First Report of Fusarium proliferatum Causing Basal Rot and Dry Rot on Onion Bulbs (Allium cepa) in Spain.

Spain is the second largest onion-producing country in Europe with 1,465,430 tons and an export value of 584 million € in 2021 (MAPA 2022). In summer 2022 rot bulb symptoms were observed in five commercial fields and during the storage of cultivars 'Orlenda', 'Veleta', 'Mallory', 'Citation' and 'Pantano' from La Roda in Albacete (Castilla-La Mancha, Spain). Approximately 20% of sampled bulbs (113 bulbs analyzed) were affected with dry scales showing brown to dark brown rot on the top and basal plate of the onion bulbs. Occasionally, white to light pink fungal mycelium was observed between rotten scales and the plate basal. Sections of dry scales (5-10 mm) of the apical and basal plate were cut and placed on potato dextrose agar (PDA) and Komada medium (Komada 1975). From 5-day-old cultures typical white to light pink mycelium with microconidia in chains formed on polyphialides and macroconidia resembling Fusarium proliferatum (Nelson et al. 1983). To confirm the pathogen identity, partial translation elongation factor 1-alpha (TEF1) and RNA polymerase II subunit 2 (RPB2) genes were amplified and sequenced using primers reported in O´Donnell et al. (1998) and Samuels et al. (2002) for TEF1 and Liu et al. (1999) for RPB2. In BLAST analyses, the sequences showed 100% identity to the corresponding region of F. proliferatum (KP964908 and JF740801). Sequences were submitted to GenBank, and registered accession numbers are OR061014-16 for TEF1 and OR061017-19 for RPB2. Pathogenicity tests were conducted by inoculating healthy onion bulbs (five replicates per treatment) on the apical and basal plate by placing a 7-day old mycelial plug (10 mm diameter) from PDA cultures. Two onion cultivars ('Pandero' by Nunhems USA and 'Mallory' by Bejo The Netherlands) were inoculated separately with three isolates (PRO1, PRO9, PRO12). Control bulbs were inoculated with sterile PDA. The experiment was carried out twice. All bulbs were placed in a moist chamber and incubated at 25°C in the dark. After 15 days, bulbs inoculated with mycelial plugs showed similar symptoms to those of the original diseased bulbs. Browning dry rot was observed on the apical and basal plate of bulbs. When bulbs were cut longitudinally inner progressing rot was observed. Control bulbs remained symptomless. In both experiments, F. proliferatum was successfully re-isolated and morphologically confirmed from symptomatic bulbs to fulfill Koch's postulates. These results confirmed that isolates PRO1, PRO9 and PRO12 were the pathogen causing basal and dry rot on onion bulbs. This pathogen has recently been identified in China on Allium cepa L. var. agrogatum (Liu et al. 2022) and Idaho on onion (Beck et al. 2020) and could become a serious threat to onion production in Spain, reducing the quality and yield of onion.

First Report of Fusarium oxysporum f. sp. lactucae Race 4 Causing Lettuce Wilt in Spain.

Spain is the fourth largest lettuce-producing country in the world and the leading European producer. Much of the production, mainly grow in open field, is dedicated to export with a value of 887 million U.S. dollars per year. In summer 2021 wilting symptoms were observed in a commercial field crop on butterhead lettuce 'Amible' in Albacete (Castilla-La Mancha, Spain). Approximately 15% of plants were affected, but losses were even more severe on subsequent crops. Vascular tissue of affected plants showed a brown to red discoloration. Sections of infected vascular tissue (3 to 5 mm long) were surface sterilized in 70% ethanol for 30 s, washed three times with sterile water, and plated on potato dextrose agar (PDA) amended with streptomycin sulfate (100 mg/liter). From 5-day-old cultures typical pale cream to purplish mycelia with microconidia, macroconidia, and chlamydospores of Fusarium oxysporum were observed. Microconidia were abundant on carnation leaf agar and measured 6.1 to 9.2 µm (mean 7.1 ± 0.7 µm; n=50)). Macroconidia were sparse, three-septate, straight to slightly curved, 23.3 to 34.8 × 4 to 5.2 µm (mean 31.5 ± 2.8 × 4.2 ± 0.3 µm; n=50). Chlamydospores were terminal and intercalary, rough walled, and measured 7.2 to 10.1 µm (mean 9.5 ± 0.6 µm; n=50) µm. DNA was extracted from three single-spore isolates using the protocol of Querol et al. (1992) and the translation elongation factor 1-α gene (TEF) was sequenced with exTEF-F/FUexTEF-R primers as described by Taylor et al. (2016). All TEF sequences (GenBank accession no. OP903519) were identical. In BLAST analyses, the isolates showed 100% identity to the corresponding region of Fusarium oxysporum f. sp. lactucae (FOL) race 4 (MK059958). All Spanish isolates were identified as FOL race 4 using a race-specific polymerase chain reaction (PCR) with the primers FPUF/FPUR (Gilardi et al. 2017), and a previously identified FOL race 4 isolate Fus 1.01 as a positive control. Pathogenicity tests were conducted to confirm the positive result of the race 4-specific PCR and to complete Koch's postulates. Three differential lettuce cultivars ('Costa Rica No. 4', 'Banchu Red Fire', and 'Romana Romabella 30 CN') provided by Rijk Zwaan (The Netherlands) were inoculated with three Spanish isolates (Al1A1, Al1D, Al2B) and the Fus 1.01 isolate used as FOL race 4 positive control (Claerbout et al., 2018). Roots of 3-week-old plants (five replicates per treatment) were dipped in a spore suspension (1 × 106 conidia/ml) for 10 min before transplanting into 250-ml pots with sterile substrate. Non-inoculated control plants were dipped in sterile water for 10 min. The experiment was carried out twice. Inoculated lettuce seedlings were planted and maintained in a growth chamber (25°C day, 18°C night). Plants were slightly watered every other day. After 21 days, wilting was observed in the cultivars 'Costa Rica No. 4' and 'Romana Romabella 30 CN'. Moreover, taproots were cut longitudinally, and vascular browning was observed in the taproot. No discoloration could be observed in the taproot of 'Banchu Red Fire' plants, coinciding with the result of isolate Fus 1.01 used as FOL race 4 positive control. Non-inoculated control plants remained healthy and vascular browning was not observed. In both experiments, F. oxysporum f.sp. lactucae was consistently reisolated using PDA medium. These results confirmed that the isolates Al1A1, Al1D and Al2B were FOL race 4. This race has recently been identified in The Netherlands (Gilardi et al. 2017), Belgium (Claerbout et al. 2018), United Kingdom, Ireland (Taylor et al. 2019) and Italy (Gilardi et al. 2019) and could become a serious threat to Spain lettuce production.

Comparison of Three Workstations For Abdominal Aortic Aneurysm Sizing: Impact in Decision Making and Graft Selection.

PURPOSE: The aim of this study was to assess the agreement of multiplanar reconstruction (MPR) and semiautomated central lumen line (CLL) analysis of abdominal aortic aneurysms (AAA), with 3 different software workstations (WS1, WS2, WS3) and 2 experienced practitioners as well as to analyze its eventual impact in graft selection. MATERIALS AND METHODS: Twenty computed tomography (CT) angiography data sets were randomly chosen from a series of 100 consecutive studies. Measurements were performed twice by each reader, in random order, and included 8 parameters (5 diameters and 3 lengths). Each observer performed a complete set of 60 studies. Intra-observer and interobserver variability for every WS was assessed. Measurements were evaluated using Bland-Altman analysis, correlation coefficients (r), and concordance correlation coefficients (CCC [95% confidence interval (CI)]). RESULTS: A high overall agreement between repeated measurements for both observers was obtained (r=0.989; CCC=0.988 [0.982-0.992] and r=0.998; CCC=0.996 [0.994-0.997], for observers 1 and 2, respectively). However, reproducibility for individual parameters was excellent for observer 2 and only moderate for observer 1. A high overall agreement was obtained for interobserver concordance (r=0.987; CCC=0.986 [0.982-0.989]). When analyzing for individual parameters, greatest interobserver differences were found at CLL measurement of the diameter of aortic neck (WS2) and bifurcation (WS1 and WS2) as well as iliac diameter in all 3 WS for both CLL and MPR. Similar differences were observed in paired comparison between WS when involving these parameters. Careful inspection of Bland-Altman charts revealed some cases of disagreement between WS and observers that would affect decision making on graft selection, changing the neck diameter to a different size, in 2 cases when measuring with WS1, and iliac diameter in 4 cases (2 of them with WS1 and 2 with WS2). Greatest discordance was observed regarding ipsilateral iliac length affecting 7 measurements that would lead to change the length of the selected limb graft (2 with WS1, 3 with WS2, and 2 with WS3). CONCLUSIONS: Although a high agreement between different observers using different WS for AAA measurements is to be expected, small differences may lead to the selection of a different graft size. The use of a single software by experienced users, and double check by a different one, may be advisable. CLINICAL IMPACT: Influence of inter and intraobserver variability in CT measurements during planning of endovascular aneurysm repair (EVAR) has been extensively reviewed. However, its impact in graft selection (final choose of diameter and lengths) has been scarcely analyzed. The results of this study suggest that, although a high agreement between different observers using different workstations for AAA measurements is to be expected, small differences may lead to the selection of a different graft size. The use of a single software by experienced users, and double check by a different one, may be advisable.

Methylene Blue functionalized carbon nanodots combined with different shape gold nanostructures for sensitive and selective SARS-CoV-2 sensing.

The development of DNA-sensing platforms based on new synthetized Methylene Blue functionalized carbon nanodots combined with different shape gold nanostructures (AuNs), as a new pathway to develop a selective and sensitive methodology for SARS-CoV-2 detection is presented. A mixture of gold nanoparticles and gold nanotriangles have been synthetized to modify disposable electrodes that act as an enhanced nanostructured electrochemical surface for DNA probe immobilization. On the other hand, modified carbon nanodots prepared a la carte to contain Methylene Blue (MB-CDs) are used as electrochemical indicators of the hybridization event. These MB-CDs, due to their structure, are able to interact differently with double and single-stranded DNA molecules. Based on this strategy, target sequences of the SARS-CoV-2 virus have been detected in a straightforward way and rapidly with a detection limit of 2.00 aM. Moreover, this platform allows the detection of the SARS-CoV-2 sequence in the presence of other viruses, and also a single nucleotide polymorphism (SNPs). The developed approach has been tested directly on RNA obtained from nasopharyngeal samples from COVID-19 patients, avoiding any amplification process. The results agree well with those obtained by RT-qPCR or reverse transcription quantitative polymerase chain reaction technique.

Evaluation of LAMP for the diagnosis of Loa loa infection in dried blood spots compared to PCR-based assays and microscopy.

BACKGROUND: Loa loa is a filarial species found exclusively in West and Central Africa. Microscopy is the traditional diagnosis method for human loiasis. Several molecular methods have developed as an alternative approach for identification of L. loa filarial parasites. OBJECTIVES: The aim of this study was to evaluate a Loa-Loop-mediated isothermal amplification (LAMP) assay to diagnose loiasis disease on dried blood spots (DBS) samples, compared to microscopy, filaria-real time-polymerase chain reaction (PCR) and nested-Loa PCR. METHODS: A total of 100 DBS samples and 100 blood smears were used for this study. DNA was extracted using saponin/Chelex method. DNA isolated was assayed by a Loa-LAMP assay in parallel to microscopy, filaria-real time PCR and nested-Loa PCR. The sensitivities and specificities of Loa-LAMP assay was computed comparing to each one of the reference methods. FINDINGS: Loa-LAMP's sensitivity was more than 90% and specificity was nearly 100% when compared to molecular methods. On the other hand, sensitivity was decreased a bit when Loa-LAMP faced microscopy, but keeping the other statistical values high. MAIN CONCLUSIONS: Loa-LAMP is an appropriate method for loiasis diagnosis in endemic areas. Though, it has disadvantages like the reagents' high price at the moment and not to be able to detect more filarial species at once.

High IGKC-Expressing Intratumoral Plasma Cells Predict Response to Immune Checkpoint Blockade.

Resistance to Immune Checkpoint Blockade (ICB) constitutes the current limiting factor for the optimal implementation of this novel therapy, which otherwise demonstrates durable responses with acceptable toxicity scores. This limitation is exacerbated by a lack of robust biomarkers. In this study, we have dissected the basal TME composition at the gene expression and cellular levels that predict response to Nivolumab and prognosis. BCR, TCR and HLA profiling were employed for further characterization of the molecular variables associated with response. The findings were validated using a single-cell RNA-seq data of metastatic melanoma patients treated with ICB, and by multispectral immunofluorescence. Finally, machine learning was employed to construct a prediction algorithm that was validated across eight metastatic melanoma cohorts treated with ICB. Using this strategy, we have unmasked a major role played by basal intratumoral Plasma cells expressing high levels of IGKC in efficacy. IGKC, differentially expressed in good responders, was also identified within the Top response-related BCR clonotypes, together with IGK variants. These results were validated at gene, cellular and protein levels; CD138+ Plasma-like and Plasma cells were more abundant in good responders and correlated with the same RNA-seq-defined fraction. Finally, we generated a 15-gene prediction model that outperformed the current reference score in eight ICB-treated metastatic melanoma cohorts. The evidenced major contribution of basal intratumoral IGKC and Plasma cells in good response and outcome in ICB in metastatic melanoma is a groundbreaking finding in the field beyond the role of T lymphocytes.

Risk patterns in food addiction: a Mexican population approach.

BACKGROUND: Food addiction (FA) is a construct that has gained interest in recent years but its relevance in Mexican population is still unexplored. AIMS: The present study has the aims of explore FA in a community of Mexican population, as well as identifying the risk patterns associated with it, in relation to the different etiological factors that have been described such as impulsivity, emotional regulation and eating styles. Furthermore, to identify a predictive model of FA severity. METHODS: The sample consisted of 160 female and male university students of Pachuca city in México, who volunteered to participate in the study. Assessment included multidimensional measures for FA, eating disorder severity, eating disorder styles, emotional regulation and impulsivity. RESULTS: A screening of FA-probable was registered for 13.8% of the sample, while 8.1% met criteria for FA-present. The FA-present group differed from FA-absent in the impulsivity levels and in emotional eating style. Patients with FA-present differed from FA-probable in the impulsivity levels. Differences between FA-probable versus FA-absent were found in the restrained eating style. Path analysis evidenced that FA severity was directly associated with older age, worse eating style profile and higher impulsivity levels, and indirectly related with the ED symptom levels. CONCLUSIONS: Our findings suggest that it is possible to establish a specific predictive model of the development of FA and its severity in Mexican population to implement adequate prevention and treatment strategies. EVIDENCE LEVEL: Level III: evidence obtained from well-designed cohort or case-control analytic studies.

Temporal evolution of the resistance genotypes of Plasmodium falciparum in isolates from Equatorial Guinea during 20 years (1999 to 2019).

BACKGROUND: Malaria is one of the deadliest diseases in the world, particularly in Africa. As such, resistance to anti-malarial drugs is one of the most important problems in terms of global malaria control. This study assesses the evolution of the different resistance markers over time and the possible influence of interventions and treatment changes that have been made in Equatorial Guinea. METHODS: A total of 1223 biological samples obtained in the period 1999 to 2019 were included in the study. Screening for mutations in the pfdhfr, pfdhps, pfmdr1, and pfcrt genes was carried out by nested PCR and restriction-fragment length polymorphisms (RFLPs), and the study of pfk13 genes was carried out by nested PCR, followed by sequencing to determine the presence of mutations. RESULTS: The partially and fully resistant haplotypes (pfdhfr + pfdhps) were found to increase over time. Moreover, in 2019, the fully resistant haplotype was found to be increasing, although its super-resistant counterpart remains much less prevalent. A continued decline in pfmdr1 and pfcrt gene mutations over time was also found. The number of mutations detected in pfk13 has increased since 2008, when artemisinin-based combination therapy (ACT) were first introduced, with more mutations being observed in 2019, with two synonymous and five non-synonymous mutations being detected, although these are not related to resistance to ACT. In addition, the non-synonymous A578S mutation, which is the most frequent on the African continent, was detected in 2013, although not in the following years. CONCLUSIONS: Withdrawal of the use of chloroquine (CQ) as a treatment in Equatorial Guinea has been shown to be effective over time, as wild-type parasite populations outnumber mutant populations. The upward trend observed in sulfadoxine-pyrimethamine (SP) resistance markers suggest its misuse, either alone or in combination with artesunate (AS) or amodiaquine (AQ), in some areas of the country, as was found in a previous study conducted by this group, which allows selective pressure from SP to continue. Single nucleotide polymorphisms (SNPs) 540E and 581G do not exceed the limit of 50 and 10%, respectively, thus meaning that SP is still effective as an intermittent preventive treatment (IPT) in this country. As for the pfk13 gene, no mutations have been detected in relation to resistance to ACT. However, in 2019 there is a greater accumulation of non-synonymous mutations compared to years prior to 2008.

Carbon nanodot-based electrogenerated chemiluminescence biosensor for miRNA-21 detection.

A simple carbon nanodot-based electrogenerated chemiluminescence biosensor is described for sensitive and selective detection of microRNA-21 (miRNA-21), a biomarker of several pathologies including cardiovascular diseases (CVDs). The photoluminescent carbon nanodots (CNDs) were obtained using a new synthesis method, simply by treating tiger nut milk in a microwave reactor. The synthesis is environmentally friendly, simple, and efficient. The optical properties and morphological characteristics of the CNDs were exhaustively investigated, confirming that they have oxygen and nitrogen functional groups on their surfaces and exhibit excitation-dependent fluorescence emission, as well as photostability. They act as co-reactant agents in the anodic electrochemiluminescence (ECL) of [Ru(bpy)3]2+, producing different signals for the probe (single-stranded DNA) and the hybridized target (double-stranded DNA). These results paved the way for the development of a sensitive ECL biosensor for the detection of miRNA-21. This was developed by immobilization of a thiolated oligonucleotide, fully complementary to the miRNA-21 sequence, on the disposable gold electrode. The target miRNA-21 was hybridized with the probe on the electrode surface, and the hybridization was detected by the enhancement of the [Ru(bpy)3]2+/DNA ECL signal using CNDs. The biosensor shows a linear response to miRNA-21 concentration up to 100.0 pM with a detection limit of 0.721 fM. The method does not require complex labeling steps, and has a rapid response. It was successfully used to detect miRNA-21 directly in serum samples from heart failure patients without previous RNA extraction neither amplification process.

Identification of Novel microRNA Profiles Dysregulated in Plasma and Tissue of Abdominal Aortic Aneurysm Patients.

microRNAs (miRNAs) are small RNAs that regulate different biological processes. Our objective was to identify miRNAs dysregulated in plasma and tissue of patients with abdominal aortic aneurysm (AAA) and explore new potential targets involved in AAA. Fifty-seven subjects were recruited for a plasma study (30 AAA patients, 16 healthy volunteers and 11 patients with atherosclerosis). The expression level of 179 miRNAs was screened in plasma from a subset of samples, and dysregulated miRNAs were validated in the entire study population. Dysregulated miRNAs were also quantified in aortic tissue of 21 AAA patients and 8 organ donors. Applying a gene set enrichment analysis, an interaction map of dysregulated miRNAs and their targets was built, and selected targets were quantified in tissue samples. miR-27b-3p and miR-221-3p were overexpressed in plasma of AAA patients compared with healthy controls, 1.6 times and 1.9 times, respectively. In AAA tissue, six miRNAs (miR-1, miR-27b-3p, miR-29b-3p, miR-133a-3p, miR-133b, and miR-195-5p) were underexpressed from 1.6 to 4.8 times and four miRNAs (miR-146a-5p, miR-21-5p, miR-144-3p, and miR-103a-3p) were overexpressed from 1.3 to 7.2 times. Thrombospondin-2, a target of miR-195-5p, was increased in AAA tissue and negatively correlated with the expression of miR-195-5p, suggesting their involvement in a common regulatory mechanism.

Fusarium proliferatum - Causal agent of garlic bulb rot in Spain: Genetic variability and mycotoxin production.

Fusarium proliferatum is a world-wide occurring fungal pathogen affecting several crops included garlic bulbs. In Spain, this is the most frequent pathogenic fungus associated with garlic rot during storage. Moreover, F. proliferatum is an important mycotoxigenic species, producing a broad range of toxins, which may pose a risk for food safety. The aim of this study is to assess the intraspecific variability of the garlic pathogen in Spain implied by analyses of translation elongation factor (tef-1α) and FUM1 gene sequences as well as the differences in growth rates. Phylogenetic characterization has been complemented with the characterization of mating type alleles as well as the species potential as a toxin producer. Phylogenetic trees based on the sequence of the translation elongation factor and FUM1 genes from seventy nine isolates from garlic revealed a considerable intraspecific variability as well as high level of diversity in growth speed. Based on the MAT alleles amplified by PCR, F. proliferatum isolates were separated into different groups on both trees. All isolates collected from garlic in Spain proved to be fumonisin B1, B2, and B3 producers. Quantitative analyses of fumonisins, beauvericin and moniliformin (common secondary metabolites of F. proliferatum) showed no correlation with phylogenetic analysis neither mycelial growth. This pathogen presents a high intraspecific variability within the same geographical region and host, which is necessary to be considered in the management of the disease.

Functionalized N95 Face Mask with a Chemical-Free Paper-Based Collector for Exhaled Breath Analysis: SARS-CoV-2 Detection with a Printed Immunosensor as a Case Study.

Exhaled breath electrochemical sensing is a promising biomedical technology owing to its portability, painlessness, cost-effectiveness, and user-friendliness. Here, we present a novel approach for target analysis in exhaled breath by integrating a comfortable paper-based collector into an N95 face mask, providing a universal solution for analyzing several biomarkers. As a model analyte, we detected SARS-CoV-2 spike protein from the exhaled breath by sampling the target analyte into the collector, followed by its detection out of the N95 face mask using a magnetic bead-based electrochemical immunosensor. This approach was designed to avoid any contact between humans and the chemicals. To simulate human exhaled breath, untreated saliva samples were nebulized on the paper collector, revealing a detection limit of 1 ng/mL and a wide linear range of 3.7-10,000 ng/mL. Additionally, the developed immunosensor exhibited high selectivity toward the SARS-CoV-2 spike protein, compared to other airborne microorganisms, and the SARS-CoV-2 nucleocapsid protein. Accuracy assessments were conducted by analyzing the simulated breath samples spiked with varying concentrations of SARS-CoV-2 spike protein, resulting in satisfactory recovery values (ranging from 97 ± 4 to 118 ± 1%). Finally, the paper-based hybrid immunosensor was successfully applied for the detection of SARS-CoV-2 in real human exhaled breath samples. The position of the collector in the N95 mask was evaluated as well as the ability of this paper-based analytical tool to identify the positive patient.

A genetic profiling guideline to support diagnosis and clinical management of lymphomas.

The new lymphoma classifications (International Consensus Classification of Mature Lymphoid Neoplasms, and 5th World Health Organization Classification of Lymphoid Neoplasms) include genetics as an integral part of lymphoma diagnosis, allowing better lymphoma subclassification, patient risk stratification, and prediction of treatment response. Lymphomas are characterized by very few recurrent and disease-specific mutations, and most entities have a heterogenous genetic landscape with a long tail of recurrently mutated genes. Most of these occur at low frequencies, reflecting the clinical heterogeneity of lymphomas. Multiple studies have identified genetic markers that improve diagnostics and prognostication, and next-generation sequencing is becoming an essential tool in the clinical laboratory. This review provides a "next-generation sequencing" guide for lymphomas. It discusses the genetic alterations of the most frequent mature lymphoma entities with diagnostic, prognostic, and predictive potential and proposes targeted sequencing panels to detect mutations and copy-number alterations for B- and NK/T-cell lymphomas.

Compelling DNA intercalation through 'anion-anion' anti-coulombic interactions: boron cluster self-vehicles as promising anticancer agents.

Anticancer drugs inhibit DNA replication by intercalating between DNA base pairs, forming covalent bonds with nucleotide bases, or binding to the DNA groove. To develop safer drugs, novel molecular structures with alternative binding mechanisms are essential. Stable boron hydrides offer a promising alternative for cancer therapy, opening up additional options like boron neutron capture therapy based on 10B and thermal neutron beams or proton boron fusion therapy using 11B and proton beams. These therapies are more efficient when the boron compound is ideally located inside cancer cells, particularly in the nucleus. Current cancer treatments often utilize small, polycyclic, aromatic, planar molecules that intercalate between ds-DNA base pairs, requiring only a spacing of approximately 0.34 nm. In this paper, we demonstrate another type of intercalation. Notably, [3,3'-Fe(1,2-C2B9H11)2]-, ([o-FESAN]-), a compact 3D molecule measuring 1.1 nm × 0.6 nm, can as well intercalate by strong non-bonding interactions preferentially with guanine. Unlike known intercalators, which are positive or neutral, [o-FESAN]- is a negative species and when an [o-FESAN]- molecule approaches the negatively charged DNA phosphate chain an anion-anion interaction consistently anti-electrostatic via Ccluster-H⋯O-P bonds occurs. Then, when more molecules approach, an elongated outstandingly self-assembled structure of [o-FESAN]--[o-FESAN]- forms moving anions towards the interthread region to interact with base pairs and form aggregates of four [o-FESAN]- anions per base pair. These aggregates, in this environment, are generated by Ccluster-H⋯O-C, N-H⋯H-B and Ccluster-H⋯H-B interactions. The ferrabis(dicarbollide) boron-rich small molecules not only effectively penetrate the nucleus but also intercalate with ds-DNA, making them promising for cancer treatment. This amphiphilic anionic molecule, used as a carrier-free drug, can enhance radiotherapy in a multimodal perspective, providing healthcare professionals with improved tools for cancer treatment. This work demonstrates these findings with a plethora of techniques.

A "signal off-on" fluorescence bioassay based on 2D-MoS2-tetrahedral DNA bioconjugate for rapid virus detection.

In this work we present the preparation of a 2D molybdenum disulphide nanosheets (2D-MoS2) and tetrahedral DNA nanostructures (TDNs) bioconjugate, and its application to the development of a bioassay for rapid and easy virus detection. The bioconjugate has been prepared by using TDNs carrying the capture probe labelled with 6-carboxyfluoresceine (6-FAM). As case of study to assess the utility of the assay developed, we have chosen the SARS-CoV-2 virus. Hence, as probe we have used a DNA sequence complementary to a region of the SARS-CoV-2 ORF1ab gene (TDN-ORF-FAM). This 6-FAM labelled capture probe is located on the top vertex of the tetrahedral DNA nanostructure, the three left vertices of TDNs have a thiol group. These TDNs are bounded to 2D-MoS2 surface through the three thiol groups, allowing the capture probe to be oriented to favour the biorecognition reaction with the analyte. This biorecognition resulting platform has finally been challenged to the detection of the SARS-CoV-2 ORF1ab gene sequence as the target model by measuring fluorescence before and after the hybridization event with a detection limit of 19.7fM. Furthermore, due to high sensitivity of the proposed methodology, it has been applied to directly detect the virus in nasopharyngeal samples of infected patients without the need of any amplification step. The developed bioassay has a wide range of applicability since it can be applied to the detection of any pathogen by changing the probe corresponding to the target sequence. Thus, a novel, hands-on strategy for rapid pathogen detection has proposed and has a high potential application value in the early diagnosis of infections causes by virus or bacteria.

Free PCR virus detection via few-layer bismuthene and tetrahedral DNA nanostructured assemblies.

In this work we describe a highly sensitive method based on a biocatalyzed electrochemiluminescence approach. The system combines, for the first time, the use of few-layer bismuthene (FLB) as a platform for the oriented immobilization of tetrahedral DNA nanostructures (TDNs) specifically designed and synthetized to detect a specific SARS-CoV-2 gene sequence. In one of its vertices, these TDNs contain a DNA capture probe of the open reading frame 1 ab (ORF1ab) of the virus, available for the biorecognition of the target DNA/RNA. At the other three vertices, there are thiol groups that enable the stable anchoring/binding to the FLB surface. This novel geometry/approach enables not only the binding of the TDNs to surfaces, but also the orientation of the capture probe in a direction normal to the bismuthine surface so that it is readily accessible for binding/recognition of the specific SARS-CoV-2 sequence. The analytical signal is based on the anodic electrochemiluminescence (ECL) intensity of luminol which, in turn, arises as a result of the reaction with H2O2, generated by the enzymatic reaction of glucose oxidation, catalyzed by the biocatalytic label avidin-glucose oxidase conjugate (Av-GOx), which acts as co-reactant in the electrochemiluminescent reaction. The method exhibits a limit of detection (LOD) of 4.31 aM and a wide linear range from 14.4 aM to 1.00 µM, and its applicability was confirmed by detecting SARS-CoV-2 in nasopharyngeal samples from COVID-19 patients without the need of any amplification process.

Bismuthene - Tetrahedral DNA nanobioconjugate for virus detection.

In this work, we present an electrochemical sensor for fast, low-cost, and easy detection of the SARS-CoV-2 spike protein in infected patients. The sensor is based on a selected combination of nanomaterials with a specific purpose. A bioconjugate formed by Few-layer bismuthene nanosheets (FLB) and tetrahedral DNA nanostructures (TDNs) is immobilized on Carbon Screen-Printed Electrodes (CSPE). The TDNs contain on the top vertex an aptamer that specifically binds to the SARS-CoV-2 spike protein, and a thiol group at the three basal vertices to anchor to the FLB. The TDNs are also marked with a redox indicator, Azure A (AA), which allows the direct detection of SARS-CoV-2 spike protein through changes in the current intensity of its electrolysis before and after the biorecognition reaction. The developed sensor can detect SARS-CoV-2 spike protein with a detection limit of 1.74 fg mL-1 directly in nasopharyngeal swab human samples. Therefore, this study offers a new strategy for rapid virus detection since it is versatile enough for different viruses and pathogens.

Rapid and simple viral protein detection by functionalized 2D MoS2/graphene electrochemiluminescence aptasensor.

In this work we present the development of an electrochemiluminescence aptasensor based on electrografting molybdenum disulphide nanosheets functionalized with diazonium salt (MoS2-N2+) upon screen-printed electrodes of graphene (SPEs GPH) for viral proteins detection. In brief, this aptasensor consists of SPEs GPH electrografted with MoS2-N2+ and modified with a thiolated aptamer, which can specifically recognize the target protein analyte. In this case, we have used SARS-CoV-2 spike protein as model protein. Electrochemiluminescence detection was performed by using the [Ru(bpy)3]2+/TPRA (tripropylamine) system, which allows the specific detection of the SARS-CoV-2 spike protein easily and rapidly with a detection limit of 9.74 fg/mL and a linear range from 32.5 fg/mL to 50.0 pg/mL. Moreover, the applicability of the aptasensor has been confirmed by the detection of the protein directly in human saliva samples. Comparing our device with a traditional saliva antigen test, our aptasensor can detect the spike protein even when the saliva antigen test gives a negative result.

Evaluation of Trichoderma spp. on Fusarium oxysporum f. sp. asparagi and Fusarium wilt Control in Asparagus Crop.

Among the key diseases affecting the asparagus crop (Asparagus officinalis L.), vascular wilting of asparagus caused by Fusarium oxysporum f. sp. asparagi stands out worldwide. This disease significantly shortens the longevity of the crop and limits economic production. Traditional control measures have been largely ineffective, and chemical control methods are difficult to apply, making biological control approaches, specifically the use of Trichoderma, an economical, effective, and risk-free alternative. This study aimed to identify the main factors that affect the efficacy of biopesticides studied as Biological Control Agents (BCAs) against Fusarium wilt in asparagus and to assess the efficacy of Trichoderma-based biopesticides under greenhouse and semi-field conditions. We evaluated the response of three Trichoderma spp. (T. atroviride, T. asperellum, and T. saturnisporum) to environmental variables, such as temperature and water activity, and their antagonistic capacity against Fusarium oxysporum f. sp. asparagi. All three Trichoderma species inhibited the growth of the pathogen in vitro. A decrease in water activity led to a greater reduction in the growth rate. The efficacy of the three biological control agents decreased with higher temperatures, resulting in minimal inhibition, particularly under conditions of restricted available water in the environment. The effect of the fungal inoculum density was also analyzed at two different temperatures. A direct correlation between the amount of inoculum and the score on the Disease Severity Index (DSI) was observed. A notable reduction in DSI was evident in treatments with high inoculum density (106 conidium/mL) for all three species of Trichoderma tested at both temperatures. In greenhouse and semi-field tests, we observed less disease control than expected, although T. asperellum and T. atroviride showed lower disease severity indices and increased the dry weight of seedlings and crowns, whereas T. saturnisporum resulted in the highest disease rate and lowest dry weight. This work highlights that the efficacy of Trichoderma as BCAs is influenced by various factors, including the quantity of soil inocula, and environmental conditions. The study findings have strong implications for selecting appropriate Trichoderma species for controlling specific pathogens under specific environmental conditions.

MoS2-Carbon Nanodots as a New Electrochemiluminescence Platform for Breast Cancer Biomarker Detection.

In this work, we present the combination of two different types of nanomaterials, 2D molybdenum disulfide nanosheets (MoS2-NS) and zero-dimensional carbon nanodots (CDs), for the development of a new electrochemiluminescence (ECL) platform for the early detection and quantification of the biomarker human epidermal growth factor receptor 2 (HER2), whose overexpression is associated with breast cancer. MoS2-NS are used as an immobilization platform for the thiolated aptamer, which can recognize the HER2 epitope peptide with high affinity, and CDs act as coreactants of the anodic oxidation of the luminophore [Ru(bpy)3]2+. The HER2 biomarker is detected by changes in the ECL signal of the [Ru(bpy)3]2+/CD system, with a low detection limit of 1.84 fg/mL and a wide linear range. The proposed method has been successfully applied to detect the HER2 biomarker in human serum samples.