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Olfaction is a well-studied sensory mechanism in Caenorhabditis elegans (C. elegans). The nematodes respond to a wide range of chemicals by either attraction, repulsion or a mixture thereof (Bargmann et al., 1993). We have used olfaction to characterize behavioural and molecular circadian rhythms in C. elegans. The circadian clock is a biological oscillator that provides an endogenous temporal structure that approximately matches the 24-hour periodicity in the environment (due to the rotational movement of the Earth). Circadian rhythms are present in most organisms from cyanobacteria to humans and they typically regulate sensory functions among many other processes. Olfaction is under circadian control in many animals (Granados-Fuentes et al., 2006; Granados-Fuentes et al., 2011; Tanoue et al., 2008; Krishnan et al., 1999). This protocol was designed to allow the assessment of olfaction for a population of worms within a short time interval, in the same plate where the worms grew (to avoid washing steps that may disturb the rhythms), and in the presence of food.

Abstract Background The identification of novel therapeutic strategies to overcome resistance to the MEK inhibitor trametinib in mutant KRAS lung adenocarcinoma (LUAD) is a challenge. This study analyzes the effects of trametinib on Id1 protein, a key factor involved in the KRAS oncogenic pathway, and investigates the role of Id1 in the acquired resistance to trametinib as well as the synergistic anticancer effect of trametinib combined with immunotherapy in KRAS-mutant LUAD. Methods We evaluated the effects of trametinib on KRAS-mutant LUAD by Western blot, RNA-seq and different syngeneic mouse models. Genetic modulation of Id1 expression was performed in KRAS-mutant LUAD cells by lentiviral or retroviral transductions of specific vectors. Cell viability was assessed by cell proliferation and colony formation assays. PD-L1 expression and apoptosis were measured by flow cytometry. The anti-tumor efficacy of the combined treatment with trametinib and PD-1 blockade was investigated in KRAS-mutant LUAD mouse models, and the effects on the tumor immune infiltrate were analyzed by flow cytometry and immunohistochemistry. Results We found that trametinib activates the proteasome-ubiquitin system to downregulate Id1 in KRAS-mutant LUAD tumors. Moreover, we found that Id1 plays a major role in the acquired resistance to trametinib treatment in KRAS-mutant LUAD cells. Using two preclinical syngeneic KRAS-mutant LUAD mouse models, we found that trametinib synergizes with PD-1/PD-L1 blockade to hamper lung cancer progression and increase survival. This anti-tumor activity depended on trametinib-mediated Id1 reduction and was associated with a less immunosuppressive tumor microenvironment and increased PD-L1 expression on tumor cells. Conclusions Our data demonstrate that Id1 expression is involved in the resistance to trametinib and in the synergistic effect of trametinib with anti-PD-1 therapy in KRAS-mutant LUAD tumors. These findings suggest a potential therapeutic approach for immunotherapy-refractory KRAS-mutant lung cancers. Graphical Abstract

Objectives: This study aimed to assess the real use of cefazolin for methicillin-susceptible Staphylococcus aureus (MSSA) infective endocarditis (IE) in the Spanish National Endocarditis Database (GAMES) and to compare it with antistaphylococcal penicillin (ASP). Methods: Prospective cohort study with retrospective analysis of a cohort of MSSA IE treated with cloxacillin and/or cefazolin. Outcomes assessed were relapse; intra-hospital, overall, and endocarditis-related mortality; and adverse events. Risk of renal toxicity with each treatment was evaluated separately. Results: We included 631 IE episodes caused by MSSA treated with cloxacillin and/or cefazolin. Antibiotic treatment was cloxacillin, cefazolin, or both in 537 (85%), 57 (9%), and 37 (6%) episodes, respectively. Patients treated with cefazolin had significantly higher rates of comorbidities (median Charlson Index 7, P

IntroductionClostridioides difficile infection (CDI) is the main cause of nosocomial diarrhoea in developed countries. Recurrent CDI (R-CDI), which affects 20%-30% of patients and significantly increases hospital stay and associated costs, is a key challenge. The main objective of this study was to explore the role of the microbiome and calprotectin levels as predictive biomarkers of R-CDI.MethodsWe prospectively (2019-2021) included patients with a primary episode of CDI. Clinical data and faecal samples were collected. The microbiome was analysed by sequencing the hypervariable V4 region of the 16S rRNA gene on an Illumina Miseq platform.ResultsWe enrolled 200 patients with primary CDI, of whom 54 developed R-CDI and 146 did not. We analysed 200 primary samples and found that Fusobacterium increased in abundance, while Collinsella, Senegalimassilia, Prevotella and Ruminococcus decreased in patients with recurrent versus non-recurrent disease. Elevated calprotectin levels correlated significantly with R-CDI (p=0.01). We built a risk index for R-CDI, including as prognostic factors age, sex, immunosuppression, toxin B amplification cycle, creatinine levels and faecal calprotectin levels (overall accuracy of 79%).DiscussionCalprotectin levels and abundance of microbial genera such as Fusobacterium and Prevotella in primary episodes could be useful as early markers of R-CDI. We propose a readily available model for prediction of R-CDI that can be applied at the initial CDI episode. The use of this tool could help to better tailor treatments according to the risk of R-CDI.

Abstract Background After embryonic development, Caenorhabditis elegans progress through for larval stages, each of them finishing with molting. The repetitive nature of C. elegans postembryonic development is considered an oscillatory process, a concept that has gained traction from regulation by a circadian clock gene homologue. Nevertheless, each larval stage has a defined duration and entails specific events. Since the overall duration of development is controlled by numerous factors, we have asked whether different rate-limiting interventions impact all stages equally. Results We have measured the duration of each stage of development for over 2500 larvae, under varied environmental conditions known to alter overall developmental rate. We applied changes in temperature and in the quantity and quality of nutrition and analysed the effect of genetically reduced insulin signalling. Our results show that the distinct developmental stages respond differently to these perturbations. The changes in the duration of specific larval stages seem to depend on stage-specific events. Furthermore, our high-resolution measurement of the effect of temperature on the stage-specific duration of development has unveiled novel features of temperature dependence in C. elegans postembryonic development. Conclusions Altogether, our results show that multiple factors fine tune developmental timing, impacting larval stages independently. Further understanding of the regulation of this process will allow modelling the mechanisms that control developmental timing.

IntroductionClostridioides difficile infection (CDI) is the main cause of nosocomial diarrhea in developed countries. A key challenge in CDI is the lack of objective methods to ensure more accurate diagnosis, especially when differentiating between true infection and colonization/diarrhea of other causes. The main objective of this study was to explore the role of the microbiome as a predictive biomarker of CDI.MethodsBetween 2018 and 2021, we prospectively included patients with CDI, recurrent CDI (R-CDI), non-CDI diarrhea (NO-CDI), colonization by C. difficile, and healthy individuals. Clinical data and fecal samples were collected. The microbiome was analyzed by sequencing the hypervariable V4 region of the 16S rRNA gene on an Illumina Miseq platform. The mothur bioinformatic pipeline was followed for pre-processing of raw data, and mothur and R were used for data analysis.ResultsDuring the study period, 753 samples from 657 patients were analyzed. Of these, 247 were from patients with CDI, 43 were from patients colonized with C. difficile, 63 were from healthy individuals, 324 were from NOCDI, and 76 were from R-CDI. We found significant differences across the groups in alpha and beta diversity and in taxonomic abundance. We identified various genera as the most significant biomarkers for CDI (Bacteroides, Proteus, Paraprevotella, Robinsoniella), R-CDI (Veillonella, Fusobacterium, Lactobacillus, Clostridium sensu stricto I), and colonization by C. difficile (Parabacteroides, Faecalicoccus, Flavonifractor, Clostridium XVIII).DiscussionWe observed differences in microbiome patterns between healthy individuals, colonized patients, CDI, R-CDI, and NOCDI diarrhea. We identified possible microbiome biomarkers that could prove useful in the diagnosis of true CDI infections. Further studies are warranted.

IntroductionSince the COVID-19 outbreak, specific mRNA-based anti-SARS-CoV-2 vaccines have been developed and distributed worldwide. Because this is the first time that mRNA vaccines have been used, there are several questions regarding their capacity to confer immunity and the durability of the specific anti-SARS-CoV-2 response. Therefore, the objective of this study was to recruit a large cohort of healthcare workers from the Gregorio Marañón Hospital vaccinated with the mRNA-1273 or BNT126b2 vaccines and to follow-up on IgG anti-RBD levels at 8 months post-vaccination.MethodsWe recruited 4,970 volunteers and measured IgG anti-RBD antibodies on days 30 and 240 post-vaccination.ResultsWe observed that both vaccines induced high levels of antibodies on day 30, while a drastic wane was observed on day 240, where mRNA-1273 vaccinated induced higher levels than BNT162b2. Stratifying by vaccine type, age, gender, and comorbidities, we identified that older mRNA-1273-vaccinated volunteers had higher antibody levels than the younger volunteers, contrary to what was observed in the BNT162b2-vaccinated volunteers.DiscussionIn conclusion, we observed that mRNA-1273 has a higher capacity to induce a humoral response than BNT162b2 and that age is a factor in the specific response.

Information on Clostridioides difficile infection (CDI) in patients with COVID-19 is scarce and points to an overall decrease of episodes during the pandemic. This situation results paradoxical, as COVID-19 patients had long periods of hospital stay and high use of antibiotics. We conducted a retrospective study from January 1st 2019 to December 31st 2020 comparing the incidence of hospital-acquired episodes of CDI (HA-CDI) among patients with and without COVID-19 admitted to our institution. During the study period, there were 47,048 patient admissions in 2019, 35,662 admissions of patients without COVID-19 in 2020 and 6,763 of COVID-19 patients. There were 68 episodes of HA-CDI in COVID-19 patients (14.75/10,000 days), 159 in 2020-non-COVID-19 patients (5.54/10,000 days) and 238 in 2019 (6.80/10,000 days). Comparison of HA-CDI in COVID-19 and non-COVID-19 patients indicates it occurs more frequently, in terms of CDI disease severity, COVID-19 does not seem to have a negative impact.

The use of venoarterial (VA) extracorporeal membrane oxygenation therapy (ECMO) in patients admitted to cardiac intensive care units (CICU) has increased. Data regarding infections in this population are scarce. In this retrospective study, we analyzed the risk factors, outcome, and predictors of in-hospital mortality due to nosocomial infections in patients with ECMO admitted to a single coronary intensive care unit between July 2013 and March 2019 treated with VA-ECMO for >48 h. From 69 patients treated with VA-ECMO >48 h, (median age 58 years), 29 (42.0%) patients developed 34 episodes of infections with an infection rate of 0.92/1000 ECMO days. The most frequent were ventilator-associated pneumonia (57.6%), tracheobronchitis (9.1%), bloodstream infections (9.1%), skin and soft tissue infections (9.1%), and cytomegalovirus reactivation (9.1%). In-hospital mortality was 47.8%, but no association with nosocomial infections was found (p = 0.75). The number of days on ECMO (OR 1.14, 95% CI 1.01–1.30, p = 0.029) and noninfectious complications were higher in the infected patients (OR: 3.8 95% CI = 1.05–14.1). A higher baseline creatinine value (OR: 8.2 95% CI = 1.12–60.2) and higher blood lactate level at 4 h after ECMO initiation (OR: 2.0 95% CI = 1.23–3.29) were significant and independent risk factors for mortality. Conclusions: Nosocomial infections in medical patients treated with VA-ECMO are very frequent, mostly Gram-negative respiratory infections. Preventive measures could play an important role for these patients.

We report a complex epidemiological scenario of a nosocomial COVID-19 outbreak in the second wave, based on WGS analysis. Initially, standard epidemiological findings led to the assumption of a homogeneous outbreak caused by a single SARS-CoV-2 strain.

In a population, chemical communication determines the response of animals to changing environmental conditions, what leads to an enhanced resistance against stressors. In response to starvation, the nematode Caenorhabditis elegans arrest post-embryonic development at the first larval stage (L1) right after hatching. As arrested L1 larvae, C. elegans become more resistant to diverse stresses, allowing them to survive for several weeks expecting to encounter more favorable conditions. L1 arrested at high densities display an enhanced resistance to starvation, dependent on soluble compounds released beyond hatching and the first day of arrest. Here, we show that this chemical communication also influences recovery after prolonged periods in L1 arrest. Animals at high density recovered faster than animals at low density. We found that the density effect on survival depends on the final effector of the insulin signaling pathway, the transcription factor DAF-16. Moreover, DAF-16 activation was higher at high density, consistent with a lower expression of the insulin-like peptide DAF-28 in the neurons. The improved recovery of animals after arrest at high density depended on soluble compounds present in the media of arrested L1s. In an effort to find the nature of these compounds, we investigated the disaccharide trehalose as putative signaling molecule, since its production is enhanced during L1 arrest and it is able to activate DAF-16. We detected the presence of trehalose in the medium of arrested L1 larvae at a low concentration. The addition of this concentration of trehalose to animals arrested at low density was enough to rescue DAF-28 production and DAF-16 activation to the levels of animals arrested at high density. However, despite activating DAF-16, trehalose was not capable of reversing survival and recovery phenotypes, suggesting the participation of additional signaling molecules. With all, here we describe a molecular mechanism underlying social communication that allows C. elegans to maintain arrested L1 larvae ready to quickly recover as soon as they encounter nutrient sources.

Abstract Background Advances in automated image-based microscopy platforms coupled with high-throughput liquid workflows have facilitated the design of large-scale screens utilising multicellular model organisms such as Caenorhabditis elegans to identify genetic interactions, therapeutic drugs or disease modifiers. However, the analysis of essential genes has lagged behind because lethal or sterile mutations pose a bottleneck for high-throughput approaches, and a systematic way to analyse genetic interactions of essential genes in multicellular organisms has been lacking. Results In C. elegans, non-conditional lethal mutations can be maintained in heterozygosity using chromosome balancers, commonly expressing green fluorescent protein (GFP) in the pharynx. However, gene expression or function is typically monitored by the use of fluorescent reporters marked with the same fluorophore, presenting a challenge to sort worm populations of interest, particularly at early larval stages. Here, we develop a sorting strategy capable of selecting homozygous mutants carrying a GFP stress reporter from GFP-balanced animals at the second larval stage. Because sorting is not completely error-free, we develop an automated high-throughput image analysis protocol that identifies and discards animals carrying the chromosome balancer. We demonstrate the experimental usefulness of combining sorting of homozygous lethal mutants and automated image analysis in a functional genomic RNA interference (RNAi) screen for genes that genetically interact with mitochondrial prohibitin (PHB). Lack of PHB results in embryonic lethality, while homozygous PHB deletion mutants develop into sterile adults due to maternal contribution and strongly induce the mitochondrial unfolded protein response (UPRmt). In a chromosome-wide RNAi screen for C. elegans genes having human orthologues, we uncover both known and new PHB genetic interactors affecting the UPRmt and growth. Conclusions The method presented here allows the study of balanced lethal mutations in a high-throughput manner. It can be easily adapted depending on the user’s requirements and should serve as a useful resource for the C. elegans community for probing new biological aspects of essential nematode genes as well as the generation of more comprehensive genetic networks.

The implementation of 1,3 β-d-glucan (BDG) has been proposed as a diagnostic tool in antifungal stewardship programs (ASPs). We aimed to analyze the influence of serum BDG in an ASP for oncologic patients and solid organ transplant (SOT) recipients. We conducted a pre–post study. In the initial period (PRE), the ASP was based on bedside advice, and this was complemented with BDG in the post-period (POST). Performance parameters of the BDG assay were determined. Antifungal (AF) use adequacy was evaluated using a point score. Clinical outcomes and AF costs were also compared before and after the intervention. Overall, 85 patients were included in the PRE-period and 112 in the POST-period. Probable or proven fungal infections were similar in both groups (54.1% vs. 57.1%; p = 0.67). The determination of BDG contributed to improved management in 75 of 112 patients (66.9%). The AF adequacy score improved in the POST-period (mean 7.75 vs. 9.29; p < 0.001). Median days of empiric AF treatment was reduced in the POST-period (9 vs. 5 days, p = 0.04). All-cause mortality (44.7% vs. 34.8%; p = 0.16) was similar in both periods. The cost of AF treatments was reduced in the POST-period with a difference of 779.6 €/patient. Our data suggest that the use of BDG was a cost-effective strategy that contributed to safely improving the results of an ASP for SOT and oncologic patients.

BACKGROUND:Cystic echinococcosis (CE) is present in all continents, except for the Antarctica. Characteristically, CE lesions are found in the liver and the lungs, but virtually any part of the body may be affected (the spleen, kidneys, heart, central nervous system, bones, among others). It is estimated that the incidence of bone involvement in CE is 0.5% to 4%. METHODOLOGY:A retrospective study was performed of patients with osseous CE treated at the National Reference Unit of Tropical Diseases of the Ramon y Cajal Hospital, Madrid, Spain, between 1989 and December 2017. Epidemiological, clinical, diagnostic and therapeutic data of patients with long-term follow-up were collected. MAIN FINDINGS:During the study period, of the 104 patients with CE, 27 exhibited bone involvement (26%). The bones most frequently affected were the spine, followed by the ribs, pelvis, femur, tibia and the scapula. The most common symptom was pain followed by medullar syndrome and pathologic fracture. In total, 81.5% of patients underwent surgery for osseous CE at least once. As many as 96% received albendazol either in (mostly long-term) monotherapy or in combination with praziquantel. CONCLUSIONS:The diagnosis and management of osseous CE is challenging. In many cases osseous CE should be considered a chronic disease and should be managed on a case-by-case basis. Lifelong follow-up should be performed for potential recurrence and sequels.

Abstract Parablechnum cordatum (Blechnaceae) exhibits significant morphological variability, with many species considered as its synonyms, and is treated as a species complex by several authors. In this study, we offer a comprehensive revision of the taxon and an updated circumscription of Parablechnum usterianum, a species frequently confused with P. cordatum, leading to a refined understanding of species delimitation. Our analysis, based on morphological and spore micro- and ultrastructural examination of Brazilian specimens within the complex, recognizes three distinct species: Parablechnum cordatum s.s., Parablechnum brasiliense, and Parablechnum regnellianum, the latter being treated as a synonym but now resurrected with a new combination. Here, we present a key to the species, along with detailed descriptions, diagnostic images, distribution maps, lectotypifications, synonymies, and nomenclatural clarifications.

The ascomycete fungus Neurospora is present in many parts of the world, in particular in tropical and subtropical areas, where it is found growing on recently burned vegetation. We have sampled the Neurospora population across Spain. The sampling sites were located in the region of Galicia (northwestern corner of the Iberian peninsula), the province of Cáceres, the city of Seville, and the two major islands of the Canary Islands archipelago (Tenerife and Gran Canaria, west coast of Africa). The sites covered a latitude interval between 27.88° and 42.74°. We have identified wild-type strains of N. discreta, N. tetrasperma, N. crassa, and N. sitophila and the frequency of each species varied from site to site. It has been shown that after exposure to light Neurospora accumulates the orange carotenoid neurosporaxanthin, presumably for protection from UV radiation. We have found that each Neurospora species accumulates a different amount of carotenoids after exposure to light, but these differences did not correlate with the expression of the carotenogenic genes al-1 or al-2. The accumulation of carotenoids in Neurospora shows a correlation with latitude, as Neurospora strains isolated from lower latitudes accumulate more carotenoids than strains isolated from higher latitudes. Since regions of low latitude receive high UV irradiation we propose that the increased carotenoid accumulation may protect Neurospora from high UV exposure. In support of this hypothesis, we have found that N. crassa, the species that accumulates more carotenoids, is more resistant to UV radiation than N. discreta or N. tetrasperma. The photoprotection provided by carotenoids and the capability to accumulate different amounts of carotenoids may be responsible, at least in part, for the distribution of Neurospora species that we have observed across a range of latitudes.

Abstract Fern spores play a vital role in dispersal. The unique features of spores, such as size, thickness, and ornamentation can aid in taxonomic decisions and division of groups. Moreover, certain species of ferns possess green spores containing chlorophyll, an uncommon feature that can reinforce the evidence of relatedness within a specific group. Lomaria spannagelii is a Neotropical species endemic to Brazil, and, even though green spores have been reported in its sister species, Lomaria nuda and Lomaria discolor, the presence of these spores has not yet been investigated in L. spannagelii. In this study, we investigated the morphology of L. spannagelii spores and sporangia, and, additionally, we determined the chromosome count of the species. Our results showed that the analyzed species has green spores, a characteristic shared with L. nuda and L. discolor, and has 2n = 64 chromosomes. This discovery provides new insights into this fern genus and emphasizes the importance of spore color and chromosome count in systematic and evolutionary studies of ferns.