Metabolomics-based chemotaxonomy of root endophytic fungi for natural products discovery.

Fungi are prolific producers of natural products routinely screened for biotechnological applications, and those living endophytically within plants attract particular attention because of their purported chemical diversity. However, the harnessing of their biosynthetic potential is hampered by a large and often cryptic phylogenetic and ecological diversity, coupled with a lack of large-scale natural products' dereplication studies. To guide efforts to discover new chemistries among root-endophytic fungi, we analyzed the natural products produced by 822 strains using an untargeted UPLC-ESI-MS/MS-based approach and linked the patterns of chemical features to fungal lineages. We detected 17 809 compounds of which 7951 were classified in 1992 molecular families, whereas the remaining were considered unique chemistries. Our approach allowed to annotate 1191 compounds with different degrees of accuracy, many of which had known fungal origins. Approximately 61% of the compounds were specific of a fungal order, and differences were observed across lineages in the diversity and characteristics of their chemistries. Chemical profiles also showed variable chemosystematic values across lineages, ranging from relative homogeneity to high heterogeneity among related fungi. Our results provide an extensive resource to dereplicate fungal natural products and may assist future discovery programs by providing a guide for the selection of target fungi.

Facultative root-colonizing fungi dominate endophytic assemblages in roots of nonmycorrhizal Microthlaspi species.

There is increasing knowledge on the diversity of root-endophytic fungi, but limited information on their lifestyles and dependence on hosts hampers our understanding of their ecological functions. We compared diversity and biogeographical patterns of cultivable and noncultivable root endophytes to assess whether their occurrence is determined by distinct ecological factors. The endophytic diversity in roots of nonmycorrhizal Microthlaspi spp. growing across Europe was assessed using high-throughput sequencing (HTS) and compared with a previous dataset based on cultivation of endophytes from the same root samples. HTS revealed a large fungal richness undetected by cultivation, but which largely comprised taxa with restricted distributions and/or low representation of sequence reads. Both datasets coincided in a consistent high representation of widespread endophytes within orders Pleosporales, Hypocreales and Helotiales, as well as similar associations of community structure with spatial and environmental conditions. Likewise, distributions of particular endophytes inferred by HTS agreed with cultivation data in suggesting individual ecological preferences. Our findings support that Microthlaspi spp. roots are colonized mostly by saprotrophic and likely facultative endophytes, and that differential niche preferences and distribution ranges among fungi importantly drive the assembly of root-endophytic communities.

Genotypic diversity in root-endophytic fungi reflects efficient dispersal and environmental adaptation.

Studying community structure and dynamics of plant-associated fungi is the basis for unravelling their interactions with hosts and ecosystem functions. A recent sampling revealed that only a few fungal groups, as defined by internal transcribed spacer region (ITS) sequence similarity, dominate culturable root endophytic communities of nonmycorrhizal Microthlaspi spp. plants across Europe. Strains of these fungi display a broad phenotypic and functional diversity, which suggests a genetic variability masked by ITS clustering into operational taxonomic units (OTUs). The aims of this study were to identify how genetic similarity patterns of these fungi change across environments and to evaluate their ability to disperse and adapt to ecological conditions. A first ITS-based haplotype analysis of ten widespread OTUs mostly showed a low to moderate genotypic differentiation, with the exception of a group identified as Cadophora sp. that was highly diverse. A multilocus phylogeny based on additional genetic loci (partial translation elongation factor 1α, beta-tubulin and actin) and amplified fragment length polymorphism profiling of 185 strains representative of the five dominant OTUs revealed a weak association of genetic differences with geography and environmental conditions, including bioclimatic and soil factors. Our findings suggest that dominant culturable root endophytic fungi have efficient dispersal capabilities, and that their distribution is little affected by environmental filtering. Other processes, such as inter- and intraspecific biotic interactions, may be more important for the local assembly of their communities.

The local environment determines the assembly of root endophytic fungi at a continental scale.

Root endophytic fungi are found in a great variety of plants and ecosystems, but the ecological drivers of their biogeographic distribution are poorly understood. Here, we investigate the occurrence of root endophytes in the non-mycorrhizal plant genus Microthlaspi, and the effect of environmental factors and geographic distance in structuring their communities at a continental scale. We sampled 52 plant populations across the northern Mediterranean and central Europe and used a cultivation approach to study their endophytic communities. Cultivation of roots yielded 2601 isolates, which were grouped into 296 operational taxonomic units (OTUs) by internal transcribed spacer sequencing of 1998 representative colonies. Climatic and spatial factors were the best descriptors of the structure of endophytic communities, outweighing soil characteristics, host genotype and geographical distance. OTU richness was negatively affected by precipitation, and the composition of communities followed latitudinal gradients of precipitation and temperature. Only six widespread OTUs belonging to the orders Pleosporales, Hypocreales and Helotiales represented about 50% of all isolates. Assessments of their individual distribution revealed particular ecological preferences or a cosmopolitan occurrence. Our findings support a strong influence of the local environment in determining root endophytic communities, and show a different niche occupancy by individual endophytes.

Effectiveness of Prophylactic Human Cytomegalovirus Hyperimmunoglobulin in Preventing Cytomegalovirus Infection following Transplantation: A Systematic Review and Meta-Analysis.

Cytomegalovirus (CMV) is a common infection occurring in patients undergoing solid organ transplantation (SOT) or hematopoietic stem cell transplantation (HSCT). CMV-specific hyperimmunoglobulin (CMVIG) has been used for the past four decades and is typically administered either prophylactically or pre-emptively. The present meta-analysis evaluated CMV infection rates in SOT patients who received prophylactic CMVIG. PubMed and the Cochrane Library were searched for studies published up to October 2021. The primary endpoint was CMV infection rate. Thirty-two SOT studies were identified (n = 1521 CMVIG-treated and n = 1196 controls). Prophylactic CMVIG treatment was often associated with a lower risk of CMV infection in transplant recipients. The average CMV infection rate was 35.8% (95% confidence interval [CI]: 33.4−38.2%) in patients treated prophylactically with CMVIG and 41.4% (95% CI: 38.6−44.2%) in the control group not receiving CMVIG (p = 0.003). Similar results were observed in analyses limited to publications evaluating currently available CMVIG products (Cytotect CP and Cytogam; p < 0.001). In combination with the established safety profile for CMVIG, these results suggest that prophylactic CMVIG treatment in patients undergoing solid organ transplantation may be beneficial, particularly in those at high risk of CMV infection or disease.

Allelic drop-out in the LDLR gene affects mutation detection in familial hypercholesterolemia.

OBJECTIVES: Familial hypercholesterolemia is a monogenic disorder caused by mutations in the LDL receptor (LDLR) gene. We observed allelic drop-out during LDLR genotyping and aimed at redesigning mutation detection. DESIGN AND METHODS: The NanoChip microelectronic array technology and PCR restriction fragment length polymorphism analysis were used. RESULTS: Allele drop-out caused false homozygous diagnoses and was overcome using PCR primers without polymorphisms in the primer binding site. CONCLUSIONS: This report presents the importance of allele drop-out in LDLR genotyping.

Development of a universal chemiluminometric genotyping method for high-throughput detection of 7 LDLR gene mutations in Greek population.

OBJECTIVES: Familial hypercholesterolemia (FH) is caused by mutations in the LDL receptor (LDLR) gene. We report the application of a universal method with high allele discrimination properties to the simultaneous genotyping of 7 LDLR mutations in Greeks, in dry-reagent format. DESIGN AND METHODS: We genotyped mutations C858A, C939A, G1285A, T1352C, G1646A, G1775A, C/T81G. Unpurified amplicons from a multiplex PCR that produced fragments encompassing all 7 mutations were subjected to probe extension reactions in the presence of fluorescein-modified dCTP, and a microtiter well-based assay of extension products with a peroxidase-antifluorescein conjugate and a chemiluminogenic substrate. We used lyophilized dry reagents and assigned genotypes by the signal ratio of normal-to-mutant-specific probe. RESULTS: We standardized the method and optimised all steps for specificity. The method was validated by genotyping blindly 119 (833 genotypings). Results were fully concordant with other methods used as standards. CONCLUSIONS: This method is accurate, simple, rapid and robust. The microtiter well format allows genotyping of a large number of samples in parallel for several mutations.

Advances in molecular techniques for the detection and quantification of genetically modified organisms.

Progress in genetic engineering has led to the introduction of genetically modified organisms (GMOs) whose genomes have been altered by the integration of a novel sequence conferring a new trait. To allow consumers an informed choice, many countries require food products to be labeled if the GMO content exceeds a certain threshold. Consequently, the development of analytical methods for GMO screening and quantification is of great interest. Exponential amplification by the polymerase chain reaction (PCR) remains a central step in molecular methods of GMO detection and quantification. In order to meet the challenge posed by the continuously increasing number of GMOs, various multiplex assays have been developed for the simultaneous amplification and/or detection of several GMOs. Classical agarose gel electrophoresis is being replaced by capillary electrophoresis (CE) systems, including CE chips, for the rapid and automatable separation of amplified fragments. Microtiter well-based hybridization assays allow high-throughput analysis of many samples in a single plate. Microarrays have been introduced in GMO screening as a technique for the simultaneous multianalyte detection of amplified sequences. Various types of biosensors, including surface plasmon resonance sensors, quartz crystal microbalance piezoelectric sensors, thin-film optical sensors, dry-reagent dipstick-type sensors and electrochemical sensors were introduced in GMO screening because they offer simplicity and lower cost. GMO quantification is performed by real-time PCR (rt-QPCR) and competitive PCR. New endogenous reference genes have been validated. rt-QPCR is the most widely used approach. Multiplexing is another trend in this field. Strategies for high-throughput multiplex competitive quantitative PCR have been reported.

The effects of fungal root endophytes on plant growth are stable along gradients of abiotic habitat conditions.

Plant symbioses with fungal root endophytes span a continuum from mutualistic to parasitic outcomes, and are highly variable depending on the genotype of each symbiont. The abiotic context in which interactions occur also seems to influence the outcome of plant-endophyte symbioses, but we lack understanding of its relative importance. We aimed to assess if changes in abiotic variables determine the effects of fungal root endophytes on plant growth. We used in vitro co-cultivation assays to test the impact of a selection of endophytic strains from diverse lineages on the growth of Arabidopsis thaliana, Microthlaspi erraticum and Hordeum vulgare along gradients of nutrient availability, light intensity or substrate pH. Most fungi showed a negative but weak effect on plant growth, whereas only a few had persistent detrimental effects across plants and conditions. Changes in abiotic factors affected plant growth but had little influence on their response to fungal inoculation. Of the factors tested, variation in nutrient availability resulted in the most variable plant-endophyte interactions, although changes were feeble and strain-specific. Our findings suggest that the effects of root endophytes on plant growth are robust to changes in the abiotic environment when these encompass the tolerance range of either symbiont.

Influence of phylogenetic conservatism and trait convergence on the interactions between fungal root endophytes and plants.

Plants associate through their roots with fungal assemblages that impact their abundance and productivity. Non-mycorrhizal endophytes constitute an important component of such fungal diversity, but their implication in ecosystem processes is little known. Using a selection of 128 root-endophytic strains, we defined functional groups based on their traits and plant interactions with potential to predict community assembly and symbiotic association processes. In vitro tests of the strains' interactions with Arabidopsis thaliana, Microthlaspi erraticum and Hordeum vulgare showed a net negative effect of fungal colonization on plant growth. The effects partly depended on the phylogenetic affiliation of strains, but also varied considerably depending on the plant-strain combination. The variation was partly explained by fungal traits shared by different lineages, like growth rates or melanization. The origin of strains also affected their symbioses, with endophytes isolated from Microthlaspi spp. populations being more detrimental to M. erraticum than strains from other sources. Our findings suggest that plant-endophyte associations are subject to local processes of selection, in which particular combinations of symbionts are favored across landscapes. We also show that different common endophytic taxa have differential sets of traits found to affect interactions, hinting to a functional complementarity that can explain their frequent co-existence in natural communities.

Diversity of exophillic acid derivatives in strains of an endophytic Exophiala sp.

Members of the fungal genus Exophiala are common saprobes in soil and water environments, opportunistic pathogens of animals, or endophytes in plant roots. Their ecological versatility could imply a capacity to produce diverse secondary metabolites, but only a few studies have aimed at characterizing their chemical profiles. Here, we assessed the secondary metabolites produced by five Exophiala sp. strains of a particular phylotype, isolated from roots of Microthlaspi perfoliatum growing in different European localities. Exophillic acid and two previously undescribed compounds were isolated from these strains, and their structures were elucidated by spectroscopic methods using MS, 1D and 2D NMR. Bioassays revealed a weak activity of these compounds against disease-causing protozoa and mammalian cells. In addition, 18 related structures were identified by UPLC/MS based on comparisons with the isolated structures. Three Exophiala strains produced derivatives containing a ß-d-glucopyranoside moiety, and their colony morphology was distinct from the other two strains, which produced derivatives lacking ß-d-glucopyranoside. Whether the chemical/morphological strain types represent variants of the same genotype or independent genetic populations within Exophiala remains to be evaluated.

Development of a three-biosensor panel for the visual detection of thrombophilia-associated mutations.

We developed a rapid and low-cost panel of three assays for visual genotyping of the three most common genetic risk factors in thrombophilia, namely, the single-point mutations in the FV (Leiden factor), FII and MTHFR genes. A triplex PCR was developed for simultaneous amplification of three fragments spanning the interrogated loci. Allele discrimination was accomplished by a 5-min primer extension reaction on each locus. Detection of the extension products was performed by means of a dual-allele dipstick-type DNA biosensor. The biosensor is disposable and allows visual detection and confirmation of the genotyping products within 15 min by hybridization without the need for specialized instruments or expensive reagents. The proposed method was evaluated by genotyping 40 samples with a variety of genotypes for all three mutations. The genotyping results were in full concordance with those obtained by restriction fragment length polymorphism analysis and sequencing.

High-throughput microtiter well-based chemiluminometric genotyping of 15 HBB gene mutations in a dry-reagent format.

BACKGROUND: Hemoglobinopathies are the most common inherited diseases worldwide. Various methods for genotyping of hemoglobin, beta (HBB) gene mutations have been reported, but there is need for a high sample-throughput, cost-effective method for simultaneous screening of several mutations. We report a method that combines the high detectability and dynamic range of chemiluminescence with the high allele-discrimination ability of probe extension reactions for simultaneous genotyping of 15 HBB mutations in a high sample-throughput, dry-reagent format. METHODS: We genotyped the HBB mutations IVSI-110G>A, CD39C>T, IVSI-1G>A, IVSI-6T>C, IVSII-745C>G, IVSII-1G>A, FSC6GAG>G-G, -101C>T, FSC5CCT>C-, IVSI-5G>A, FSC8AAG>-G, -87C>G, IVSII-848C>A, term+6C>G, and HbS (cd6GAG>GTG). The method used comprises the following: (a) duplex PCR that produces fragments encompassing all 15 mutations, (b) probe extension reactions in the presence of fluorescein-modified dCTP, using unpurified amplicons, and (c) microtiter well-based assay of extension products with a peroxidase-antifluorescein conjugate and a chemiluminogenic substrate. We used lyophilized dry reagents to simplify the procedure and assigned the genotype by the signal ratio of the normal-to-mutant-specific probe. RESULTS: We standardized the method by analyzing 60 samples with known genotypes and then validated by blindly genotyping 115 samples with 45 genotypes. The results were fully concordant with sequencing. The reproducibility (including PCR, probe extension reaction, and chemiluminometric assay) was studied for 20 days, and the CVs were 11%-19%. CONCLUSIONS: This method is accurate, reproducible, and cost-effective in terms of equipment and reagents. The application of the method is simple, rapid, and robust. The microtiter well format allows genotyping of a large number of samples in parallel for several mutations.

Rapid genotyping of CYP2D6, CYP2C19 and TPMT polymorphisms by primer extension reaction in a dipstick format.

In recent years an increasing amount of interest has been directed at the study and routine testing of polymorphisms responsible for variations in drug metabolism. Most of the current methods involve either time-consuming electrophoresis steps or specialized and expensive equipment. In this context, we have developed a rapid, simple and robust method for genotyping of CYP2D6*3, CYP2D6*4, CYP2C19*2, CYP2C19*3 and TPMT*2 single nucleotide polymorphisms (SNP). Genomic DNA is isolated from whole blood and the segments that span the SNP of interest are amplified by PCR. The products are subjected directly (without purification) to two primer extension (PEXT) reactions (three cycles each) using normal and mutant primers in the presence of biotin-dUTP. The PEXT primers contain a (dA)(30) segment at the 5' end. The PEXT products are detected visually by a dry-reagent dipstick-type assay in which the biotinylated extension products are captured from immobilized streptavidin on the test zone of the strip and detected by hybridization with oligo(dT)-functionalized gold nanoparticles. Patient samples (76 variants in total) were genotyped and the results were fully concordant with those obtained by direct DNA sequencing.

One-step purification and refolding of recombinant photoprotein aequorin by immobilized metal-ion affinity chromatography.

A hexahistidine tag was fused to the N-terminus of apoaequorin. A suitable vector encoding the fusion protein was constructed and used for transformation of Escherichia coli JM109 cells. Apoaequorin was overexpressed under the control of tac promoter. It was found, however, that most of the protein existed in the form of inclusion bodies. Inclusion bodies were solubilized with urea, followed by purification and refolding of (His)(6)-apoaequorin in a single chromatographic step by immobilized metal-ion affinity chromatography using Ni(2+)-nitrilotriacetic acid agarose. The purity, as determined by SDS-PAGE analysis, was greater than 80%. The yield was 0.7-1 mg apoaequorin from a 50 ml bacterial culture. The kinetics of light emission of purified aequorin upon addition of Ca(2+) was typical of the commercial aequorin. The luminescence of the purified aequorin was a linear function of its concentration extending over six orders of magnitude. As low as 0.5 attomoles purified aequorin gave a signal-to-noise ratio of 1.8.

Detection of transgenes in soybean via a polymerase chain reaction and a simple bioluminometric assay based on a universal aequorin-labeled oligonucleotide probe.

The recombinant photoprotein aequorin was used as a reporter in highly sensitive and automatable hybridization assays for the analysis of transgenic sequences in genetically modified organisms (GMO). The terminator of the nopaline synthase gene (NOS) from Agrobacterium tumefaciens and the 35S promoter sequence were detected in genetically modified soybean. The endogenous, soybean-specific, lectin gene was also detected for confirmation of the integrity of extracted DNA. A universal detection reagent was produced through conjugation of aequorin to the oligonucleotide (dA)(30). Biotinylated (through PCR) products for the three target sequences were captured onto streptavidin-coated wells, and one strand was removed by NaOH treatment. The immobilized single-stranded DNAs were then hybridized with oligonucleotide probes consisting of a target-specific segment and a poly(dT) tail. This allowed the subsequent determination of all hybrids through the use of the (dA)(30)-aequorin conjugate as a universal reagent. The bound aequorin was measured by adding Ca(2+) and integrating the light emission for 3 s. As low as 2 pM (100 amol per well) of amplified DNA was detectable for all three targets, with a signal-to-background ratio of about 2. The analytical range extended up to 2000 pM. As low as 0.05% GMO content in soybean can be detected with a signal-to-background ratio of 8.2. The overall repeatability of the proposed assay, including DNA extraction, PCR, and hybridization assay, ranged from 7.5-19.8%. The use of a (dA)(30)-aequorin conjugate renders the assay configuration general for any target DNA, provided that the specific probe carries a poly(dT) tail.

Affinity capture-facilitated preparation of aequorin- oligonucleotide conjugates for rapid hybridization assays.

We report a general procedure for the preparation of biomolecular conjugates that combine the molecular recognition properties of oligonucleotides with the high detectability of the photoprotein aequorin. Central to the conjugation protocols is the use of recombinant aequorin fused to a hexahistidine tag. In one protocol, an amino-modified oligonucleotide was treated with a homobifunctional cross-linker carrying two N-hydroxysuccinimide ester groups, and the derivative was allowed to react with (His)(6)-aequorin. A second strategy involved the introduction of protected sulfhydryl groups into (His)(6)-aequorin and subsequent reaction with a heterobifunctional linker containing a N-hydroxysuccinimide and a maleimide group. The strong, but reversible, binding of (His)(6)-aequorin to Ni(2+)-nitrilotriacetic acid agarose enabled the rapid and effective removal of the unreacted oligonucleotide, which otherwise diminishes the performance of the hybridization assay by competing with the conjugate for the complementary target sequence. Aequorin-oligo conjugates prepared by affinity capture showed similar performance with those purified by anion-exchange HPLC. The conjugates were applied to the development of rapid bioluminometric hybridization assays. The analytical range extended from 2 to 2000 pmol/L of target DNA. The reproducibility was less than 10%. The conjugate obtained from a reaction of 10 nmol of (His)(6)-aequorin is sufficient for about 5000 hybridization assays. The proposed conjugation strategy is general because a variety of reporter proteins can be fused to hexahistidine tag by using suitable vectors that are commercially available.

Oligonucleotide-functionalized gold nanoparticles as probes in a dry-reagent strip biosensor for DNA analysis by hybridization.

The highly specific molecular recognition properties of oligonucleotides are combined with the unique optical properties of gold nanoparticles for the development of a dry-reagent strip-type biosensor that enables visual detection of double stranded DNA within minutes. The assay does not require instrumentation and avoids the multiple incubation and washing steps performed in most current assays. Gold nanoparticle reporters with oligo(dT) attached to their surface form an integral part of the strip. Biotinylated PCR products (233 bp or 495 bp) are hybridized (5 min) with a poly(dA)-tailed oligo and applied on the strip, which is then immersed in the appropriate buffer. As the buffer migrates upward, it rehydrates the nanoparticles that are linked to the target DNA through poly(dA)/(dT) hybridization. Capture of the hybrids by immobilized streptavidin in the test zone of the strip generates a characteristic red band. A second red band is formed, by hybridization, in the control zone of the strip to indicate proper test performance. The sensor offers at least 8 times higher detectability than ethidium bromide staining of agarose gels and provides confirmation of the amplified fragments. Quantitative data are obtained by densitometric analysis of the bands. As low as 2 fmol of amplified DNA were detectable by the strip sensor. Also, 500 copies of prostate-specific antigen cDNA were detected by combining PCR and the strip sensor. The sensor was used successfully for detection of hepatitis C virus in plasma samples from 20 patients. The strip detected 16 out of 16 positive samples and gave no signal for 4 samples that were negative for the virus. To our knowledge, this is the first dry-reagent system that makes use of oligonucleotide-conjugated gold nanoparticles as probes.