Plant volatile-triggered defense in citrus against biotic stressors.

Plants employ sophisticated defense mechanisms, including releasing volatile organic compounds, to defend against biotic and abiotic stresses. These compounds play a crucial role in plant defense by attracting natural enemies and facilitating communication between plants to activate defense mechanisms. However, there has been no research on how exposure to these compounds activates defense mechanisms in citrus plants. To elucidate the underlying mechanisms governing citrus defensive activation, we conducted a molecular analysis of the rootstock Citrange carrizo [a hybrid of Citrus sinensis × Poncirus trifoliata] in response to defense activation by the volatile (Z)-3-hexenyl propanoate [(Z)-3-HP], utilizing a groundbreaking transcriptomic analysis involving the genomes of both parental species. Our results revealed significant gene expression changes, notably the overexpression of genes related to plant immunity, antioxidant activity, defense against herbivores, and tolerance to abiotic stress. Significantly, P. trifoliata contributed most notably to the hybrid's gene expression profile in response to (Z)-3-HP. Additionally, plants exposed to (Z)-3-HP repelled several citrus pests, attracted natural predators, and led to diminished performance of two key citrus pests. Our study emphasizes the complex molecular basis of volatile-triggered defenses in citrus and highlights the potential of plant volatiles in pest control strategies.

Trigonelline is an NAD+ precursor that improves muscle function during ageing and is reduced in human sarcopenia.

Mitochondrial dysfunction and low nicotinamide adenine dinucleotide (NAD+) levels are hallmarks of skeletal muscle ageing and sarcopenia1-3, but it is unclear whether these defects result from local changes or can be mediated by systemic or dietary cues. Here we report a functional link between circulating levels of the natural alkaloid trigonelline, which is structurally related to nicotinic acid4, NAD+ levels and muscle health in multiple species. In humans, serum trigonelline levels are reduced with sarcopenia and correlate positively with muscle strength and mitochondrial oxidative phosphorylation in skeletal muscle. Using naturally occurring and isotopically labelled trigonelline, we demonstrate that trigonelline incorporates into the NAD+ pool and increases NAD+ levels in Caenorhabditis elegans, mice and primary myotubes from healthy individuals and individuals with sarcopenia. Mechanistically, trigonelline does not activate GPR109A but is metabolized via the nicotinate phosphoribosyltransferase/Preiss-Handler pathway5,6 across models. In C. elegans, trigonelline improves mitochondrial respiration and biogenesis, reduces age-related muscle wasting and increases lifespan and mobility through an NAD+-dependent mechanism requiring sirtuin. Dietary trigonelline supplementation in male mice enhances muscle strength and prevents fatigue during ageing. Collectively, we identify nutritional supplementation of trigonelline as an NAD+-boosting strategy with therapeutic potential for age-associated muscle decline.

Dysimmunity in common variable immunodeficiency is associated with alterations in oral, respiratory, and intestinal microbiota.

Common variable immunodeficiency (CVID) is the most common symptomatic primary immunodeficiency characterized by decreased immunoglobulins and recurrent infections. Its aetiology remains unknown, and some patients present with severe non-infectious autoimmune or inflammatory complications with elevated associated morbimortality. Recently, intestinal dysbiosis has been proposed as a driver of immune dysregulation. In this study, we assessed the oral, respiratory, and gastrointestinal microbiota of 41 CVID patients (24 with dysimmune and 17 with infection complications) and 15 healthy volunteers using 16S rRNA gene sequencing to explore associations between microbiome profiles and CVID phenotypes. Profound differences in the composition of the microbiota in saliva, sputum, and stool were detected between dysimmune CVID patients and healthy individuals. Globally, respiratory species diversity and faecal bacterial richness were lower in CVID individuals with immune complications. Although a single species could not be identified as a robust predictor of dysimmunity, a combination of around 5-7 bacterial species in each type of sample could predict this severe phenotype with an accuracy of around 90% in the study population. Our study provides new insights into these previously unexplored but highly interrelated ecological niches among themselves and with patient profiles. Our data suggest that this disease-related systemic dysbiosis could be implicated in the immune dysregulation associated with severe cases of CVID.

Safety and immunogenicity of the protein-based PHH-1V compared to BNT162b2 as a heterologous SARS-CoV-2 booster vaccine in adults vaccinated against COVID-19: a multicentre, randomised, double-blind, non-inferiority phase IIb trial.

Background: A SARS-CoV-2 protein-based heterodimer vaccine, PHH-1V, has been shown to be safe and well-tolerated in healthy young adults in a first-in-human, Phase I/IIa study dose-escalation trial. Here, we report the interim results of the Phase IIb HH-2, where the immunogenicity and safety of a heterologous booster with PHH-1V is assessed versus a homologous booster with BNT162b2 at 14, 28 and 98 days after vaccine administration. Methods: The HH-2 study is an ongoing multicentre, randomised, active-controlled, double-blind, non-inferiority Phase IIb trial, where participants 18 years or older who had received two doses of BNT162b2 were randomly assigned in a 2:1 ratio to receive a booster dose of vaccine-either heterologous (PHH-1V group) or homologous (BNT162b2 group)-in 10 centres in Spain. Eligible subjects were allocated to treatment stratified by age group (18-64 versus ≥65 years) with approximately 10% of the sample enrolled in the older age group. The primary endpoints were humoral immunogenicity measured by changes in levels of neutralizing antibodies (PBNA) against the ancestral Wuhan-Hu-1 strain after the PHH-1V or the BNT162b2 boost, and the safety and tolerability of PHH-1V as a boost. The secondary endpoints were to compare changes in levels of neutralizing antibodies against different variants of SARS-CoV-2 and the T-cell responses towards the SARS-CoV-2 spike glycoprotein peptides. The exploratory endpoint was to assess the number of subjects with SARS-CoV-2 infections ≥14 days after PHH-1V booster. This study is ongoing and is registered with ClinicalTrials.gov, NCT05142553. Findings: From 15 November 2021, 782 adults were randomly assigned to PHH-1V (n = 522) or BNT162b2 (n = 260) boost vaccine groups. The geometric mean titre (GMT) ratio of neutralizing antibodies on days 14, 28 and 98, shown as BNT162b2 active control versus PHH-1V, was, respectively, 1.68 (p < 0.0001), 1.31 (p = 0.0007) and 0.86 (p = 0.40) for the ancestral Wuhan-Hu-1 strain; 0.62 (p < 0.0001), 0.65 (p < 0.0001) and 0.56 (p = 0.003) for the Beta variant; 1.01 (p = 0.92), 0.88 (p = 0.11) and 0.52 (p = 0.0003) for the Delta variant; and 0.59 (p ≤ 0.0001), 0.66 (p < 0.0001) and 0.57 (p = 0.0028) for the Omicron BA.1 variant. Additionally, PHH-1V as a booster dose induced a significant increase of CD4+ and CD8+ T-cells expressing IFN-γ on day 14. There were 458 participants who experienced at least one adverse event (89.3%) in the PHH-1V and 238 (94.4%) in the BNT162b2 group. The most frequent adverse events were injection site pain (79.7% and 89.3%), fatigue (27.5% and 42.1%) and headache (31.2 and 40.1%) for the PHH-1V and the BNT162b2 groups, respectively. A total of 52 COVID-19 cases occurred from day 14 post-vaccination (10.14%) for the PHH-1V group and 30 (11.90%) for the BNT162b2 group (p = 0.45), and none of the subjects developed severe COVID-19. Interpretation: Our interim results from the Phase IIb HH-2 trial show that PHH-1V as a heterologous booster vaccine, when compared to BNT162b2, although it does not reach a non-inferior neutralizing antibody response against the Wuhan-Hu-1 strain at days 14 and 28 after vaccination, it does so at day 98. PHH-1V as a heterologous booster elicits a superior neutralizing antibody response against the previous circulating Beta and the currently circulating Omicron BA.1 SARS-CoV-2 variants in all time points assessed, and for the Delta variant on day 98 as well. Moreover, the PHH-1V boost also induces a strong and balanced T-cell response. Concerning the safety profile, subjects in the PHH-1V group report significantly fewer adverse events than those in the BNT162b2 group, most of mild intensity, and both vaccine groups present comparable COVID-19 breakthrough cases, none of them severe. Funding: HIPRA SCIENTIFIC, S.L.U.

Influence of Different Rootstocks on Fruit Quality and Primary and Secondary Metabolites Content of Blood Oranges Cultivars.

Blood oranges have high concentrations of bioactive compounds that are beneficial to health. In Europe, the cultivation of blood oranges is increasing due to their excellent nutritional properties. In Citrus crops, rootstocks play an important role in juice and can increase the content of bioactive compounds. The morphological, qualitative and nutritional parameters were analyzed in cultivars 'Tarocco Ippolito', 'Tarocco Lempso', 'Tarocco Tapi' and 'Tarocco Fondaconuovo' grafted onto Citrus macrophylla and Citrus reshni. 'Tarocco Lempso' grafted onto Citrus macrophylla obtained the highest values of weight (275.78 g), caliber (81.37 mm and 76.79 mm) and juice content (162.11 g). 'Tarocco Tapi' grafted onto Citrus reshni obtained the most interesting qualitative parameters (15.40 °Brix; 12.0 MI). 'Tarocco Lempso' grafted onto Citrus reshni obtained the most intense red juice (a* = 9.61). Overall, the highest concentrations of primary metabolites were in proline, aspartate, citric acid, and sucrose. The results showed that 'Tarocco Ippolito' juice grafted onto Citrus reshni had the highest levels of total hydroxycinnamic acids (263.33 mg L-1), total flavones (449.74 mg L-1) and total anthocyanins (650.42 mg L-1). To conclude, 'Tarocco Lempso' grafted onto Citrus macrophylla obtained the best values of agronomic parameters, and the cultivars grafted onto Citrus reshni obtained significantly higher concentrations in primary and secondary metabolites.

Effect of Rootstock on the Volatile Profile of Mandarins.

Mandarin production has increased in recent years, especially for fresh consumption, due to its ease of peeling, its aroma, and its content of bioactive compounds. In this sense, aromas play a fundamental role in the sensory quality of this fruit. The selection of the appropriate rootstock is crucial for the success of the crop and its quality. Therefore, the objective of this study was to identify the influence of 9 rootstocks ("Carrizo citrange", "Swingle citrumelo CPB 4475", "Macrophylla", "Volkameriana", "Forner-Alcaide 5", "Forner-Alcaide V17", "C-35", "Forner-Alcaide 418", and "Forner-Alcaide 517") on the volatile composition of "Clemenules" mandarin. For this, the volatile compounds of mandarin juice were measured using headspace solid-phase micro-extraction in a gas chromatograph coupled to a mass spectrometer (GC-MS). Seventy-one volatile compounds were identified in the analyzed samples, with limonene being the main compound. The results obtained showed that the rootstock used in the cultivation of mandarins affects the volatile content of the juice, with "Carrizo citrange", "Forner-Alcaide 5", "Forner-Alcaide 418", and "Forner-Alcaide 517" being those that presented the highest concentration.

Safety perception in the operating environment: The nurses' perspective versus that of the surgeons.

Background: Adverse effects due to surgery occur in 25% of patients, and the patient safety perception seems to differ between nurses and surgeons in the operating room (OR). This difference can be attributed to lack of communication. However, our hospital has not conducted any studies on patient safety climate (PSC) in the OR. Aims: To determine if the perception of PSC of nurses and surgeons in the OR diverges and understand whether these differences could be explained by communication gap. Methods: A total of 42 perioperative nurses and 44 surgeons in the OR of a tertiary hospital answered the Spanish version of the US Hospital Survey on PSC. This was an observational, cross-sectional study with descriptive statistics and a non-parametric test. Results: Nurses had a worse perception of the dimensions of overall safety, leader expectations, teamwork within units, feedback, staffing, and hospital management (p < .05). Although no differences were found concerning organizational learning/continuous improvement, communication openness, nonpunitive responses, and teamwork across hospital unit dimensions, the findings suggest that the nurses' perception was worse than that of the surgeons. Conclusions: In general, OR nurses have worse PSC than surgeons, mainly in the areas where communication it's important. Our study has provided the data that will enable the hospital management team to make decisions to improve the PSC in the operating room area. We recommend a more active presence of nurses in directive teams.

Current clinical spectrum of common variable immunodeficiency in Spain: The multicentric nationwide GTEM-SEMI-CVID registry.

Common variable immunodeficiency (CVID) constitutes a heterogenic group of primary immunodeficiency disorders with a wide-ranging clinical spectrum. CVID-associated non-infectious morbidity constitutes a major challenge requiring a full understanding of its pathophysiology and its clinical importance and global variability, especially considering the broad clinical, genetic, and regional heterogeneity of CVID disorders. This work aimed to develop a nationwide, multicenter, retrospective study over a 3-year period describing epidemiological, clinical, laboratory, therapeutic, and prognostic features of 250 CVID patients in Spain. The mean diagnostic delay was around 10 years and most patients initially presented with infectious complications followed by non-infectious immune disorders. However, infectious diseases were not the main cause of morbimortality. Non-infectious lung disease was extraordinarily frequent in our registry affecting approximately 60% of the patients. More than one-third of the patients in our cohort showed lymphadenopathies and splenomegaly in their follow-up, and more than 33% presented immune cytopenias, especially Evans' syndrome. Gastrointestinal disease was observed in more than 40% of the patients. Among biopsied organs in our cohort, benign lymphoproliferation was the principal histopathological alteration. Reaching 15.26%, the global prevalence of cancer in our registry was one of the highest reported to date, with non-Hodgkin B lymphoma being the most frequent. These data emphasize the importance of basic and translational research delving into the pathophysiological pathways involved in immune dysregulation and diffuse lymphocytic infiltration. This would reveal new tailored strategies to reduce immune complications, and the associated healthcare burden, and ensure a better quality of life for CVID patients.

Nicotinamide riboside kinases regulate skeletal muscle fiber-type specification and are rate-limiting for metabolic adaptations during regeneration.

Nicotinamide riboside kinases (NRKs) control the conversion of dietary Nicotinamide Riboside (NR) to NAD+, but little is known about their contribution to endogenous NAD+ turnover and muscle plasticity during skeletal muscle growth and remodeling. Using NRK1/2 double KO (NRKdKO) mice, we investigated the influence of NRKs on NAD+ metabolism and muscle homeostasis, and on the response to neurogenic muscle atrophy and regeneration following muscle injury. Muscles from NRKdKO animals have altered nicotinamide (NAM) salvage and a decrease in mitochondrial content. In single myonuclei RNAseq of skeletal muscle, NRK2 mRNA expression is restricted to type IIx muscle fibers, and perturbed NAD+ turnover and mitochondrial metabolism shifts the fiber type composition of NRKdKO muscle to fast glycolytic IIB fibers. NRKdKO does not influence muscle atrophy during denervation but alters muscle repair after myofiber injury. During regeneration, muscle stem cells (MuSCs) from NRKdKO animals hyper-proliferate but fail to differentiate. NRKdKO also alters the recovery of NAD+ during muscle regeneration as well as mitochondrial adaptations and extracellular matrix remodeling required for tissue repair. These metabolic perturbations result in a transient delay of muscle regeneration which normalizes during myofiber maturation at late stages of regeneration via over-compensation of anabolic IGF1-Akt signaling. Altogether, we demonstrate that NAD+ synthesis controls mitochondrial metabolism and fiber type composition via NRK1/2 and is rate-limiting for myogenic commitment and mitochondrial maturation during skeletal muscle repair.

Nicotinamide Riboside and Dihydronicotinic Acid Riboside Synergistically Increase Intracellular NAD+ by Generating Dihydronicotinamide Riboside.

Through evolution, eukaryote organisms have developed the ability to use different molecules as independent precursors to generate nicotinamide adenine dinucleotide (NAD+), an essential molecule for life. However, whether these different precursors act in an additive or complementary manner is not truly well understood. Here, we have evaluated how combinations of different NAD+ precursors influence intracellular NAD+ levels. We identified dihydronicotinic acid riboside (NARH) as a new NAD+ precursor in hepatic cells. Second, we demonstrate how NARH, but not any other NAD+ precursor, can act synergistically with nicotinamide riboside (NR) to increase NAD+ levels in cultured cells and in mice. Finally, we demonstrate that the large increase in NAD+ prompted by the combination of these two precursors is due to their chemical interaction and conversion to dihydronicotinamide riboside (NRH). Altogether, this work demonstrates for the first time that NARH can act as a NAD+ precursor in mammalian cells and how different NAD+ precursors can interact and influence each other when co-administered.

From the bacterial citrus microbiome to the selection of potentially host-beneficial microbes.

Citrus is the most cultivated fruit crop worldwide. The modern citrus industry needs new bioproducts to overcome phytopathological threats, tolerate stresses and increase yield and quality. Mutualistic microbes from roots significantly impact host physiology and health and are a potentially beneficial resource. The bacterial microbiome can be surveyed to select potentially host-beneficial microbes. To achieve this goal, a prevalent "core-citrus" bacterial microbiome was obtained by picking those operational taxonomic units (OTUs) shared among samples within and across two Citrus rootstock genotypes grown in the same soil for more than 20 years. A sub-selection of main OTUs from the defined "core-citrus" microbiome was made based on abundance, host-enriched versus bulk soil, and rhizosphere-indicator species. In parallel, an extensive census of the cultivable microbiota was performed to collect a large number of bacterial citrus isolates. Metataxonomic data were linked to cultured microbes, matching 16S rRNA gene sequences from bacterial isolates with those counterpart OTU reference sequences from the selected bacterial "core-citrus" microbiome. This approach allowed selection of potentially host-beneficial bacteria to mine for agricultural probiotics in future biotechnological applications required for the citrus industry.

Comparative transcriptomic analyses of citrus cold-resistant vs. sensitive rootstocks might suggest a relevant role of ABA signaling in triggering cold scion adaption.

BACKGROUND: The citrus genus comprises a number of sensitive tropical and subtropical species to cold stress, which limits global citrus distribution to certain latitudes and causes major economic loss. We used RNA-Seq technology to analyze changes in the transcriptome of Valencia delta seedless orange in response to long-term cold stress grafted on two frequently used citrus rootstocks: Carrizo citrange (CAR), considered one of the most cold-tolerant accessions; C. macrophylla (MAC), a very sensitive one. Our objectives were to identify the genetic mechanism that produce the tolerant or sensitive phenotypes in citrus, as well as to gain insights of the rootstock-scion interactions that induce the cold tolerance or sensitivity in the scion. RESULTS: Plants were kept at 1 ºC for 30 days. Samples were taken at 0, 15 and 30 days. The metabolomic analysis showed a significant increase in the concentration of free sugars and proline, which was higher for the CAR plants. Hormone quantification in roots showed a substantially increased ABA concentration during cold exposure in the CAR roots, which was not observed in MAC. Different approaches were followed to analyze gene expression. During the stress treatment, the 0-15-day comparison yielded the most DEGs. The functional characterization of DEGs showed enrichment in GO terms and KEGG pathways related to abiotic stress responses previously described in plant cold adaption. The DEGs analysis revealed that several key genes promoting cold adaption were up-regulated in the CAR plants, and those repressing it had higher expression levels in the MAC samples. CONCLUSIONS: The metabolomic and transcriptomic study herein performed indicates that the mechanisms activated in plants shortly after cold exposure remain active in the long term. Both the hormone quantification and differential expression analysis suggest that ABA signaling might play a relevant role in promoting the cold hardiness or sensitiveness of Valencia sweet orange grafted onto Carrizo citrange or Macrophylla rootstocks, respectively. Our work provides new insights into the mechanisms by which rootstocks modulate resistance to abiotic stress in the production variety grafted onto them.

WHO Ordinal Scale and Inflammation Risk Categories in COVID-19. Comparative Study of the Severity Scales.

BACKGROUND: The WHO ordinal severity scale has been used to predict mortality and guide trials in COVID-19. However, it has its limitations. OBJECTIVE: The present study aims to compare three classificatory and predictive models: the WHO ordinal severity scale, the model based on inflammation grades, and the hybrid model. DESIGN: Retrospective cohort study with patient data collected and followed up from March 1, 2020, to May 1, 2021, from the nationwide SEMI-COVID-19 Registry. The primary study outcome was in-hospital mortality. As this was a hospital-based study, the patients included corresponded to categories 3 to 7 of the WHO ordinal scale. Categories 6 and 7 were grouped in the same category. KEY RESULTS: A total of 17,225 patients were included in the study. Patients classified as high risk in each of the WHO categories according to the degree of inflammation were as follows: 63.8% vs. 79.9% vs. 90.2% vs. 95.1% (p<0.001). In-hospital mortality for WHO ordinal scale categories 3 to 6/7 was as follows: 0.8% vs. 24.3% vs. 45.3% vs. 34% (p<0.001). In-hospital mortality for the combined categories of ordinal scale 3a to 5b was as follows: 0.4% vs. 1.1% vs. 11.2% vs. 27.5% vs. 35.5% vs. 41.1% (p<0.001). The predictive regression model for in-hospital mortality with our proposed combined ordinal scale reached an AUC=0.871, superior to the two models separately. CONCLUSIONS: The present study proposes a new severity grading scale for COVID-19 hospitalized patients. In our opinion, it is the most informative, representative, and predictive scale in COVID-19 patients to date.

Integrating Clinics, Laboratory, and Imaging for the Diagnosis of Common Variable Immunodeficiency-Related Granulomatous-Lymphocytic Interstitial Lung Disease.

Background: Granulomatous-lymphocytic interstitial lung disease (GLILD) is a distinct clinic-radio-pathological interstitial lung disease (ILD) that develops in 9% to 30% of patients with common variable immunodeficiency (CVID). Often related to extrapulmonary dysimmune disorders, it is associated with long-term lung damage and poorer clinical outcomes. The aim of this study was to explore the potential use of the integration between clinical parameters, laboratory variables, and developed CT scan scoring systems to improve the diagnostic accuracy of non-invasive tools. Methods: A retrospective cross-sectional study of 50 CVID patients was conducted in a referral unit of primary immune deficiencies. Clinical variables including demographics and comorbidities; analytical parameters including immunoglobulin levels, lipid metabolism, and lymphocyte subpopulations; and radiological and lung function test parameters were collected. Baumann's GLILD score system was externally validated by two observers in high-resolution CT (HRCT) scans. We developed an exploratory predictive model by elastic net and Bayesian regression, assessed its discriminative capacity, and internally validated it using bootstrap resampling. Results: Lymphadenopathies (adjusted OR 9.42), splenomegaly (adjusted OR 6.25), Baumann's GLILD score (adjusted OR 1.56), and CD8+ cell count (adjusted OR 0.9) were included in the model. The larger range of values of the validated Baumann's GLILD HRCT scoring system gives it greater predictability. Cohen's κ statistic was 0.832 (95% CI 0.70-0.90), showing high concordance between both observers. The combined model showed a very good discrimination capacity with an internally validated area under the curve (AUC) of 0.969. Conclusion: Models integrating clinics, laboratory, and CT scan scoring methods may improve the accuracy of non-invasive diagnosis of GLILD and might even preclude aggressive diagnostic tools such as lung biopsy in selected patients.

Clinical evaluation of antiseptic mouth rinses to reduce salivary load of SARS-CoV-2.

Most public health measures to contain the COVID-19 pandemic are based on preventing the pathogen spread, and the use of oral antiseptics has been proposed as a strategy to reduce transmission risk. The aim of this manuscript is to test the efficacy of mouthwashes to reduce salivary viral load in vivo. This is a multi-centre, blinded, parallel-group, placebo-controlled randomised clinical trial that tests the effect of four mouthwashes (cetylpyridinium chloride, chlorhexidine, povidone-iodine and hydrogen peroxide) in SARS-CoV-2 salivary load measured by qPCR at baseline and 30, 60 and 120 min after the mouthrinse. A fifth group of patients used distilled water mouthrinse as a control. Eighty-four participants were recruited and divided into 12-15 per group. There were no statistically significant changes in salivary viral load after the use of the different mouthwashes. Although oral antiseptics have shown virucidal effects in vitro, our data show that salivary viral load in COVID-19 patients was not affected by the tested treatments. This could reflect that those mouthwashes are not effective in vivo, or that viral particles are not infective but viral RNA is still detected by PCR. Viral infectivity studies after the use of mouthwashes are therefore required. ( https://clinicaltrials.gov/ct2/show/NCT04707742 ; Identifier: NCT04707742).

A Method to Monitor the NAD+ Metabolome-From Mechanistic to Clinical Applications.

Nicotinamide adenine dinucleotide (NAD+) and its reduced form (NADH) are coenzymes employed in hundreds of metabolic reactions. NAD+ also serves as a substrate for enzymes such as sirtuins, poly(ADP-ribose) polymerases (PARPs) and ADP-ribosyl cyclases. Given the pivotal role of NAD(H) in health and disease, studying NAD+ metabolism has become essential to monitor genetic- and/or drug-induced perturbations related to metabolic status and diseases (such as ageing, cancer or obesity), and its possible therapies. Here, we present a strategy based on liquid chromatography-tandem mass spectrometry (LC-MS/MS), for the analysis of the NAD+ metabolome in biological samples. In this method, hydrophilic interaction chromatography (HILIC) was used to separate a total of 18 metabolites belonging to pathways leading to NAD+ biosynthesis, including precursors, intermediates and catabolites. As redox cofactors are known for their instability, a sample preparation procedure was developed to handle a variety of biological matrices: cell models, rodent tissues and biofluids, as well as human biofluids (urine, plasma, serum, whole blood). For clinical applications, quantitative LC-MS/MS for a subset of metabolites was demonstrated for the analysis of the human whole blood of nine volunteers. Using this developed workflow, our methodology allows studying NAD+ biology from mechanistic to clinical applications.

Chagas disease screening in pregnant Latin American women: Adherence to a systematic screening protocol in a non-endemic country.

BACKGROUND: Chagas disease (CD) is a chronic parasitic disease caused by Trypanosoma cruzi and is endemic to continental Latin America. In Spain, the main transmission route is congenital. We aimed to assess adherence to regional recommendations of universal screening for CD during pregnancy in Latin American women in the province of Alicante from 2014 to 2018. METHODOLOGY/PRINCIPAL FINDINGS: Retrospective quality study using two data sources: 1) delivery records of Latin American women that gave birth in the 10 public hospitals of Alicante between January 2014 and December 2018; and 2) records of Chagas serologies carried out in those centers between May 2013 and December 2018. There were 3026 deliveries in Latin American women during the study period; 1178 (38.9%) underwent CD serology. Screening adherence ranged from 17.2% to 59.3% in the different health departments and was higher in Bolivian women (48.3%). Twenty-six deliveries (2.2%) had a positive screening; CD was confirmed in 23 (2%) deliveries of 21 women. Bolivians had the highest seroprevalence (21/112; 18.7%), followed by Colombians (1/333; 0.3%) and Ecuadorians (1/348; 0.3%). Of 21 CD-positive women (19 Bolivians, 1 Colombian, 1 Ecuadorian), infection was already known in 12 (57.1%), and 9 (42.9%) had already been treated. Only 1 of the 12 untreated women (8.3%) was treated postpartum. Follow-up started in 20 of the 23 (87.0%) neonates but was completed only in 11 (47.8%); no cases of congenital transmission were detected. Among the 1848 unscreened deliveries, we estimate 43 undiagnosed cases of CD and 1 to 2 undetected cases of congenital transmission. CONCLUSIONS/SIGNIFICANCE: Adherence to recommendations of systematic screening for CD in Latin American pregnant women in Alicante can be improved. Strategies to strengthen treatment of postpartum women and monitoring of exposed newborns are needed. Currently, there may be undetected cases of congenital transmission in our province.

Rootstock effect on fruit quality, anthocyanins, sugars, hydroxycinnamic acids and flavanones content during the harvest of blood oranges 'Moro' and 'Tarocco Rosso' grown in Spain.

The physico-chemical quality parameters (external and internal color, firmness, acidity, total soluble solids, anthocyanins, sugars, hydroxycinnamic acids and flavanones) of 'Moro' and 'Tarocco Rosso' blood oranges grafted onto eight different rootstocks at three harvest time were studied. The rootstocks were 'Carrizo', 'C-35', 'Cleopatra' mandarin, 'Citrus volkameriana', 'Citrus macrophylla', 'Swingle' citrumelo, 'Forner-Alcaide 5' and 'Forner-Alcaide 13'. All studied parameters were highly rootstock/scion-dependent and showed changes throughout harvest. The content of the main anthocyanins revealed their relation with internal fruit color in both cultivars. The rootstocks that led to fruit with the lowest anthocyanins displayed the least sucrose content. The differences detected in the amount of hydroxycinnamic acids (chlorogenic, ferulic and sinapic) and flavanones (hesperidin, narirutin and didymin) related to anthocyanins content, explained phenylpropanoid pathway.

Biomarker-based validity of a food frequency questionnaire estimating intake in Brazilian children and adolescents.

This study evaluated the validity of nutrient and food group intakes estimated by an FFQ against biomarkers. A 71-item semiquantitative FFQ was administered to 210 Brazilian children and adolescents aged 9-13 years. Intakes were correlated with biomarkers in plasma and red blood cells. Correlations between nutrients and their biomarkers were presented for animal protein, myristic acid (C14:0), EPA, DHA, ß-carotene, folate, and vitamins B3, B5 and B6. Food groups and biomarkers were correlated as follows: fish products with EPA and DHA; milk and dairy with C14:0, pyridoxal 5'-phosphate and vitamin B12; total vegetables and dark green and orange vegetables with ß-carotene; 5-methyltetrahydrofolate with green vegetables; and flour products with para-aminobenzoylglutamic acid. This FFQ is a valid tool for ranking Brazilian children and adolescents according to their intake of several nutrients and food groups.

Predicting Clinical Outcome with Phenotypic Clusters in COVID-19 Pneumonia: An Analysis of 12,066 Hospitalized Patients from the Spanish Registry SEMI-COVID-19.

(1) Background: Different clinical presentations in COVID-19 are described to date, from mild to severe cases. This study aims to identify different clinical phenotypes in COVID-19 pneumonia using cluster analysis and to assess the prognostic impact among identified clusters in such patients. (2) Methods: Cluster analysis including 11 phenotypic variables was performed in a large cohort of 12,066 COVID-19 patients, collected and followed-up from 1 March to 31 July 2020, from the nationwide Spanish Society of Internal Medicine (SEMI)-COVID-19 Registry. (3) Results: Of the total of 12,066 patients included in the study, most were males (7052, 58.5%) and Caucasian (10,635, 89.5%), with a mean age at diagnosis of 67 years (standard deviation (SD) 16). The main pre-admission comorbidities were arterial hypertension (6030, 50%), hyperlipidemia (4741, 39.4%) and diabetes mellitus (2309, 19.2%). The average number of days from COVID-19 symptom onset to hospital admission was 6.7 (SD 7). The triad of fever, cough, and dyspnea was present almost uniformly in all 4 clinical phenotypes identified by clustering. Cluster C1 (8737 patients, 72.4%) was the largest, and comprised patients with the triad alone. Cluster C2 (1196 patients, 9.9%) also presented with ageusia and anosmia; cluster C3 (880 patients, 7.3%) also had arthromyalgia, headache, and sore throat; and cluster C4 (1253 patients, 10.4%) also manifested with diarrhea, vomiting, and abdominal pain. Compared to each other, cluster C1 presented the highest in-hospital mortality (24.1% vs. 4.3% vs. 14.7% vs. 18.6%; p < 0.001). The multivariate study identified age, gender (male), body mass index (BMI), arterial hypertension, chronic obstructive pulmonary disease (COPD), ischemic cardiopathy, chronic heart failure, chronic hepatopathy, Charlson's index, heart rate and respiratory rate upon admission >20 bpm, lower PaO2/FiO2 at admission, higher levels of C-reactive protein (CRP) and lactate dehydrogenase (LDH), and the phenotypic cluster as independent factors for in-hospital death. (4) Conclusions: The present study identified 4 phenotypic clusters in patients with COVID-19 pneumonia, which predicted the in-hospital prognosis of clinical outcomes.