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Until there is an effective implementation of COVID-19 vaccination program, a robust testing strategy, along with prevention measures, will continue to be the most viable way to control disease spread. Such a strategy should rely on disparate diagnostic tests to prevent a slowdown in testing due to lack of materials and reagents imposed by supply chain problems, which happened at the beginning of the pandemic. In this study, we have established a single-tube test based on RT-LAMP that enables the visual detection of less than 100 viral genome copies of SARS-CoV-2 within 30 min. We benchmarked the assay against the gold standard test for COVID-19 diagnosis, RT-PCR, using 177 nasopharyngeal RNA samples. For viral loads above 100 copies, the RT-LAMP assay had a sensitivity of 100% and a specificity of 96.1%. Additionally, we set up a RNA extraction-free RT-LAMP test capable of detecting SARS-CoV-2 directly from saliva samples, albeit with lower sensitivity. The saliva was self-collected and the collection tube remained closed until inactivation, thereby ensuring the protection of the testing personnel. As expected, RNA extraction from saliva samples increased the sensitivity of the test. To lower the costs associated with RNA extraction, we performed this step using an alternative protocol that uses plasmid DNA extraction columns. We also produced the enzymes needed for the assay and established an in-house-made RT-LAMP test independent of specific distribution channels. Finally, we developed a new colorimetric method that allowed the detection of LAMP products by the visualization of an evident color shift, regardless of the reaction pH.

Liquid-liquid phase separation (LLPS) in living cells provides innovative pathways for synthetic compartmentalized catalytic systems. While LLPS has been explored for enhancing enzyme catalysis, its potential application to catalytic peptides remains unexplored. Here, we demonstrate the use of coacervation, a key LLPS feature, to constrain the conformational flexibility of catalytic peptides, resulting in structured domains that enhance peptide catalysis. Using the flexible catalytic peptide P7 as a model, we induce reversible biomolecular coacervates with structured peptide domains proficient in hydrolyzing phosphate ester molecules and selectively sequestering phosphorylated proteins. Remarkably, these coacervate-based microreactors exhibit a 15,000-fold increase in catalytic efficiency compared to soluble peptides. Our findings highlight the potential of a single peptide to induce coacervate formation, selectively recruit substrates, and mediate catalysis, enabling a simple design for low-complexity, single peptide-based compartments with broad implications. Moreover, LLPS emerges as a fundamental mechanism in the evolution of chemical functions, effectively managing conformational heterogeneity in short peptides and providing valuable insights into the evolution of enzyme activity and catalysis in prebiotic chemistry. This study shows that liquid-liquid phase separation enhances the catalytic efficiency of peptides by up to 15,000-fold through the formation of peptide coacervates. These microreactors can also selectively recruit phosphorylated proteins, providing insights into the evolution of enzymatic activity.

PurposeTo study weight loss, comorbidity remission, complications, and nutritional deficits after duodenal switch (DS) and single-anastomosis DS with sleeve gastrectomy (SADI-S).Material and MethodsRetrospective review of patients submitted to DS or SADI-S for morbid obesity in a single university hospital.ResultsFour hundred forty patients underwent DS (n = 259) or SADI-S (n = 181). Mean preoperative body mass index (BMI) was 50.8 ± 6.4Kg/m2. Mean follow-up was 56.1 ± 37.2 months for DS and 27.2 ± 18.9 months for SADI-S. Global mean excess weight loss was 77.4% at 2 years similar for SADI-S and DS, and 72.1% at 10 years after DS. Although early complications were similar in SADI-S and DS (13.3% vs. 18.9%, p = n.s.), long-term complications and vitamin and micronutrient deficiencies were superior after DS. Rate of comorbidities remission was 85.2% for diabetes, 63.9% for hypertension, 77.6% for dyslipidemia, and 82.1% for sleep apnea, with no differences between both techniques. In patients with initial BMI > 55 kg/m2 (n = 91), DS achieved higher percentage of BMI < 35 kg/m2 (80% vs. 50%, p = 0.025) and higher rate of diabetes remission (100% vs. 75%, p = 0050).ConclusionsDS and SADI-S showed similar weight loss and comorbidity remission rates at 2 years. In patients with initial BMI > 55 kg/m2, DS obtained better BMI control at 2 years and better diabetes remission, but more long-term complications and supplementation needs.

Summary The Health Promoting University (HPU) concept encourages universities to incorporate health into the university culture, processes and policies in an effort to promote the health of the university community. Universities worldwide have adopted the approach and a framework for action has been developed to guide universities to become a HPU. However, information on how universities translate the framework into actions is scarce. This study explored the way in which 54 universities from 25 countries across the world implemented the HPU framework. An online questionnaire was used to assess the action areas and items of work addressed by the universities and to determine their adherence to the components of the HPU framework: use of the whole systems approach; multiservice collaboration; recognition by the university authorities; funding availability; membership of a HPU network and evaluation of the initiative. The results showed that these components were addressed by most universities. A Multi Correspondence and cluster analysis identified four types of universities based on the implementation of the components: ‘emerging’ HPUs that are not recognized by the university authorities and tend to not apply the whole systems approach or evaluation of the initiative, and ‘established’ HPUs that are recognized by the authorities, apply the whole systems approach and evaluate the initiative but that differ with regard to funding and membership of a HPU network. These results demonstrate that universities implement the HPU framework for action differently in order to become a Health Promoting University.

Bacteria that reside in the gastrointestinal tract of healthy humans are essential for our health, sustenance and well-being. About 50–60% of those bacteria have the ability to produce resilient spores that are important for the life cycle in the gut and for host-to-host transmission. A genomic signature for sporulation in the human intestine was recently described, which spans both commensals and pathogens such as Clostridioides difficile and contains several genes of unknown function. We report on the characterization of a signature gene, CD25890 , which, as we show is involved in the control of sporulation initiation in C. difficile under certain nutritional conditions. Spo0A is the main regulatory protein controlling entry into sporulation and we show that an in-frame deletion of CD25890 results in increased expression of spo0A per cell and increased sporulation. The effect of CD25890 on spo0A is likely indirect and mediated through repression of the sinRR ´ operon. Deletion of the CD25890 gene, however, does not alter the expression of the genes coding for the cytotoxins or the genes involved in biofilm formation. Our results suggest that CD25890 acts to modulate sporulation in response to the nutrients present in the environment.

Endosporulation is an ancient bacterial developmental program that culminates with the differentiation of a highly resistant endospore. In the model organism Bacillus subtilis, gene expression in the forespore and in the mother cell, the two cells that participate in endospore development, is governed by cell type-specific RNA polymerase sigma subunits. σ(F) in the forespore, and σ(E) in the mother cell control early stages of development and are replaced, at later stages, by σ(G) and σ(K), respectively. Starting with σ(F), the activation of the sigma factors is sequential, requires the preceding factor, and involves cell-cell signaling pathways that operate at key morphological stages. Here, we have studied the function and regulation of the sporulation sigma factors in the intestinal pathogen Clostridium difficile, an obligate anaerobe in which the endospores are central to the infectious cycle. The morphological characterization of mutants for the sporulation sigma factors, in parallel with use of a fluorescence reporter for single cell analysis of gene expression, unraveled important deviations from the B. subtilis paradigm. While the main periods of activity of the sigma factors are conserved, we show that the activity of σ(E) is partially independent of σ(F), that σ(G) activity is not dependent on σ(E), and that the activity of σ(K) does not require σ(G). We also show that σ(K) is not strictly required for heat resistant spore formation. In all, our results indicate reduced temporal segregation between the activities of the early and late sigma factors, and reduced requirement for the σ(F)-to-σ(E), σ(E)-to-σ(G), and σ(G)-to-σ(K) cell-cell signaling pathways. Nevertheless, our results support the view that the top level of the endosporulation network is conserved in evolution, with the sigma factors acting as the key regulators of the pathway, established some 2.5 billion years ago upon its emergence at the base of the Firmicutes Phylum.

A genomic signature for endosporulation includes a gene coding for a protease, YabG, which in the model organism Bacillus subtilis is involved in assembly of the spore coat. We show that in the human pathogen Clostridioidesm difficile , YabG is critical for the assembly of the coat and exosporium layers of spores. YabG is produced during sporulation under the control of the mother cell-specific regulators σ E and σ K and associates with the spore surface layers. YabG shows an N-terminal SH3-like domain and a C-terminal domain that resembles single domain response regulators, such as CheY, yet is atypical in that the conserved phosphoryl-acceptor residue is absent. Instead, the CheY-like domain carries residues required for activity, including Cys207 and His161, the homologues of which form a catalytic diad in the B . subtilis protein, and also Asp162. The substitution of any of these residues by Ala, eliminates an auto-proteolytic activity as well as interdomain processing of CspBA, a reaction that releases the CspB protease, required for proper spore germination. An in-frame deletion of yabG or an allele coding for an inactive protein, yabG C207A , both cause misassemby of the coat and exosporium and the formation of spores that are more permeable to lysozyme and impaired in germination and host colonization. Furthermore, we show that YabG is required for the expression of at least two σ K -dependent genes, cotA , coding for a coat protein, and cdeM , coding for a key determinant of exosporium assembly. Thus, YabG also impinges upon the genetic program of the mother cell possibly by eliminating a transcriptional repressor. Although this activity has not been described for the B . subtilis protein and most of the YabG substrates vary among sporeformers, the general role of the protease in the assembly of the spore surface is likely to be conserved across evolutionary distance.

Oral vaccines are highly demanded by the aquaculture sector, to allow mass delivery of antigens without using the expensive and labor-intensive injectable vaccines. These later require individual handling of fish, provoking stress-related mortalities. One possible strategy to create injection-free vaccine delivery vehicles is the use of bacterial spores, extremely resistant structures with wide biotechnological applications, including as probiotics, display systems, or adjuvants. Bacterial spores, in particular those of Bacillus subtilis , have been shown to behave as mucosal vaccine adjuvants in mice models. However, such technology has not been extensively explored against fish bacterial disease. In this study, we used a laboratory strain of B. subtilis , for which a variety of genetic manipulation tools are available, to display at its spores surface either a Vibrio antigenic protein, OmpK, or the green fluorescence protein, GFP. When previously vaccinated by immersion with the OmpK- carrying spores, zebrafish survival upon a bacterial challenge with V. anguillarum and V. parahaemolyticus , increased up to 50 - 90% depending on the pathogen targeted. Further, we were able to detect anti-GFP-antibodies in the serum of European seabass juveniles fed diets containing the GFP-carrying spores and anti- V. anguillarum antibodies in the serum of European seabass juveniles fed the OmpK-carrying spores containing diet. More important, seabass survival was increased from 60 to 86% when previously orally vaccinated with in-feed OmpK- carrying spores. Our results indicate that B. subtilis spores can effectively be used as antigen-carriers for oral vaccine delivery in fish.

Background There has been a rise in endometrial cancer (EC) incidence leading to increased mortality. To counter this trend, improving the stratification of post-surgery recurrence risk and anticipating disease relapse and treatment resistance is essential. Liquid biopsy analyses offer a promising tool for these clinical challenges, though the best strategy for applying them in EC must be defined. This study was designed to determine the value of cfDNA/ctDNA monitoring in improving the clinical management of patients with localized and recurrent disease. Methods Plasma samples and uterine aspirates (UA) from 198 EC patients were collected at surgery and over time. The genetic landscape of UAs was characterized using targeted sequencing. Total cfDNA was analyzed for ctDNA presence based on the UA mutational profile. Results High cfDNA levels and detectable ctDNA at baseline correlated with poor prognosis for DFS (p-value < 0.0001; HR = 9.25) and DSS (p-value < 0.0001; HR = 11.20). This remained clinically significant when stratifying tumors by histopathological risk factors. Of note, cfDNA/ctDNA analyses discriminated patients with early post-surgery relapse and the ctDNA kinetics served to identify patients undergoing relapse before any clinical evidence emerged. Conclusions This is the most comprehensive study on cfDNA/ctDNA characterization in EC, demonstrating its value in improving risk stratification and anticipating disease relapse in patients with localized disease. CtDNA kinetics assessment complements current strategies to monitor the disease evolution and the treatment response. Therefore, implementing cfDNA/ctDNA monitoring in clinical routines offers a unique opportunity to improve EC management. Translational relevance The study demonstrates that high levels of cfDNA and detectable ctDNA at baseline are strong indicators of poor prognosis. This enables more accurate risk stratification beyond traditional histopathological factors, allowing clinicians to identify high-risk patients who may benefit from more aggressive treatment and closer monitoring. Moreover, longitudinal analysis of cfDNA/ctDNA can detect disease recurrence months before clinical symptoms or imaging evidence appear. This early warning system offers a significant advantage in clinical practice, providing a window of opportunity for early intervention and potentially improving patient outcomes.

Purpose A novel robotic platform—Hugo™ RAS (robotic-assisted surgery) system—has been introduced with several innovations that may prove advantageous for surgeons, such as an open console and four interchangeable modular arms. Our study aims to evaluate this platform’s safety, efficacy, and potential impact on the surgical treatment of colorectal pathology. Methods Patients underwent robotic-assisted colorectal procedures with the Hugo™ RAS system at the General University Hospital of Elche from October 2023 to July 2024. Patient characteristics, intraoperative and postoperative variables, and robotic technical issues were recorded. Results Forty consecutive patients were included (14 right, 13 left, and 8 rectum neoplasms; 4 left diverticulitis; and 1 ileocecal Crohn’s disease). The patients’ characteristics were as follows: median age, 69.5 years; 24 males and 16 females; 45% ASA III–IV; and Charlson Comorbidity Index > 5:42.5%. We recorded four medical (2 anemia, 1 phlebitis, and 1 admission to the intensive care unit) and three surgical (1 hematoma of the incision, 1 intestinal occlusion, and 1 dehiscence of the anastomosis) postoperative complications. We had no conversions neither open nor laparoscopic surgery. The average hospital stay was 3 days, with no mortality or readmission. Conclusions The Hugo™ RAS system is safe and feasible for colorectal procedures. The modularity of the arms provides the versatility of configurations adjusted depending on the patient’s body features and the surgeon’s preferences and greater adaptability to operating rooms. The open console is highly comfortable and ergonomic for the surgeon, allowing communication with the operating room environment. Trial registration NCT06512480