Large DNA Research Articles

Unveiling novel Neocosmospora species from Thai mangroves as potent biocontrol agents against Colletotrichum species.

Neocosmospora species are saprobes, endophytes, and pathogens belonging to the family Nectriaceae. This study aims to investigate the taxonomy, biosynthetic potential and application of three newly isolated Neocosmospora species from mangrove habitats in the southern part of Thailand using phylogeny, bioactivity screening, genome sequencing and bioinformatics analysis. Detailed descriptions, illustrations, and a multi-locus phylogenetic tree with large subunit ribosomal DNA (LSU), internal transcribed spacer (ITS), translation elongation factor 1-alpha (ef1-α), and RNA polymerase II second largest subunit (RPB2) regions showing the placement of three fungal strains, MFLUCC 17-0253, MFLUCC 17-0257, and MFLUCC 17-0259 clustered within the Neocosmospora clade with strong statistical support. Fungal crude extracts of the new species Neocosmospora mangrovei MFLUCC 17-0253 exhibited strong antifungal activity to control Colletotrichum truncatum CG-0064, while N. ferruginea MFLUCC 17- 0259 exhibited only moderate antifungal activity toward C. acutatum CC-0036. Thus, N. mangrovei MFLUCC 17-0253 was sequenced by Oxford nanopore technology. The bioinformatics analysis revealed that 49.17 Mb genome of this fungus harbors 41 potential biosynthetic gene clusters. Two fungal isolates of Neocosmospora and a new species of N. mangrovei were reported in this study. These fungal strains showed activity against pathogenic fungi causing anthracnose in chili. In addition, full genome sequencing and bioinformatics analysis of N. mangrovei MFLUCC 17-0253 was obtained.

Novel betaherpesviruses and gammaherpesviruses in bats from central China

Herpesviruses are large double-stranded DNA viruses that cause infections in animals and humans with a characteristic of latent infectious within specific tissues. Bats are natural hosts of variety human-infecting viruses and recently have been described as hosts for herpesviruses in several countries around the world. In this study we collected 140 insectivorous bats in the neighboring urban areas of Wuhan City, Hubei Province in the central China between 2020 and 2021. Nested PCR targeting the dpol gene sequence indicated that a total of 22 individuals (15.7% of the sample) tested positive for herpesvirus with 4 strains belonging to the genus Betaherpesvirus and the remaining 18 strains classified as Gammahersvirus. Furthermore, the herpesvirus prevalence in Rhinolophus pusillus was higher at 26.3%, compared to 8.4% in Myotis davidii. The RP701 strain from R. pusillus was the predominant gammaherpesvirus strain detected in bats, accounting for 94.4% (17/18) of all strains. The variations in γ-herpesviruses genomic sequences was evident in phylogenetic tree, where RP701 strain was clustered together with ruminant γ-herpesviruses, while MD704 strain formed a distinct clade with a hedgehog γ-herpesvirus. Four betaherpesviruses exclusively identified from M. davidii, with nucleotide identities ranging from 79.7 to 82.6% compared to known betaherpesviruses. Our study provided evidence that M. davidii can sever as natural host for β-herpesviruses, which extended the host species range. In conclusion, we found that bats from central China harbored novel β-herpesviruses and γ-herpesviruses which were phylogenetically related to ruminant γ-herpesvirus and hedgehog γ-herpesvirus. Our study indicates that bats are natural hosts of β- and γ-herpesviruses and further studies are needed to determine whether there is cross-species transmission of herpesviruses between bats and other animals, or humans.

Scanning Electron Microscopy Imaging of Large DNA Molecules Using a Metal-Free Electro-Stain Composed of DNA-Binding Proteins and Synthetic Polymers.

This paper presents the first scanning electron microscopy (SEM)-based DNA imaging in biological samples. This novel approach incorporates a metal-free electro-stain reagent, formulated by combining DNA-binding proteins and synthetic polymers to enhance the visibility of 2-nm-thick DNA under SEM. Notably, DNA molecules stain with proteins and polymers appear as dark lines under SEM. The resulting DNA images exhibit a thickness of 15.0±4.0nm. As SEM is the primary platform, it integrates seamlessly with various chemically functionalized large surfaces with the aid of microfluidic devices. The approach allows high-resolution imaging of various DNA structures including linear, circular, single-stranded DNA and RNA, originating from nuclear and mitochondrial genomes. Furthermore, quantum dots are successfully visualized as bright labels that are sequence-specifically incorporated into DNA molecules, which highlights the potential for SEM-based optical DNA mapping. In conclusion, DNA imaging using SEM with the novel electro-stain offers electron microscopic resolution with the ease of optical microscopy.

PSI-5 Evaluation of EhV (surrogate for African swine fever virus) inactivation in corn- and soybean-based feed ingredients and diets at various times and temperatures during storage

Abstract African swine fever virus (ASFV) is a member of the nucleocytoplasmic large DNA viruses (NCLDVs) and remains stable in a variety of environments, including animal feed ingredients as shown in previous laboratory experiments and simulations. Emiliania huxleyi virus (EhV) is another member of NCLDV, which has a restricted host range limited to a species of marine algae called Emiliania huxleyi. This algal NCLDV has many similar morphological and physical characteristics to ASFV which makes it a safe and reliable surrogate for studying ASFV survival and inactivation in feed ingredient matrices in-situ under various time and temperature storage conditions. The objective of the present study was to investigate the inactivation of ASFV in various feedstuffs under a wide range of time and temperature conditions during storage using EhV as a surrogate. Corn- and soybean-based feed ingredients including corn, corn fermented protein, distiller dried grains with solubles, extruded soybean meal, high protein distiller’s grains, solvent-extracted soybean meal, soybean hulls, complete feed, and a positive control (no matrix) were inoculated with EhV (1 × 108 viruses/mL). Negative controls were inoculated with medium containing no EhV for each matrix. To assess the effects of different times and temperatures of storage, all samples were stored at temperatures of 4, 24, and 34 °C for 1, 5, 60, and 120 d. For each timepoint and temperature combination, samples were evaluated for virus presence (qPCR) and viability using a previously validated viability qPCR (v-qPCR) method. On average, we observed a 0.2 log reduction in viable viral DNA content over the 120-d period, with a 0.1 log reduction across varying temperatures. No reductions in EhV viability exceeding 1 log were detected for any matrix and the positive control at any time and temperature combination evaluated. No EhV was recovered from the negative control samples. Given that the experimental error was calculated to be within the range of 1 log, we concluded that storing EhV at 4, 24, or 34 °C for up to 120 d did not result in the inactivation of EhV in any of the ingredients, complete feed, or the control with no matrix. These results demonstrate that ASFV-like NCLDVs is extremely resilient and can maintain viability in various feed matrices during long-term storage and suggest that extending storage time alone may not be an effective mitigation practice against African swine fever virus.

Heteromultivalent DNA Enhances the Assembly Yield of Hybrid Nanoparticles and Facilitates Dynamic Disassembly for Bioanalysis Using ICP-MS.

To obtain enhanced physical and biological properties, various nanoparticles are typically assembled into hybrid nanoparticles through the binding of multiple homologous DNA strands to their complementary counterparts, commonly referred to as homomultivalent assembly. However, the poor binding affinity and limited controllability of homomultivalent disassembly restrict the assembly yield and dynamic functionality of the hybrid nanoparticles. To achieve a higher binding affinity and flexible assembly choice, we utilized the paired heteromultivalency DNA to construct hybrid nanoparticles and demonstrate their excellent assembly characteristics and dynamic applications. Specifically, through heteromultivalency, DNA-functionalized magnetic beads (MBs) and gold nanoparticles (AuNPs) were efficiently assembled. By utilizing ICP-MS, the assembly efficiency of AuNPs on MBs was directly monitored, enabling quantitative analysis and optimization of heteromultivalent binding events. As a result, the enhanced assembly yield is primarily attributed to the fact that heteromultivalency allows for the maximization of effective DNA probes on the surface of nanoparticles, eliminating steric hindrance interference. Subsequently, with external oligonucleotides as triggers, it was revealed that the disassembly mechanism of hybrid nanoparticles was initiated, which was based on an increased local concentration rather than toehold-mediated displacement of paired heteromultivalency DNA probes. Capitalizing on these features, an output platform was then established based on ICP-MS signals that several Boolean operations and analytical applications can be achieved by simply modifying the design sequences. The findings provide new insights into DNA biointerface interaction, with potential applications to complex logic operations and the construction of large DNA nanostructures.

Targeted genome-modification tools and their advanced applications in crop breeding.

Crop improvement by genome editing involves the targeted alteration of genes to improve plant traits, such as stress tolerance, disease resistance or nutritional content. Techniques for the targeted modification of genomes have evolved from generating random mutations to precise base substitutions, followed by insertions, substitutions and deletions of small DNA fragments, and are finally starting to achieve precision manipulation of large DNA segments. Recent developments in base editing, prime editing and other CRISPR-associated systems have laid a solid technological foundation to enable plant basic research and precise molecular breeding. In this Review, we systematically outline the technological principles underlying precise and targeted genome-modification methods. We also review methods for the delivery of genome-editing reagents in plants and outline emerging crop-breeding strategies based on targeted genome modification. Finally, we consider potential future developments in precise genome-editing technologies, delivery methods and crop-breeding approaches, as well as regulatory policies for genome-editing products.

'Gel-Stacks' gently confine or reversibly immobilize arrays of single DNA molecules for manipulation and study.

Large DNA molecules (>20kb) are difficult analytes prone to breakage during serial manipulations and cannot be 'rescued' as full-length amplicons. Accordingly, to present, modify and analyze arrays of large, single DNA molecules, we created an easily realizable approach offering gentle confinement conditions or immobilization via spermidine condensation for controlled delivery of reagents that support live imaging by epifluorescence microscopy termed 'Gel-Stacks.' Molecules are locally confined between two hydrogel surfaces without covalent tethering to support time-lapse imaging and multistep workflows that accommodate large DNA molecules. With a thin polyacrylamide gel layer covalently bound to a glass surface as the base and swappable, reagent-infused, agarose slabs on top, DNA molecules are stably presented for imaging during reagent delivery by passive diffusion.

AMPLON: Amplifying DNA with Multiarm Priming and Looping Optimization of Nucleic Acid.

Nucleic acid amplification, the bedrock of biotechnology and molecular diagnostics, surges in applications-especially isothermal approaches-heightening the demand for advanced and precisely engineered methods. Here, a novel approach for amplifying DNA with multiarm priming and looping optimization of nucleic acid (AMPLON) is presented. AMPLON relies on a novel polymeric material with unique set of multiarm polyethylene glycol-DNA primers for efficient DNA amplification under isothermal conditions. Each arm carries single-stranded DNA complementing the sense or antisense sequence of the target DNA. The amplification reaction begins with antisense arms binding to the target DNA, forming a template for sense-carrying arms to direct multiarm large DNA amplicon synthesis through successive DNA looping and unlooping steps. Using human immunodeficiency virus type 1 (HIV-1) as a model clinical target, AMPLON exhibits high sensitivity, detecting target concentrations as low as 100 copies mL-1. Compared to a quantitative real-time polymerase chain reaction assay using sensitive primers, AMPLON reliably identifies HIV-1 RNA in plasma samples (n = 20) with a significant agreement rate of 95%. With its ability to achieve highly specific and sensitive target amplification within 30 min, AMPLON holds immense potential to transform the field of nucleic acid research and unleashing new possibilities in medicine and biotechnology.

Ascochyta erotica sp. nov. Pathogenic on Convolvulus arvensis

Convolvulus arvensis is an herbaceous dicotyledonous plant in the Convolvulaceae family that is native to Europe and Asia. It is a perennial soboliferous plant and is one of the most harmful weeds. This weed is successful in many types of climates, including temperate, tropical, and Mediterranean climates, but it is most troublesome for agriculture throughout the temperate zone. In this study, several pathogenic isolates were collected from this host. The internal transcribed spacer (ITS) and large subunit (28S) or ribosomal DNA, partial DNA-directed RNA polymerase II subunit (rpb2), and β-tubulin (tub2) genes were amplified and sequenced for all the isolates studied. Further, both a multilocus phylogenetic analysis of DNA sequences and an analysis of morphological features were implemented. Based on the results obtained, all the studied isolates were found to be distinct from any described species in the genus Ascochyta and are, therefore, described here as a new species Ascochyta erotica sp. nov. The pathogenicity of A. erotica sp. nov. was also tested and confirmed on leaf segments of C. arvensis.

Spray-drying of PEI-/PPI-based nanoparticles for DNA or siRNA delivery

Spray-drying of nucleic acid-based drugs designed for gene therapy or gene knockdown is associated with many advantages including storage stability and handling as well as the possibility of pulmonary application. The encapsulation of nucleic acids in nanoparticles prior to spray-drying is one strategy for obtaining efficient formulations. This, however, strongly relies on the definition of optimal nanoparticles, excipients and spray-drying conditions. Among polymeric nanoparticles, polyethylenimine (PEI)-based complexes with or without chemical modifications have been described previously as very efficient for gene or oligonucleotide delivery. The tyrosine-modification of linear or branched low molecular weight PEIs, or of polypropylenimine (PPI) dendrimers, has led to high complex stability, improved cell uptake and transfection efficacy as well as high biocompatibility.In this study, we identify optimal spray-drying conditions for PEI-based nanoparticles containing large plasmid DNA or small siRNAs, and further explore the spray-drying of nanoparticles containing chemically modified polymers. Poly(vinyl alcohol) (PVA), but not trehalose or lactose, is particularly well-suited as excipient, retaining or even enhancing transfection efficacies compared to fresh complexes. A big mesh size is critically important as well, while the variation of the spray-drying temperature plays a minor role. Upon spray-drying, microparticles in a ∼ 3.3 – 8.5 µm size range (laser granulometry) are obtained, dependent on the polymers. Upon their release from the spray-dried material, the nanoparticles show increased sizes and markedly altered zeta potentials as compared to their fresh counterparts. This may contribute to their high efficacy that is seen also after prolonged storage of the spray-dried material.We conclude that these spray-dried systems offer a great potential for the preparation of nucleic acid drug storage forms with facile reconstitution, as well as for their direct pulmonary application as dry powder.

Identification and assessment of antifungal susceptibility of Candida species based on bronchoalveolar lavage in immunocompromised and critically ill patients

Background and Objectives: The presence of fungi in the respiratory tract as mycobiome, particularly Candida species (spp.), remains a serious problem due to increasing numbers of immunocompromised patients. The confirmed reliable ex- istence of these pathogens due to frequent colonization is essential. This investigation aimed to recognize Candida spp. among isolates from bronchoalveolar lavage of immunocompromised and critically ill patients and to evaluate their suscep- tibility to antimycotic drugs.
 Materials and Methods: Bronchoalveolar lavage fluid was collected from 161 hospitalized patients presenting with sus- pected respiratory fungal infection /colonization. The specimens were examined by standard molecular and mycological assays. Candida spp. were recognized with sequence assessment of the D1-D2 section of the large subunit ribosomal DNA. The susceptibility of Candida isolates to common antimycotic drugs was distinguished by standard broth microdilution.
 Results: Seventy-one clinical isolates of Candida spp. were recognized. Candida albicans was the most frequent, followed by C. glabrata, C. krusei (Pichia kudriavzevii), C. dubliniensis, C. parapsilosis, and C. tropicalis. We found 5.1% of C. albi- cans isolates and 8% of C. glabrata isolates to show resistance to fluconazole. The whole of the Candida spp. were sensitive to amphotericin B and caspofungin.
 Conclusion: This study demonstrated that C. albicans and C. glabrata are the most common isolates of bronchoalveolar lavage fluid in patients, and the drug susceptibility screening confirmed that amphotericin B and caspofungin are effective against Candida spp. but some C.  glabrata and C. albicans isolates showed resistance to fluconazole.

A Robust and Efficient Method to Purify DNA-Scaffolded Nanostructures by Gravity-Driven Size Exclusion Chromatography.

In recent decades, nucleic acid self-assemblies have emerged as popular nanomaterials due to their programmable and robust assembly, prescribed geometry, and versatile functionality. However, it remains a challenge to purify large quantities of DNA nanostructures or DNA-templated nanocomplexes for various applications. Commonly used purification methods are either limited by a small scale or incompatible with functionalized structures. To address this unmet need, we present a robust and scalable method of purifying DNA nanostructures by Sepharose resin-based size exclusion. The resin column can be manually packed in-house with reusability. The separation is driven by a low-pressure gravity flow in which large DNA nanostructures are eluted first followed by smaller impurities of ssDNA and proteins. We demonstrated the efficiency of the method for purifying DNA origami assemblies and protein-immobilized DNA nanostructures. Compared to routine agarose gel electrophoresis that yields 1 μg or less of purified products, this method can purify ∼100-1000 μg of DNA nanostructures in less than 30 min, with the overall collection yield of 50-70% of crude preparation mixture. The purified nanocomplexes showed more precise activity in evaluating enzyme functions and antibody-triggered activation of complement protein reactions.

Chemical unclonable functions based on operable random DNA pools

Physical unclonable functions (PUFs) based on unique tokens generated by random manufacturing processes have been proposed as an alternative to mathematical one-way algorithms. However, these tokens are not distributable, which is a disadvantage for decentralized applications. Finding unclonable, yet distributable functions would help bridge this gap and expand the applications of object-bound cryptography. Here we show that large random DNA pools with a segmented structure of alternating constant and randomly generated portions are able to calculate distinct outputs from millions of inputs in a specific and reproducible manner, in analogy to physical unclonable functions. Our experimental data with pools comprising up to >1010 unique sequences and encompassing >750 comparisons of resulting outputs demonstrate that the proposed chemical unclonable function (CUF) system is robust, distributable, and scalable. Based on this proof of concept, CUF-based anti-counterfeiting systems, non-fungible objects and decentralized multi-user authentication are conceivable.

Deconvoluting the Effect of Cell-Penetrating Peptides for Enhanced and Controlled Insertion of Large-Scale DNA Nanopores.

DNA nanopores have emerged as powerful tools for molecular sensing, but the efficient insertion of large DNA nanopores into lipid membranes remains challenging. In this study, we investigate the potential of cell-penetrating peptides (CPPs), specifically SynB1 and GALA, to enhance the insertion efficiency of large DNA nanopores. We constructed SynB1- or GALA-functionalized DNA nanopores with an 11 nm inner diameter and visualized and quantified their membrane insertion using a TIRF microscopy-based single-liposome assay. The results demonstrated that incorporating an increasing number of SynB1 or GALA peptides into the DNA nanopore significantly enhanced the membrane perforation. Kinetic analysis revealed that the DNA nanopore scaffold played a role in prearranging the CPPs, which facilitated membrane interaction and pore formation. Notably, the use of pH-responsive GALA peptides allowed highly efficient and pH-controlled insertion of large DNA pores. Furthermore, single-channel recording elucidated that the insertion process of single GALA-modified nanopores into planar lipid bilayers was dynamic, likely forming transient large toroidal pores. Overall, our study highlights the potential of CPPs as insertion enhancers for DNA nanopores, which opens avenues for improved molecule sensing and the controlled release of cargo molecules.

Oxytetracycline hyper-production through targeted genome reduction of Streptomyces rimosus.

Most biosynthetic gene clusters (BGC) encoding the synthesis of important microbial secondary metabolites, such as antibiotics, are either silent or poorly expressed; therefore, to ensure a strong pipeline of novel antibiotics, there is a need to develop rapid and efficient strain development approaches. This study uses comparative genome analysis to instruct rational strain improvement, using Streptomyces rimosus, the producer of the important antibiotic oxytetracycline (OTC) as a model system. Sequencing of the genomes of two industrial strains M4018 and R6-500, developed independently from a common ancestor, identified large DNA rearrangements located at the chromosome end. We evaluated the effect of these genome deletions on the parental S. rimosus Type Strain (ATCC 10970) genome where introduction of a 145 kb deletion close to the OTC BGC in the Type Strain resulted in massive OTC overproduction, achieving titers that were equivalent to M4018 and R6-500. Transcriptome data supported the hypothesis that the reason for such an increase in OTC biosynthesis was due to enhanced transcription of the OTC BGC and not due to enhanced substrate supply. We also observed changes in the expression of other cryptic BGCs; some metabolites, undetectable in ATCC 10970, were now produced at high titers. This study demonstrated for the first time that the main force behind BGC overexpression is genome rearrangement. This new approach demonstrates great potential to activate cryptic gene clusters of yet unexplored natural products of medical and industrial value.IMPORTANCEThere is a critical need to develop novel antibiotics to combat antimicrobial resistance. Streptomyces species are very rich source of antibiotics, typically encoding 20-60 biosynthetic gene clusters (BGCs). However, under laboratory conditions, most are either silent or poorly expressed so that their products are only detectable at nanogram quantities, which hampers drug development efforts. To address this subject, we used comparative genome analysis of industrial Streptomyces rimosus strains producing high titers of a broad spectrum antibiotic oxytetracycline (OTC), developed during decades of industrial strain improvement. Interestingly, large-scale chromosomal deletions were observed. Based on this information, we carried out targeted genome deletions in the native strain S. rimosus ATCC 10970, and we show that a targeted deletion in the vicinity of the OTC BGC significantly induced expression of the OTC BGC, as well as some other silent BGCs, thus suggesting that this approach may be a useful way to identify new natural products.

DULCITRANSVERSOTREMA N. GEN. (DIGENEA: TRANSVERSOTREMATIDAE) AND FIRST MORPHOLOGICAL AND NUCLEOTIDE-BASED CONFIRMATION OF THE LIFE CYCLE OF A TRANSVERSOTREMATID IN NORTH AMERICA.

We propose Dulcitransversotrema n. gen. for the freshwater transversotrematids Dulcitransversotrema patialense (Soparkar, 1924) n. comb. and Dulcitransversotrema chauhani (Agrawal and Singh, 1960) n. comb. Dulcitransversotrema is unique by the combination of lacking an oral sucker and an extensively coiled uterus as well as having testes that are not deeply lobed and that abut or nearly abut the inner posterolateral margins of the digestive tract (vs. deeply lobed testes that abut the cyclocoel region of the digestive tract only or that do not abut the ceca), a pre-oral genital pore (vs. post-oral genital pore), an ovary that is anterior to and abuts or nearly abuts the sinistral testis (vs. an ovary that is separated from the sinistral testis by some distance or having an ovary that is median and immediately pre-testicular), non-embryonated eggs (vs. embryonated eggs), typically having none or sparse vitelline follicles between the testes and ceca (vs. having a wholly inter-cecal vitellarium comprising a pair of highly compacted clusters of vitelline follicles anterolateral to the testes or having numerous vitelline follicles between the testes and ceca), an oblong, median, and primarily inter-testicular vitelline reservoir (vs. an extensively elongate, pre-testicular vitelline reservoir that arches anteriorly around the sinistral testis), and a subterminal excretory pore opening on the dorsal body surface between the level of the cyclocoel and posterior body end (vs. a terminal excretory pore). We describe the redia and cercaria of Dulcitransversotrema cf. patialense from the red-rimmed melania snail, Melanoides tuberculata (Müller, 1774) (Cerithioidea: Thiaridae), and their corresponding adult from beneath the scales of the zebrafish, Danio rerio (Hamilton, 1822) (Cypriniformes: Danionidae). All hosts were sampled from a spring-fed earthen pond at a private aquaculture facility near Ruskin, Florida. No transversotrematid life cycle was known from North America previously. The large subunit ribosomal DNA and ribosomal internal transcribed spacer 2 sequences of D. cf. patialense from Florida were most similar to those from D. patialense infecting red-rimmed melania from Mayagüez, Puerto Rico and differed by 134 and 69 nucleotides, respectively. Both phylogenetic analyses recovered Dulcitransversotrema as monophyletic and sister to a clade comprising Transversotrema spp. plus Crusziella formosa Cribb, Bray, and Barker, 1992.

A commentary of “Novel approaches in precise genome manipulations from single nucleotides to large DNA segments”: Top 10 Scientific Advances of 2023, China

A commentary of “Novel approaches in precise genome manipulations from single nucleotides to large DNA segments”: Top 10 Scientific Advances of 2023, China

Isolation of viruses, including mollivirus, with the potential to infect Acanthamoeba from a Japanese warm temperate zone.

Acanthamoeba castellanii is infected with diverse nucleocytoplasmic large DNA viruses. Here, we report the co-isolation of 12 viral strains from marine sediments in Uranouchi Inlet, Kochi, Japan. Based on the morphological features revealed by electron microscopy, these isolates were classified into four viral groups including Megamimiviridae, Molliviridae, Pandoraviridae, and Pithoviridae. Genomic analyses indicated that these isolates showed high similarities to the known viral genomes with which they are taxonomically clustered, and their phylogenetic relationships were also supported by core gene similarities. It is noteworthy that Molliviridae was isolated from the marine sediments in the Japanese warm temperate zone because other strains have only been found in the subarctic region. Furthermore, this strain has 19 and 4 strain-specific genes found in Mollivirus sibericum and Mollivirus kamchatka, respectively. This study extends our knowledge about the habitat and genomic diversity of Molliviridae.

DNA barcoding insufficiently identifies European wild bees (Hymenoptera, Anthophila) due to undefined species diversity, genus-specific barcoding gaps and database errors.

Recent declines in insect abundances, especially populations of wild pollinators, pose a threat to many natural and agricultural ecosystems. Traditional species monitoring relies on morphological character identification and is inadequate for efficient and standardized surveys. DNA barcoding has become a standard approach for molecular identification of organisms, aiming to overcome the shortcomings of traditional biodiversity monitoring. However, its efficacy depends on the completeness of reference databases. Large DNA barcoding efforts are (almost entirely) lacking in many European countries and such patchy data limit Europe-wide analyses of precisely how to apply DNA barcoding in wild bee identification. Here, we advance towards an effective molecular identification of European wild bees. We conducted a high-effort survey of wild bees at the junction of central and southern Europe and DNA barcoded all collected morphospecies. For global analyses, we complemented our DNA barcode dataset with all relevant European species and conducted global analyses of species delimitation, general and genus-specific barcoding gaps and examined the error rate in DNA data repositories. We found that (i) a sixth of all specimens from Slovenia could not be reliably identified, (ii) species delimitation methods show numerous systematic discrepancies, (iii) there is no general barcoding gap across all bees and (iv) the barcoding gap is genus specific, but only after curating for errors in DNA data repositories. Intense sampling and barcoding efforts in underrepresented regions and strict curation of DNA barcode repositories are needed to enhance the use of DNA barcoding for the identification of wild bees.

Abstract 6938: Identification of distinct tumor-TME ecomodules in glioma from neurofibromatosis type 1

Abstract Neurofibromatosis type 1 (NF-1) is the most common cancer predisposition syndrome in which 15-20% of affected individuals develop glioma. Large scale DNA and RNA bulk profiling showed the molecular complexity of NF-1 glioma with the tumor cellular ecosystem constituted by multiple malignant phenotypes and heterogenous immune microenvironment. However, the composition and function of infiltrating cells was hidden in the bulk tumor, and the extended granularity of NF-1 glioma tumor microenvironment (TME) remained still unexplored. Here, we collected glioma samples from 46 NF-1 patients including 22 high-grade (HGG) and 24 low-grade (LGG) tumors, and we analyzed their gene expression by single nuclei RNA sequencing. A total of 239,044 single cells were classified into tumor and non-tumor components by integrating multiple computational approaches (including genomic copy number inference, gene signature enrichment, and clustering). We defined the pattern of intra-tumor heterogeneity of NF-1 glioma cells using non-negative matrix factorization and derived 7 malignant meta-programs (MPs) that we respectively defined as Neuronal-like, EMT, Astrocyte-like, Dividing Radial Glia-like, Ependymal-like, Immune, and Glycolytic/Hypoxic-like. These MPs recapitulated normal brain cell subtypes, thereby reflecting broad cell plasticity. The non-tumor cell compartment (121,364 cells, 51%) was dissected for the characterization of the cell types that populate the TME of NF-1 glioma. We identified different subpopulations exhibiting specific immune functions within myeloid and lymphoid components. Different glioma ecomodules were highlighted by comparing the relative composition of the TME across the tumors. Recruitment and activation of cytotoxic CD8+ T cells and natural killers by an active crosstalk with dendritic and pro-inflammatory myeloid cells defined an immune-supportive phenotype that could mediate a potential anti-tumor response in low-grade NF-1 glioma (LGG immune high). Conversely, regulatory T cell infiltration and effector T cell exhaustion induced immune suppression in a low-grade glioma immune dysfunctional ecomodule. The absence of lymphocytes characterized a large set of cold tumors, mostly including high-grade glioma. Together, the complex interplay of tumor cell states with different TME compartments elucidated the existence of separate ecomodules in NF-1 glioma, with the LGG immune high TME associated with Neuronal-like and the LGG immune dysfunctional with Ependymal-like tumor cells. The Ependymal-like state also exhibits maximal association with brain-specific normal cells, including oligodendrocytes, neurons and astrocytes, whereas the HGG are enriched with Dividing Radial Glia- and Glycolytic/Hypoxic-like tumor cell states. The elucidation of different ecomodules provides novel insights for the application of targeted therapies in NF-1 glioma patients. Citation Format: Luciano Garofano, Fulvio D'Angelo, Michael Oh, Michele Ceccarelli, Franck Bielle, Marc Sanson, Anna Lasorella, Antonio Iavarone. Identification of distinct tumor-TME ecomodules in glioma from neurofibromatosis type 1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6938.