities and health managers to give a faster and more efficient response to the pandemic situation, and also allowed important industrial companies to adapt their production to each situation. The wastewater-based epidemiology program developed in our metropolitan area of A Coruña (Spain) during the SARS-CoV-2 pandemic served as a powerful early warning system combining statistical models with mutations and viral load monitoring in wastewater over time., (© 2023. The Author(s).)

Background: CART therapy has produced a paradigm shift in the treatment of relapsing FL patients. Strategies to optimize disease surveillance after these therapies are increasingly necessary. This study explores the potential value of ctDNA monitoring with an innovative signature of personalized trackable mutations., Method: Eleven FL patients treated with anti-CD19 CAR T-cell therapy were included. One did not respond and was excluded. Genomic profiling was performed before starting lymphodepleting chemotherapy to identify somatic mutations suitable for LiqBio-MRD monitoring. The dynamics of the baseline mutations (4.5 per patient) were further analyzed on 59 cfDNA follow-up samples. PET/CT examinations were performed on days +90, +180, +365, and every six months until disease progression or death., Results: After a median follow-up of 36 months, all patients achieved a CR as the best response. Two patients progressed. The most frequently mutated genes were CREBBP, KMT2D and EP300. Simultaneous analysis of ctDNA and PET/CT was available for 18 time-points. When PET/CT was positive, two out of four ctDNA samples were LiqBio-MRD negative. These two negative samples corresponded to women with a unique mesenteric mass in two evaluations and never relapsed. Meanwhile, 14 PET/CT negative images were mutation-free based on our LiqBio-MRD analysis (100%). None of the patients had a negative LiqBio-MRD test by day +7. Interestingly, all durably responding patients had undetectable ctDNA at or around three months after infusion. Two patients presented discordant results by PET/CT and ctDNA levels. No progression was confirmed in these cases. All the progressing patients were LiqBio-MRD positive before progression., Conclusion: This is a proof-of-principle for using ctDNA to monitor response to CAR T-cell therapy in FL. Our results confirm that a non-invasive liquid biopsy MRD analysis may correlate with response and could be used to monitor response. Harmonized definitions of ctDNA molecular response and pinpointing the optimal timing for assessing ctDNA responses are necessary for this setting. If using ctDNA analysis, we suggest restricting follow-up PET/CT in CR patients to a clinical suspicion of relapse, to avoid false-positive results., Competing Interests: AMM, SD, YRH, LR and MR are are employees of Altum Sequencing Co. RA, JM-L, and SB are equity shareholders of Altum Sequencing Co. CW is an employee of Hosea Precision Medical Technology Co. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Jiménez-Ubieto, Martín-Muñoz, Poza, Dorado, García-Ortiz, Revilla, Sarandeses, Ruiz-Heredia, Baumann, Rodríguez, Calbacho, Sánchez, Pina, García-Sancho, Figaredo, Rufián, Rodríguez, Carneros, Martínez-Laperche, Bastos-Oreiro, Wang, Cedena, Rapado, de Toledo, Valeri, Ayala, Martínez-López and Barrio.)

Objectives: The IPSS-M is a recently published score for risk stratification in myelodysplastic syndromes (MDS), based on clinical and molecular data. We aimed to evaluate its relevance on treatment choice in a real-life setting., Methods: We retrospectively collected clinical, cytogenetic and molecular data from 166 MDS patients. We calculated IPSS-R and IPSS-M scores and compared Overall Survival (OS) and Leukemia Free Survival (LFS). We also analyzed which patients would have been affected by the re-stratification in terms of clinical management., Results: We found that 86.1% of the patients had at least one genetic alteration. The most frequent mutated genes were SF3B1 (25.9%), DNMT3A (16.8%) and ASXL1 (14.4%). IPSS-M re-stratified 48.2% of the patients, of which 16.9% were downgraded and 31.3% were upgraded. IPSS-M improved outcome prediction, with a Harrell's c-index of 0.680 vs 0.626 for OS and 0.801 vs 0.757 for LFS. In 22.2% of the cohort, the reclassification of the IPSS-M could potentially affect clinical management; 17.4% of the patients would be eligible for treatment intensification and 4.8% for treatment reduction., Conclusions: IPSS-M implementation in clinical practice could imply different treatment approaches in a significant number of patients. Our work validates IPSS-M in an external cohort and confirms its applicability in a real-life setting., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Zamanillo, Poza, Ayala, Rapado, Martinez-Lopez and Cedena.)

Background: Currently, many central auditory processing disorder screening tests are available for children, and serious games (SGs) are frequently used as a tool for the diagnosis of different neural deficits and disorders in health care. However, it has not been possible to find a proposal that unifies both ideas. In addition, the validation and improvement of SGs, in general, does not take into account the player-game interaction, thus omitting valuable information about the playability and usability of the game., Objective: This study presented Amalia's Planet, a game conceived for use in school environments, which allows a first assessment of a child through their performance of the proposed tasks related to different aspects of auditory performance. In addition, the game defines a series of events in relation to the execution of the tasks, which were evaluated for the subsequent optimization of its performance and the improvement of its usability., Methods: Using screening tools based on the use of SG technologies, a total of 87 school-age children were evaluated to test the various hypotheses proposed in this study. By grouping users according to whether they had personal history of hearing pathologies, the discriminant power, playability, and usability of the final solution were examined using traditional statistical techniques and process mining (PM) algorithms., Results: With a confidence level of 80% for test 2 (P=.19), there was no statistical evidence to reject the null hypothesis that a player's performance is affected by whether the player had a previous auditory pathology. Furthermore, the tool allowed the screening of 2 players initially categorized as healthy because of their low level of performance in the tests and the similarity of their behavior with that of the group of children with a previous pathology. With regard to the validation of the proposed solution, the use of PM techniques made it possible to detect the existence of events that lasted too long, which can lead to player frustration, and to discover small structural flaws in the game., Conclusions: SGs seem to be an appropriate tool for the screening of children at risk of central auditory processing disorder. Moreover, the set of PM techniques provides a reliable source of information regarding the playability and usability of the solution to the development team, allowing its continuous optimization., (©Ana-Marta Gabaldón-Pérez, María Dolón-Poza, Martina Eckert, Nuria Máximo-Bocanegra, María-Luisa Martín-Ruiz, Iván Pau De La Cruz. Originally published in JMIR Serious Games (https://games.jmir.org), 26.04.2023.)

In the present study, we screened 84 Follicular Lymphoma patients for somatic mutations suitable as liquid biopsy MRD biomarkers using a targeted next-generation sequencing (NGS) panel. We found trackable mutations in 95% of the lymph node samples and 80% of the liquid biopsy baseline samples. Then, we used an ultra-deep sequencing approach with 2 · 10 -4 sensitivity (LiqBio-MRD) to track those mutations on 151 follow-up liquid biopsy samples from 54 treated patients. Positive LiqBio-MRD at first-line therapy correlated with a higher risk of progression both at the interim evaluation (HR INT 11.0, 95% CI 2.10-57.7, p = 0.005) and at the end of treatment (HR EOT , HR 19.1, 95% CI 4.10-89.4, p < 0.001). Similar results were observed by PET/CT Deauville score, with a median PFS of 19 months vs. NR (p < 0.001) at the interim and 13 months vs. NR (p < 0.001) at EOT. LiqBio-MRD and PET/CT combined identified the patients that progressed in less than two years with 88% sensitivity and 100% specificity. Our results demonstrate that LiqBio-MRD is a robust and non-invasive approach, complementary to metabolic imaging, for identifying FL patients at high risk of failure during the treatment and should be considered in future response-adapted clinical trials., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Background: The methodology described in previous literature for Monkeypox virus (MPXV) sequencing shows low efficiency when using metagenomic approaches. The aim of the present study was to evaluate a new fine-tuned method for extraction and enrichment of genomic MPXV DNA using clinical samples and to compare it to a non-enrichment metagenomic approach., Results: A new procedure that allows sample enrichment in MPXV DNA, avoiding wasting the sequencing capacity in human DNA, was designed. This procedure consisted of host DNA depletion using a saponin/NaCl combination treatment and DNase, together with high g-force centrifugations. After typical quality control, samples using the enrichment method contained around 96% of reads not classified as human DNA, while the non-enrichment protocol showed around 5-10%. When reads not belonging to Orthopoxvirus were removed, enriched samples kept about 50% of the original read counts, while non-enriched ones kept only 2-7%., Conclusions: Results showed a very significant improvement in sequencing efficiency, increasing the number of reads belonging to MPXV, the depth of coverage and the trustworthiness of the consensus sequences. This, in turn, allows for more samples to be included in a single cartridge, reducing costs and time to diagnosis, which can be very important factors when dealing with a contagious disease., (© 2023. The Author(s).)

In the last decades, personalized medicine has been increasing its presence in different fields of medicine, including ophthalmology. A new factor that can help us direct medicine towards the challenge of personalized treatments is the microbiome. The gut microbiome plays an important role in controlling immune response, and dysbiosis has been associated with immune-mediated diseases such as non-infectious uveitis (NIU). In this review, we gather the published evidence, both in the pre-clinical and clinical studies, that support the possible role of intestinal dysbiosis in the pathogenesis of NIU, as well as the modulation of the gut microbiota as a new possible therapeutic target. We describe the different mechanisms that have been proposed to involve dysbiosis in the causality of NIU, as well as the potential pharmacological tools that could be used to modify the microbiome (dietary supplementation, antibiotics, fecal microbiota transplantation, immunomodulators, or biologic drugs) and, consequently, in the control of the NIU. Furthermore, there is increasing scientific evidence suggesting that the treatment with anti-TNF not only restores the composition of the gut microbiota but also that the study of the composition of the gut microbiome will help predict the response of each patient to anti-TNF treatment.

Background: Hypertension is a prevalent condition among SARS-CoV-2 infected patients. Whether renin-angiotensin-aldosterone system (RAAS) inhibitors are beneficial or harmful is controversial., Methods: We have performed a national retrospective, nonexperimental comparative study from two tertiary hospitals to evaluate the impact of chronic use of RAAS inhibitors in hypertensive COVID-19 patients. A meta-analysis was performed to strengthen our findings., Results: Of 849 patients, 422 (49.7%) patients were hypertensive and 310 (73.5%) were taking RAAS inhibitors at baseline. Hypertensive patients were older, had more comorbidities, and a greater incidence of respiratory failure (-0.151 [95% CI -0.218, -0.084]). Overall mortality in hypertensive patients was 28.4%, but smaller among those with prescribed RAAS inhibitors before (-0.167 [95% CI -0.220, -0.114]) and during hospitalization (0.090 [-0.008,0.188]). Similar findings were observed after two propensity score matches that evaluated the benefit of angiotensin-converting enzyme inhibitors and angiotensin receptor blockers among hypertensive patients. Multivariate logistic regression analysis of hypertensive patients found that age, diabetes mellitus, C-reactive protein, and renal failure were independently associated with all-cause mortality. On the contrary, ACEIs decreased the risk of death (OR 0.444 [95% CI 0.224-0.881]). Meta-analysis suggested a protective benefit of RAAS inhibitors (OR 0.6 [95% CI 0.42-0.8]) among hypertensive COVID-19., Conclusion: Our data suggest that RAAS inhibitors may play a protective role in hypertensive COVID-19 patients. This finding was supported by a meta-analysis of the current evidence. Maintaining these medications during hospital stay may not negatively affect COVID-19 outcomes., (© 2021 Elsevier España, S.L.U. All rights reserved.)

The quantification of the SARS-CoV-2 RNA load in wastewater has emerged as a useful tool to monitor COVID-19 outbreaks in the community. This approach was implemented in the metropolitan area of A Coruña (NW Spain), where wastewater from a treatment plant was analyzed to track the epidemic dynamics in a population of 369,098 inhabitants. Viral load detected in the wastewater and the epidemiological data from A Coruña health system served as main sources for statistical models developing. Regression models described here allowed us to estimate the number of infected people (R 2  = 0.9), including symptomatic and asymptomatic individuals. These models have helped to understand the real magnitude of the epidemic in a population at any given time and have been used as an effective early warning tool for predicting outbreaks in A Coruña municipality. The methodology of the present work could be used to develop a similar wastewater-based epidemiological model to track the evolution of the COVID-19 epidemic anywhere in the world where centralized water-based sanitation systems exist., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

Acinetobacter baumannii is a multidrug-resistant pathogen that represents a serious threat to global health. A. baumannii possesses a wide range of virulence factors that contribute to the bacterial pathogenicity. Among them, the siderophore acinetobactin is one of the most important, being essential for the development of the infection. In this study we performed an in-depth analysis of the acinetobactin cluster in the strain A. baumannii ATCC 17978. For this purpose, nineteen individual isogenic mutant strains were generated, and further phenotypical analysis were performed. Individual mutants lacking the biosynthetic genes entA, basG , basC , basD , and basB showed a significant loss in virulence, due to the disruption in the acinetobactin production. Similarly, the gene bauA , coding for the acinetobactin receptor, was also found to be crucial for the bacterial pathogenesis. In addition, the analysis of the Δ basJ/ Δ fbsB double mutant strain demonstrated the high level of genetic redundancy between siderophores where the role of specific genes of the acinetobactin cluster can be fulfilled by their fimsbactin redundant genes. Overall, this study highlights the essential role of entA , basG , basC , basD , basB and bauA in the pathogenicity of A. baumannii and provides potential therapeutic targets for the design of new antivirulence agents against this microorganism., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Conde-Pérez, Vázquez-Ucha, Álvarez-Fraga, Ageitos, Rumbo-Feal, Martínez-Guitián, Trigo-Tasende, Rodríguez, Bou, Jiménez, Beceiro and Poza.)