DNA Sequence Research Articles

Towards global insect biomonitoring with frugal methods.

None of the global targets for protecting nature are currently met, although humanity is critically dependent on biodiversity. A significant issue is the lack of data for most biodiverse regions of the planet where the use of frugal methods for biomonitoring would be particularly important because the available funding for monitoring is insufficient, especially in low-income countries. We here discuss how three approaches to insect biomonitoring (computer vision, lidar, DNA sequences) could be made more frugal and urge that all biomonitoring techniques should be evaluated for global suitability before becoming the default in high-income countries. This requires that techniques popular in high-income countries should undergo a phase of 'innovation through simplification' before they are implemented more broadly. We predict that techniques that acquire raw data at low cost and are suitable for analysis with AI (e.g. images, lidar-signals) will be particularly suitable for global biomonitoring, while techniques that rely heavily on patented technologies may be less promising (e.g. DNA sequences). We conclude the opinion piece by pointing out that the widespread use of AI for data analysis will require a global strategy for providing the necessary computational resources and training. This article is part of the theme issue 'Towards a toolkit for global insect biodiversity monitoring'.

Genome editing: An insight into disease resistance, production efficiency, and biomedical applications in livestock.

One of the primary concerns for the survival of the human species is the growing demand for food brought on by an increasing global population. New developments in genome-editing technology present promising opportunities for the growth of wholesome and prolific farm animals. Genome editing in large animals is used for a variety of purposes, including biotechnology to improve food production, animal health, and pest management, as well as the development of animal models for fundamental research and biomedicine. Genome editing entails modifying genetic material by removing, adding, or manipulating particular DNA sequences from a particular locus in a way that does not happen naturally. The three primary genome editors are CRISPR/Cas 9, TALENs, and ZFNs. Each of these enzymes is capable of precisely severing nuclear DNA at a predetermined location. One of the most effective inventions is base editing, which enables single base conversions without the requirement for a DNA double-strand break (DSB). As reliable methods for precise genome editing in studies involving animals, cytosine and adenine base editing are now well-established. Effective zygote editing with both cytosine and adenine base editors (ABE) has resulted in the production of animal models. Both base editors produced comparable outcomes for the precise editing of point mutations in somatic cells, advancing the field of gene therapy. This review focused on the principles, methods, recent developments, outstanding applications, the advantages and disadvantages of ZFNs, TALENs, and CRISPR/Cas9 base editors, and prime editing in diverse lab and farm animals. Additionally, we address the methodologies that can be used for gene regulation, base editing, and epigenetic alterations, as well as the significance of genome editing in animal models to better reflect real disease. We also look at methods designed to increase the effectiveness and precision of gene editing tools. Genome editing in large animals is used for a variety of purposes, including biotechnology to improve food production, animal health, and pest management, as well as the development of animal models for fundamental research and biomedicine. This review is an overview of the existing knowledge of the principles, methods, recent developments, outstanding applications, the advantages and disadvantages of zinc finger nucleases (ZFNs), transcription-activator-like endonucleases (TALENs), and clustered regularly interspaced short palindromic repeats associated protein 9 (CRISPR/Cas 9), base editors and prime editing in diverse lab and farm animals, which will offer better and healthier products for the entire human race.

Pan-Cancer Interrogation of B7-H3 (CD276) as an Actionable Therapeutic Target across Human Malignancies.

B7-H3 (CD276) is a transmembrane glycoprotein of the B7 immune checkpoint superfamily that has emerged as a promising therapeutic target. To better understand the applicability of B7-H3-directed therapies, we analyzed 156,791 samples comprising 50 cancer types to interrogate the clinical, genomic, transcriptomic, and immunological correlates of B7-H3 mRNA expression. DNA (592-gene/whole-exome) and RNA (whole-transcriptome) sequencing was performed from samples submitted to Caris Life Sciences (Phoenix, AZ). B7-H3 high versus low expression was based on top and bottom quartiles for each cancer type. Patients' overall survival was determined from insurance claims data. Pathway analysis was performed using Gene Set Enrichment Analyses (GSEA). Immune cell fractions were inferred using quanTIseq. B7-H3 is expressed across several human malignancies including prostate, pancreatic, ovarian, and lung cancers. High B7-H3 expression is associated with differences in overall survival, possibly indicating a prognostic role of B7-H3 for some cancers. When examining molecular features across all cancer types, we did not identify recurrent associations between B7-H3 expression and genetic alterations in TP53, RB1, and KRAS. However, we find consistent enrichment of EMT, Wnt, TGF-beta, and Notch signaling pathways. Additionally, tumors with high B7-H3 expression are associated with greater proportions of M1 macrophages, but lower fractions of CD8+ T cells. We have begun to define the genomic, transcriptomic, clinical, and immunological features associated with B7-H3 expression in 50 cancer types. We report novel clinical and molecular features of B7-H3-high tumors which may inform how current B7-H3 therapeutics should be deployed and prioritized.

Primary Cutaneous NUT Carcinoma: Clinicopathologic and Genetic Study of 4 Cases.

WHO Classification of Skin Tumors, fifth edition (2023) has newly described primary cutaneous NUT carcinoma; however, information on this cancer type remains scarce. Herein, we performed clinicopathologic and genetic analyses of 4 cases. Four elderly women (median age 77y, range: 68 to 82y) were included. The median tumor size was 12.5 (10 to 40mm). Tumors were located on the scalp, temple, thigh, and palm. Two (50%) patients presented with regional lymph node metastases. Neither distant metastasis nor mortality was observed during patient follow-up of 10.5 (3 to 15) months. Sanger, panel DNA and whole-exome RNA sequencing revealed BRD3::NUTM1 (n=2) and BRD4::NUTM1 (n=2) fusions. Histology of BRD3-rearranged tumors revealed an epidermal connection, relatively small tumor nests, and ductal or intracytoplasmic luminal formation, whereas that of BRD4-rearranged tumors revealed large solid nests comprising discohesive tumor cells. NUT, cytokeratins, p63, EMA, TRPS1, c-MYB, CD56, and INSM1 were immunoexpressed to varying degrees in all (100%) tumors. Furthermore, diffuse SOX10 expression was common (3/4, 75%). The literature review of five previously described cases revealed women predominance, no recurrence, frequent BRD3::NUTM1 fusions, and histology of ductoglandular structures. Our study findings and literature suggest elderly women predominance, relatively frequent BRD3::NUTM1 fusions, histopathologic ductoglandular differentiation, absence of abrupt keratinisation, and a characteristic immunoprofile in primary cutaneous NUT carcinoma, unlike in that of other organ. No distant metastasis or disease-associated mortality was seen in all cases with limited follow-up.

Selective Capture and Manipulation of DNA through Double Charged Nanopores.

In the past few decades, nanometer-scale pores have been employed as powerful tools for sensing biological molecules. Owing to its unique structure and properties, solid-state nanopores provide interesting opportunities for the development of DNA sequencing technology. Controlling DNA translocation in nanopores is an important means of improving the accuracy of sequencing. Here we present a proof of principle study of accelerating DNA captured across targeted graphene nanopores using surface charge density and find the intrinsic mechanism of the combination of electroosmotic flow induced by charges of nanopore and electrostatic attraction/repulsion between the nanopore and ssDNA. The theoretical study performed here provides a new means for controlling DNA transport dynamics and makes better and cheaper application of graphene in molecular sequencing.

Identification of a candidate gene for the I locus determining the dominant white bulb color in onion (Allium cepa L.).

Through a map-based cloning approach, a gene coding for an R2R3-MYB transcription factor was identified as a causal gene for the I locus controlling the dominant white bulb color in onion. White bulb colors in onion (Allium cepa L.) are determined by either the C or I loci. The causal gene for the C locus was previously isolated, but the gene responsible for the I locus has not been identified yet. To identify candidate genes for the I locus, an approximately 7-Mb genomic DNA region harboring the I locus was obtained from onion and bunching onion (A. fistulosum) whole genome sequences using two tightly linked molecular markers. Within this interval, the AcMYB1 gene, known as a positive regulator of anthocyanin production, was identified. No polymorphic sequences were found between white and red AcMYB1 alleles in the 4,860-bp full-length genomic DNA sequences. However, a 4,838-bp LTR-retrotransposon was identified in the white allele, in the 79-bp upstream coding region from the stop codon. The insertion of this LTR-retrotransposon created a premature stop codon, resulting in the replacement of 26 amino acids with seven different residues. A molecular marker was developed based on the insertion of this LTR-retrotransposon to genotype the I locus. A perfect linkage between bulb color phenotypes and marker genotypes was observed among 5,303 individuals of segregating populations. The transcription of AcMYB1 appeared to be normal in both red and white onions, but the transcription of CHS-A, which encodes chalcone synthase and is involved in the first step of the anthocyanin biosynthesis pathway, was inactivated in the white onions. Taken together, an aberrant AcMYB1 protein produced from the mutant allele might be responsible for the dominant white bulb color in onions.

Intergeneric hybrid origin of the invasive tetraploid Cirsium vulgare.

The invasive tetraploid Cirsium vulgare hybridizes with both Cirsium and Lophiolepis. Its conflicted position in molecular phylogenies, and its peculiar combination of morphological, anatomical, and genomic features that are alternatively shared with representatives of Cirsium or Lophiolepis, strongly suggest its intergeneric hybrid origin. Genetic relationships of C. vulgare (8 samples) with genus Lophiolepis (11 species) and other representatives of genus Cirsium (12 species) were evaluated using restriction site-associated DNA sequencing (RADseq) and examined using analytical and imaging approaches, such as NeighborNet, Heatmap, and STRUCTURE, to identify nuclear genomes admixture. Estimation of the intensity of spontaneous hybridization within and between Cirsium and Lophiolepis was based on herbarium revisions and published data for all reported hybrids pertinent to taxa currently included in Cirsium or Lophiolepis. The genome of any examined Cirsium species is more similar to C. vulgare than to any Lophiolepis species, and vice versa. The nuclear genome of the tetraploid C. vulgare is composed of two equivalent parts, each attributable either to Lophiolepis or to Cirsium; the organellar RADseq data clustered C. vulgare with the genus Cirsium. Spontaneous hybridization between Cirsium and Lophiolepis is significantly less intensive than within these genera. Our analyses provide compelling evidence that the invasive species C. vulgare has an allotetraploid intergeneric origin, with the maternal parent from Cirsium and the paternal from Lophiolepis. For the purpose of delimiting monophyletic genera, we propose keeping Lophiolepis separate from Cirsium and segregating C. vulgare into the hybridogenous genus Ascalea.

Uncovering DNA methylation landscapes to decipher evolutionary footprints of phenotypic diversity in chickpea.

Genetic diversity and environmental factors are long believed to be the dominant contributor to phenotypic diversity in crop plants. However, it has been recently established that, besides genetic variation, epigenetic variation, especially variation in DNA methylation, plays a significant role in determining phenotypic diversity in crop plants. Therefore, assessing DNA methylation diversity in crop plants becomes vital, especially in the case of crops like chickpea, which has a narrow genetic base. Thus, in the present study, we employed whole-genome bisulfite sequencing to assess DNA methylation diversity in wild and cultivated (desi and kabuli) chickpea. This revealed extensive DNA methylation diversity in both wild and cultivated chickpea. Interestingly, the methylation diversity was found to be significantly higher than genetic diversity, suggesting its potential role in providing vital phenotypic diversity for the evolution and domestication of the Cicer gene pool. The phylogeny based on DNA methylation variation also indicates a potential complementary role of DNA methylation variation in addition to DNA sequence variation in shaping chickpea evolution. Besides, the study also identified diverse epi-alleles of many previously known genes of agronomic importance. The Cicer MethVarMap database developed in this study enables researchers to readily visualize methylation variation within the genes and genomic regions of their interest (http://223.31.159.7/cicer/public/). Therefore, epigenetic variation like DNA methylation variation can potentially explain the paradox of high phenotypic diversity despite the narrow genetic base in chickpea and can potentially be employed for crop improvement.

117 Gene editing on the farm: Current status and future prospects

Abstract Livestock with enhanced disease resistance genes offer both health advantages to animals and economic opportunities for farmers aiming to meet the rising demand for affordable, high-quality protein. This study builds upon prior research that utilized gene editing to modify the porcine CD163 gene, demonstrating resistance to the RNA virus responsible for porcine reproductive and respiratory syndrome (PRRS). To amplify the impact of this disease resistance trait, it is crucial for it to be present in commercially significant breeding populations, facilitating the widespread production and distribution of resistant pigs. In pursuit of this objective, a groundbreaking gene editing program was implemented, leveraging CRISPR-Cas technology, next-generation DNA sequencing, in vitro and in vivo off-target analyses, whole-genome sequencing (WGS), and a simplified breeding strategy. This innovative approach aimed to introduce a single modified CD163 allele into four genetically elite and diverse porcine lines, rendering the cells and animals resistant to PRRS virus (PRRSV) infection. The resulting founder population is currently undergoing large-scale phenotype testing, multiplication through classical breeding methods, ongoing genetic refinement, and poised for commercialization pending regulatory approvals. This presentation will provide insights into the process and data associated with this scaled production, marking a significant stride towards the realization of commercially elite PRRS virus resistant pigs.

Genetic Screening—Emerging Issues

In many countries, some form of genetic screening is offered to all or part of the population, either in the form of well-organized screening programs or in a less formalized way. Screening can be offered at different phases of life, such as preconception, prenatal, neonatal and later in life. Screening should only be offered if the advantages outweigh the disadvantages. Technical innovations in testing and treatment are driving changes in the field of prenatal and neonatal screening, where many jurisdictions have organized population-based screening programs. As a result, a greater number and wider range of conditions are being added to the programs, which can benefit couples’ reproductive autonomy (preconception and prenatal screening) and improve early diagnosis to prevent irreversible health damage in children (neonatal screening) and in adults (cancer and cascade screening). While many developments in screening are technology-driven, citizens may also express a demand for innovation in screening, as was the case with non-invasive prenatal testing. Relatively new emerging issues for genetic screening, especially if testing is performed using DNA sequencing, relate to organization, data storage and interpretation, benefit–harm ratio and distributive justice, information provision and follow-up, all connected to acceptability in current healthcare systems.

Taxonomy and phylogeny of the family Suberitidae (Porifera: Demospongiae) in California

This study presents a comprehensive taxonomic revision of the family Suberitidae (Porifera: Demospongiae) for California, USA. We include the three species previously known from the region, document two additional species previously known from other regions, and formally describe four new species as Pseudosuberites latke sp. nov., Suberites californiana sp. nov., Suberites kumeyaay sp. nov., and Suberites agaricus sp. nov. Multi-locus DNA sequence data is presented for seven of the nine species, and was combined with all publicly available data to produce the most comprehensive global phylogeny for the family to date. By integrating morphological and genetic data, we show that morphological characters may be sufficient for regional species identification but are likely inadequate for global classification into genera that reflect the evolutionary history of the family. We therefore propose that DNA sequencing is a critical component to support future taxonomic revisions.

Diagnostic value of microbial cell-free DNA sequencing for suspected invasive fungal infections: a retrospective multicenter cohort study

Abstract Background An early diagnosis and treatment of invasive fungal disease(IFD) is associated with improved outcome, but the moderate sensitivity of non-invasive diagnostic tests makes this challenging. Invasive diagnostic procedures like bronchoalveolar lavage(BAL) have a higher yield but are not without risk. The detection and sequencing of microbial cell-free DNA(mcfDNA) may facilitate a non-invasive diagnosis. Materials In a prospective observational study, we collected plasma in the 120 hours preceding or following a BAL in patients with hematological malignancies suspected for a pulmonary IFD. The EORTC/MSGERC2020 criteria were used for IFD classification. Sequencing was performed by Karius(Redwood City, California) using their Karius Test® (KT) on plasma and a ‘research use only test'(RUO-KT) on BAL fluid if available. Cases with a probable/proven IFD were identified based on standard diagnostic tests on serum and BAL (microscopy, PCR, GM, culture) and used to calculate the sensitivity, specificity and additional diagnostic value of the KT. Results Of 106 patients enrolled, 39 (37%) had a proven/probable invasive aspergillosis (IA), 7 (7%) a non-Aspergillus IFD and 4 (4%) a mixed IFD. The KT detected fungal mcfDNA in 29 (28%) patients. Compared with usual diagnostic tests, the sensitivity and specificity were 44.0%(95%CI:31.2–57.7%) and 96.6%(95%CI:88.5–99.1%). Sensitivity of the KT was higher in non-Aspergillus IFD (Mucorales:2/3, Pneumocystis jirovecii:3/5). On BAL, the sensitivity was 72.2% (95%CI:62.1–96.3%) and specificity 83.3%(95%CI:49.1–87.5%). Conclusions Sequencing of mcfDNA may facilitate a non-invasive diagnosis of IFD in particular non-Aspergillus IFD. However, on plasma and similar to currently available diagnostics, it cannot be used as a “rule-out” test.

Quercetin Exhibits Preferential Binding Interaction by Selectively Targeting HRAS1 I-Motif DNA-Forming Promoter Sequences.

I-Motif (iM) DNA structures represent among the most significant noncanonical nucleic acid configurations. iM-forming DNA sequences are found in an array of vital genomic locations and are particularly frequent in the promoter islands of various oncogenes. Thus, iM DNA is a crucial candidate for anticancer medicines; therefore, binding interactions between iM DNA and small molecular ligands, such as flavonoids, are critically important. Extensive sets of spectroscopic strategies and thermodynamic analysis were utilized in the present investigation to find out the favorable interaction of quercetin (Que), a dietary flavonoid that has various health-promoting characteristics, including anticancer properties, with noncanonical iM DNA structure. Spectroscopic studies and thermal analysis revealed that Que interacts preferentially with HRAS1 iM DNA compared with VEGF, BCL2 iM, and duplex DNA. Que, therefore, emerged as a suitable natural-product-oriented antagonist for targeting HRAS1 iM DNA. The innovative spectroscopic as well as mechanical features of Que and its specific affinity for HRAS1 iM may be useful for therapeutic applications and provide crucial insights for the design of compounds with remarkable medicinal properties.

Nuclear sequences of mitochondrial origin in domestic yak

Mitochondrial DNA sequences are frequently transferred into the nuclear genome, generating nuclear mitochondrial DNA sequences (NUMTs). Here, we analysed, for the first time, NUMTs in the domestic yak genome. We obtained 499 alignment matches covering 340.2 kbp of the yak nuclear genome. After a merging step, we identified 167 NUMT regions with a total length of ~ 503 kbp, representing 0.02% of the nuclear genome. We discovered copies of all mitochondrial regions and found that most NUMT regions are intergenic or intronic and mostly untranscribed. 98 different NUMT regions from domestic yak showed high homology with cow and/or wild yak genomes, suggesting selection or hybridization between domestic/wild yak and cow. To rule out the possibility that the identified NUMTs could be artifacts of the domestic yak genome assembly, we validated experimentally five NUMT regions by PCR amplification. As NUMT regions show high similarity to the mitochondrial genome can potentially pose a risk to domestic yak DNA mitochondrial studies, special care is therefore needed to select primers for PCR amplification of mitochondrial DNA sequences.

Allelic differences in the key genes of betalain biosynthesis in table beet accessions with contrasting root color from the VIR collection

Background. Table beet (Beta vulgaris L.) contains a great amount of naturally red-colored betanins. A number of enterprises are forced to import foreign dyes because the reserves of domestic plant raw materials are insufficient. With this in view, the development of cultivars with high betalain pigment content is now required.Materials and methods. Allelic differences were screened in the key genes of the betalain biosynthesis pathway among table beet accessions with various root color from the VIR collection using the Sanger DNA sequencing method.Results and discussion. For the first time we identified a nonsense mutation in the CYP76AD1 gene in cv. ‘Serdolik’ with yellow flesh; it led to the truncation of the functional P450 domain. We suggested that the detected polymorphism correlated with phenotypic switching because the well-known role of CYP76AD1 was essential for the red betalain accumulation. Moreover, a number of missense mutations in cv. ‘Avalanche’ in the first exon of the BvDODA1 gene were found. These mutations were probably associated with the expression of the uncolored phenotype. An in silico analysis revealed highly homologous copies of CYP76AD5, located tandemly on chromosome 9. Attention should be paid to these copies, together with the CYP76AD6 gene, as they seem the most preferable targets for a knockout to increase the red pigment content.Conclusion. It is possible to identify the best table beet accessions for further genome editing among a previously selected high-betanin group. In addition, this study revealed the allelic differences in the key genes of the betalain biosynthesis pathway. These results will be useful for the development of DNA molecular markers to facilitate the selection of table beet forms with required properties.

Interaction of sound-audition traits between eared insects and arthropodophagous bats: using a DNA approach to assess diet

Abstract Arthropod–bat interactions are often considered as a base model for studying factors underlying predator–prey coevolutionary processes. Bats developed ultrasonic echolocation to hunt, and in response some arthropods developed defense mechanisms such as ultrasonic hearing, allowing them to elude bat predators. The present study analyzes the feeding patterns of bats, focusing on sonic-auditory sensory mechanisms in predator–prey interactions. Next-generation DNA sequence data from fecal samples were used to analyze the diet of 17 bat species from Mexico. Arthropod prey taxa were classified according to their auditory traits, and echolocation data were recompiled from literature review. We: (i) classified arthropod families according to their hearing ability; (ii) estimated arthropod taxon richness and proportion in the diet of each bat species; and (iii) used multidimensional scaling, principal component analysis, and regression to analyze prey consumption patterns in relation to their auditory traits and in relation to echolocation characteristics of bats. Finally, we analyzed the relationship between foraging time and auditory characteristics of prey. Families with hearing organs correspond to the orders Lepidoptera and Orthoptera. We registered 20 families of Lepidoptera and 5 of Orthoptera—7 and 3 with hearing organs, respectively. Of these orders, families lacking ears were recorded in the diet of a few bat species. Our results support the allotonic frequency hypothesis predicting a difference in emission frequency intervals between predator and prey. However, we found that the consumption of earless moths is less frequent and is related to diurnal and twilight activity—hence, their consumption is limited to bat species foraging early. Results indicate bats feed on arthropod prey successfully despite the ultrasonic hearing ability of the prey. These results may be due to counteradaptations that allow maintenance of an asymmetric “arms race” between bats and eared insects that favors the predator.

First report of Colletotrichum queenslandicum associated with chilli anthracnose in Mauritius.

Globally, chilli (Capsicum annuum L.) is one of the most economically important and widely cultivated crop which elicits ethnomedicinal and nutritional potential as well as enhancing the taste and aroma of foods (Ayob et al., 2022; Kiran et al., 2020). Anthracnose disease is regarded as a prime constraint in chilli production, leading to enormous losses in tropical and subtropical countries. In September 2022, chilli fruit displaying sunken, shriveled and dark bown to black lesion with abundant acervuli on the surface was obtained from Flacq, Mauritius. From the symptomatic tissue, small pieces of the diseased tissue were excised, surface-disinfected using 1% sodium hypochlorite, twice rinsed using sterilized distilled water, air-dried and plated on PDA. After 7 days of incubation at room temperature, white to greyish white colony with dense white cottony aerial mycelium was recovered. Out of two isolates, CHF and CH10, the latter was considered for morphological and molecular characterization. The observed conidia (n=30) were unicellular, straight, cylindrical with rounded ends and slight constriction near the centre and had average length and width of 20.5 µm and 6 µm, respectively. For growth rate measurement of the isolate, two 5×5 mm of fungal agar plugs were taken from growing edge of colony, inoculated at centre of individual PDA plate and incubated at room temperature with a natural light/dark cycle. The diameter of the cultures were measured perpendicularly for a period of 7 days and the growth rate was calculated as 7-day average of daily growth (mm day-1). The growth rate of the fungal isolate (CH10) was 13.5 mm day-1 on PDA. Based on the morphological characters, the isolate was classified within the C. gloeosporioides species complex. For precise identification of the isolate, DNA was extracted from fungal mycelium using traditional DNA isolation methods (Ranghoo and Hyde, 2000), followed by PCR amplification and DNA sequencing using primer pairs ITS4/ITS5 (White et al., 1990), GDF/GDR and T1/Bt2b (Gan et al., 2016), respectively. ITS gene sequence (600 bp) confirmed that the isolate was Colletotrichum, with 99.83% similarity to KR704204 while GADPH (277 bp), TUB2 (733 bp) and ApMat (801 bp) gene sequences showed 99.64 to 100% similarity to C. queenslandicum with GenBank reference sequences, KT372374, KU221378 and MG674932 respectively. The gene sequences of isolate CH10 were deposited in GenBank database under the following accession numbers OR681557 (ITS), OR233734 (GADPH), OR475575 (TUB2) and PP622748 (ApMat). Koch's postulates were confirmed by spraying disease-free chilli plants with 10µL of conidial suspension (1 × 106 spores/ml) prepared from 7 days old colony of isolate CH10. Healthy chilli plants inoculated with sterile distilled water served as a negative control experiment. The plants were grown in pots in a moist chamber at 25˚C. After 5 days post-inoculation, anthracnose symptoms were developed on test plants while the control plant remained asymptomatic. The original isolate was successfully recovered from the test fruits, thus fulfilling Koch's postulates. The experiment was repeated thrice and revealed the same results. To the best of our knowledge, this is the first record of C. queenslandicum in Mauritius and is the first time to report anthracnose of chilli caused by this fungus. Colletotrichum queenslandicum has previously been reported in Europe, Mexico, US, Puerto Rico, Australia, Fiji, Brazil, Indonesia and China. Furthermore, the latter was associated with papaya, avocado, cashew, coffee, Persian lime, Licania tomentosa, white mangrove, lychee, mango, Nephelium lappaceum, olive, passionfruit, Dracaena cambodiana and Syzygium australe (Câmara and Vieira, 2022; Shidiq et al., 2024; Wang et al., 2022). This study will allow local farmers training and extension facilities to increase awareness among farmers about this disease-causing agent and allow them to take necessary measures for building up chilli crops resilience against this new and emerging pathogen in Mauritius.

Click Chemistry-Based Force Spectroscopy Revealed Enhanced Binding Dynamics of Phosphorylated HMGB1 to Cisplatin-DNA.

Cisplatin, a cornerstone in cancer chemotherapy, is known for its DNA-binding capacity and forms lesions that lead to cancer cell death. However, the repair of these lesions compromises cisplatin's effectiveness. This study investigates how phosphorylation of HMGB1, a nuclear protein, modifies its binding to cisplatin-modified DNA (CP-DNA) and thus protects it from repair. Despite numerous methods for detecting protein-DNA interactions, quantitative approaches for understanding their molecular mechanism remain limited. Here, we applied click chemistry-based single-molecule force spectroscopy, achieving high-precision quantification of the interaction between phosphorylated HMGB1 and CP-DNA. This method utilizes a synergy of click chemistry and enzymatic ligation for precise DNA-protein immobilization and interaction in the system. Our results revealed that HMGB1 binds to CP-DNA with a significantly high rupture force of ∼130 pN, stronger than most natural DNA-protein interactions and varying across different DNA sequences. Moreover, Ser14 is identified as the key phosphorylation site, enhancing the interaction's kinetic stability by 35-fold. This increase in stability is attributed to additional hydrogen bonding suggested by molecular dynamics (MD) simulations. Our findings not only reveal the important role of phosphorylated HMGB1 in potentially improving cisplatin's therapeutic efficacy but also provide a precise method for quantifying protein-DNA interactions.

Abstract PO2-14-08: Development and characterization of breast cancer organoids representing epithelial heterogeneity and drug response

Abstract Organoids serve as an important preclinical model in multiple cancers including breast cancer, demonstrating preservation of biological features and feasibility of drug screening, with reasonable cost and high scalability. It has been reported that patient-derived organoids (PDOs) can be robustly derived from both primary and metastatic breast tumors, with high resemblance to source tissues in histological, genomic, and transcriptomic features, as well as consistency in drug response compared to xeno-transplantations or patients (Sachs et al, 2018). Since 2018, the Institute for Precision Medicine (IPM) has developed a panel of breast cancer organoids. The current inventory now consists of 128 PDOs from 94 primary tumors, 12 metastases, 12 rapid-autopsy tumors, and 10 normal breast tissues with a 60% success rate of culture; as well as 33 organoids from animal models including 11 patient-derived xenograft organoids (PDXOs), 15 rat (RDO) and 7 mouse (MDO) tumor organoids with 85% success rate. For PDOs, the protocol includes in-lab processing of fresh, deidentified tissue within 60 minutes of surgical operation, thanks to close collaboration with surgeons, consented patients, and an institutional biospecimen core. BC organoids have been comprehensively characterized in terms of morphology, histology, genomics, transcriptomics, and functional experiments. Key biological features are preserved. PDOs (n=56) exhibit a mutational spectrum consistent with human breast cancers. ER expression is detectable in a subset of cultures with robust estradiol response indicated by GREB1 expression; and PDOs from invasive lobular carcinoma (ILC) mostly showed more discohesive structures than PDOs from no special type (NST), with clear E-cadherin loss under immunofluorescence. We have also successfully constructed transcriptionally and genetic modified BC PDOs with shRNA and CRISPR, which showed replicable performance in proliferation assay, drug response, and signaling by western blot. Of note, longitudinal tracking of PDOs with single cell RNA-sequencing (scRNA-seq) revealed well-preserved heterogeneity compared to primary tumor, while transcriptomic clonality was mildly decreased in later passages from pilot data. Future work will include expansion of current collection from various sources, longitudinal characterization with scRNA-seq and DNA sequencing, as well as more functional studies. We aim to construct a robust resource of well-characterized and reliably-usable model bank for preclinical breast cancer research. Citation Format: Daniel Brown, Kai Ding, Fangyuan Chen, Jian Chen, Steffi Oesterreich, Peter Lucas, Priscilla McAuliffe, Jennifer Atkinson, ADRIAN LEE. Development and characterization of breast cancer organoids representing epithelial heterogeneity and drug response [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-14-08.

Abstract PO3-23-11: Multiomics analysis of ER+ breast cancer with matched primaries, lymph node metastases and recurrent cancers on adjuvant endocrine therapy

Abstract Background: 80% of all breast cancers (BCs) are ER-positive (ER+). A significant number develop endocrine therapy resistance (ETR) . Clones develop in lymph nodes (LNs) which differ from the primary and these are implicated in ETR. A multiomics analysis of primary ER+ breast cancers, matched LN metastasis (LN+), if present, and matched recurrences (primary, nodal or distant) has been performed. Patients and Methods: The patients included are: Cohort A: 95 patients with matched primary and recurrent ER+ BC on different endocrine therapies. 35% had involved LNs at diagnosis. Recurrences: local in 59/95, concurrent local and nodal in 33/95, LN-only in 3/95. Median time to recurrence 4.5 years. Cohort B: 18 patients with ER+ LN+ BC who developed multiple recurrences (local and/or nodal, >3 consecutive recurrences/patient) on different lines of sequential endocrine therapy. Cohort C: 7 patients with ER+ breast cancer with multiple involved LNs treated with 3-6 months of neoadjuvant ET before surgery. Biopsies of primary and matched LNs performed at diagnosis and throughout treatment (3-4 timepoints/patient). Clinical response by serial ultrasound. Primary and LN samples (mean 12 samples/patient) were analysed together with normal DNA. Targeted RNAseq and DNA exome sequencing and whole-genome expression analysis was performed. Somatic mutations and copy number alterations (CNA) were determined, and differential gene expression analysis was performed. Validation of pathways implicated in ETR was by spatial NanoString GeoMx. All patients have long term follow-up. Results: Cohort A: A mean of 6 potential driver mutations identified by DNA sequencing in each primary BC (range 1-16). Most commonly mutated genes were PIK3CA, TP53, ERBB4, MAP3K1 and ERBB3. Multiple aberrations and a highly diverse mutational landscape were observed in all recurrences. In the majority of recurrences significant changes in variant allele frequency (VAF) were identified across several ETR-implicated genes and in over 50% new gains/losses were identified. Most frequently, losses were seen in ERBB3, ARID1A, HRAS and FOXA1 with gains in CDH1, NF1, PIK3CA, ESR1, TBX3 and GATA3 in recurrences vs primaries. Transcriptomic analysis revealed differentially expressed pathways including ER, HER2, GATA3, AKT, RAS and p63 signalling. Integrated analysis revealed several driver mutations with corresponding changes in upstream pathway activity. 33 patients had concurrent local and nodal recurrences and 52% had similar somatic mutation profiles in both with only minor variance in VAF. In 48%, 1 or more new potential driver mutations was identified in axillary nodes compared to recurrent primary cancers with losses in AKT1, CDH1 and TP53 being most common. Multiomics profiling of Cohorts B and C is complete and analysis underway. Discussion: This multiomics study is the largest cohort to-date of matched early and recurrent ER+/HER2- cancers. It sheds new light on the complex somatic and transcriptomic changes in local and nodal recurrent disease. The mechanisms of ETR are diverse and underlying mechanisms and clinically meaningful biomarkers including potentially actionable mutations and targets have been identified. Cohort C will uniquely capture and compare clonal evolution of breast cancer in primary and multiple matched lymph during neoadjuvant ET. Citation Format: Carlos Martinez-Perez, Charlene Kay, Rebecca Swan, Lorna Renshaw, J Michael Dixon, Arran K Turnbull. Multiomics analysis of ER+ breast cancer with matched primaries, lymph node metastases and recurrent cancers on adjuvant endocrine therapy [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-23-11.