High Degree Of Polymorphism Research Articles

Полиморфные варианты генов цитокинов в популяциях Арктической зоны России: предрасположенность к заболеваниям

The strategy for the development of the Arctic zone of Russia is primarily aimed at improving the quality of life of the Arctic population, health saving and reducing morbidity. The climatic and geographical conditions of the Arctic zone are extremely uncomfortable for living and cause the development of a number of diseases, as well as the polysyndrome of “northern stress”. Moreover, the Arctic population is affected by persistent organic pollutants that enter the body through a traditional diet and contribute to the development of oncological diseases by suppressing the functions of the immune system. Cytokines — proteins encoded by genes with a high degree of polymorphism, responsible for the nature of inflammatory processes, for the effectiveness of defense functions of the body in response to infections and the oncological process development — are one of the main mediators of the immune system. The distribution of polymorphisms in the cytokine genes produced by different types of cells of immune system (rs2069762 IL2, rs2243250 IL4, rs2069812 IL5, rs1800872 IL10, rs1800925 IL13, rs2275913 IL17A, rs7044343 IL33) in the populations of Nenets, Dolgan-Nganasans and Slavs was studied. Analysis of the results showed that the frequency of TG and GG genotypes rs2069762 IL2, CT genotype rs2243250 IL4, CT genotype rs2069812 IL5, TG genotype rs1800872 IL10, CC genotype rs1800925 IL13, GA genotype rs2275913 IL17A, CC rs7044343 IL33 genotype is significantly higher in Arctic populations compared to Slavs, and may be a potential genetic marker of disease development. The studied mutations are associated with the expression level of the corresponding cytokines and their production, which entails changes in the functioning of the cytokine network. It can be concluded that the indigenous inhabitants of the Russian Arctic have genetically determined rapid development of immune reactions, protection to the development of allergic diseases and resistance to the formation of malignant tumours in comparison with Slavs, i.e. the immigrant population.

Microsatellites for butterfly conservation: historical challenges, current relevance, and a guide to implementation

We are nearing the 20-year anniversary of a groundbreaking paper which details how microsatellite marker development in Lepidoptera is “extremely difficult for no apparent reason.” How far have we come in these past 20 years? Microsatellites are still the marker of choice in many population genetics studies for their ease of use, high degrees of polymorphism, species-specificity, and low cost. The rise of next-generation sequencing technologies (e.g. 454, Illumina, PacBio, etc.) has greatly advanced our abilities to generate many microsatellite markers per species. In this paper, we summarize the improvements in marker development using next-generation technology. Using case studies, we review the use and implementation of microsatellite markers in different conservation programs. Lastly, we provide a guide to data interpretation of microsatellite data generated for butterflies, with the goal of supporting student researchers and conservation practitioners in evaluating the meaning in their data.

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Abstract Major histocompatibility complex (MHC) genes are among the most polymorphic and fastest evolving genes in vertebrate genomes with more than 1,000 alleles found at some human MHC loci. In this study, MHCII genes are explored in Atlantic herring, a species that comprises one of the biggest populations among vertebrates and occurs in huge shoals providing a potential target for pathogens using whole genome, long read sequencing of 14 individuals from three different geographic regions. The analysis identified a total of nine MHC class II loci present on three chromosomes. Some of the genes show extremely high nucleotide diversity (π = 10-13%) compared with the genome average (0.3%) as well as strong signatures of positive selection (dN/dS >> 1). Furthermore, we showed a highly significant nonrandom association of alpha and beta genes, strongly suggesting coevolution of alpha and beta alleles forming the peptide binding domain of MHC class II molecules. To the best of our knowledge this is the first genetic study of MHC polymorphism in a non-model organism based on whole genome, long-read sequencing. It demonstrates that the Atlantic herring MHC class II genes are among the most polymorphic so far described. The very high degree of polymorphisms is most likely explained by the combination of the large population size minimizing loss of genetic diversity due to drift and pathogen-driven selection.

Polymorphism of IL4 and IL13 anti-inflammatory cytokine genes in indigenous populations of the russian Arctic

Living conditions in the Arctic zone are extremely unfavorable for living and contribute to the development of a number of diseases due to suppression of the functions of the immune system. Cytokines are one of the main mediators of the immune system; they are encoded by genes with a high degree of polymorphism. This study examined the distribution of genetic variants in the cytokine genes (rs2243250 IL4, rs1800925 IL13) among the Nenets, Dolgan-Nganasan and Slavs. It was previously established that these mutations are associated with the level of expression of these interleukins and their production, which leads to changes in the impairment of the immune response to the influence of the pathogen. The study showed the predominance of genotypes CT and TT of the rs2243250 IL4 polymorphism, and genotype CC of the rs1800925 IL13 polymorphism in the Nenets and Dolgan-Nganasan populations. The results obtained suggest that the indigenous inhabitants of the Russian Arctic have a genetically determined rapid immune response and protection against the development of allergic diseases compared to the Slavs.

THE GENETIC DIVERSITY OF MAIZE INBRED LINES GROWN IN IRAQ USING SIMPLE SEQUENCE RECURRENT (SSR) MARKERS

This study was conducted to determine the genetic variability among maize inbred lines grown in Iraq by using Simple Sequence Recurrent (SSR) markers. For estimating the genetic relationships, 10 SSR primers were used with 10 maize inbred lines. Two hundred and sixteen alleles (bands) with a range of 13 to 41 were identified for the SSR loci. Polymorphism information content (PIC) of the 10 SSR loci ranged from 0.60% to 100%. Primers ‘umc1566’, ‘umc1542’, and ‘umc2189’ showed the maximum PIC value; by contrast, ‘umc2225’showed the lowest PIC value. The 10 maize inbreds were clustered based on the matrix of genetic similarity using the UPGMA algorithm. Cluster analysis placed the inbreds lines in three clusters based on SSR data. The studied inbreds lines divided to groups and subgroups with cluster analysis that revealed agreement with their geographical origin. The results indicated that SSR marker has a high degree of polymorphism that allows efficient identification of maize genotypes, and this could be used in determining their heterotic groups.

High-resolution characterization of KIR genes polymorphism in healthy subjects from the Bulgarian population-A pilot study.

Although killer-cell immunoglobulin-like receptor (KIR) gene content has been widely studied in health and disease, with the advancement of next-generation sequencing (NGS) technology the high-resolution characterization of this complex gene region has become achievable. KIR allele-level diversity has lately been described across human populations. The present study aimed to analyze for the first time the allele-level polymorphism of nine KIR genes in 155 healthy, unrelated individuals from the Bulgarian population by applying NGS. The highest degree of polymorphism was detected for the KIR3DL3 gene with 40 observed alleles at five-digit resolution in total, 22 of which were common. On the other hand, the KIR3DS1 gene was found to have the lowest degree of polymorphism among the studied KIR genes with one common allele: KIR3DS1*01301 (31.6%). To better understand KIR allelic associations and patterns in Bulgarians, we have estimated the pairwise linkage disequilibrium (LD) for the 10 KIR loci, where KIR2DL3*00501 allele was found in strong LD with KIR2DL1*00101 (D' = 1.00, R2 = 0.742). This is the first study investigating KIR polymorphism at the allele level in a population from the South-East European region. Considering the effect of the populationally shaped KIR allelic polymorphism on NK cell function, this data could lead to a better understanding of the genetic heterogeneity of this region and can be carried into clinical practice by improvement of the strategies taken for NK-mediated diseases.

Exploring methodologies in Cannabis tissue-culture and genetic transformation: Opportunities and obstacles

In recent years, the growing interest in Cannabis sativa L., particularly its medicinal and aromatic properties, has propelled advancements in its tissue culture and genetic transformation techniques. This review delineates the significant strides and persistent challenges in the field, offering a comprehensive overview of the current methodologies and their implications. It discusses the synergistic effects of Thidiazuron (TDZ) and Naphthaleneacetic acid (NAA) in the Murashige and Skoog (MS) medium as well as the use of meta-topolin (mT). This synthetic cytokinin (mT) facilitates a high induction frequency and many shoots per explant. It introduces a time-efficient and resource-optimized pathway for Cannabis micropropagation and germplasm conservation. The genetic transformation in Cannabis was predominantly facilitated through Agrobacterium mediated transformation, a cornerstone technique that enabled the integration of foreign genes into the plant genome. Regulatory implications associated with gene editing in Cannabis sativa are highlighted. Despite these advancements, the field grapples with several challenges, including the recalcitrant nature of Cannabis, especially regarding in vitro propagation or genetic transformation, the genotypic specificity of regeneration protocols, and the reproducibility of existing methods. The complexity of the Cannabis genome, characterized by a high degree of polymorphism and multiple copies of specific genes, further exacerbates these challenges. Moreover, the current research landscape is marred by a lack of standardized protocols and variable responses among different Cannabis varieties, necessitating more robust and universally applicable protocols. This review underscores the pressing need for further research to optimize protocols for higher efficiency and to develop suitable systems for in-vitro plantlet regeneration. Cannabis sativa, Genetic transformation, Hemp, Regeneration, Tissue culture

Synthesis of Quantum-Confined Borophene Nanoribbons.

Borophene nanoribbons (BNRs) are one-dimensional strips of atomically thin boron expected to exhibit quantum-confined electronic properties that are not present in extended two-dimensional borophene. While the parent material borophene has been experimentally shown to possess anisotropic metallicity and diverse polymorphic structures, the atomically precise synthesis of nanometer-wide BNRs has not yet been achieved. Here, we demonstrate the synthesis of multiple BNR polymorphs with well-defined edge configurations within the nanometer-scale terraces of vicinal Ag(977). Through atomic-scale imaging, spectroscopy, and first-principles calculations, the synthesized BNR polymorphs are characterized and found to possess distinct edge structures and electronic properties. For single-phase BNRs, v1/6-BNRs and v1/5-BNRs adopt reconstructed armchair edges and sawtooth edges, respectively. In addition, the electronic properties of single-phase v1/6-BNRs and v1/5-BNRs are dominated by Friedel oscillations and striped moiré patterns, respectively. On the other hand, mixed-phase BNRs possess quantum-confined states with increasing nodes in the electronic density of states at elevated biases. Overall, the high degree of polymorphism and diverse edge topologies in borophene nanoribbons provide a rich quantum platform for studying one-dimensional electronic states.

Evaluation of the phenotypic polymorphism of the pattern of Leptinotarsa decemlineata Say pronotum in the conditions of Kamianka-Buzka city (Lviv Oblast)

Aim. The Colorado potato beetle population is characterized by a high degree of polymorphism. This provides a high level of adaptability to the most diverse distribution conditions. The aim is to the study of the variability of the pattern of Leptinotarsa decemlineata pronotum in June 2021 and June 2022. Methods. The analysis of the phenotypic variability of L. decemlineata population of Kamianka-Buzka city based on studying the presence or absence of certain phens, their number and placement according to Tauer method. Results. 22 variations of phens belonging to A, B, C, D, E, G, K, L, M, P groups were found. The dominant were phens of A, B, C, D, E, F groups. The most variable were the phens of A and E groups. The phenotypic structure of the colorado beetle population was determined according to Kohmaniuk’s classification. It was found that the dominant morphs in 2021 and 2022 were U (42 % and 27 %), UP (13 % and 18 %) and V (23 % and 21 %), respectively. Conclusions. The analysis of the frequency of phenoforms according to the Fasulati method showed that in the population of 2021 and 2022, phenoforms 3, 6 and 9 were dominant. They form the basis of the population and ensure the resistance of the population to the insecticides. The study of the intrapopulation diversity indicates an unbalanced population structure of L. decemlineata.

Comparative Phylogeography, Historical Demography, and Population Genetics of Three Common Coastal Fauna in Spartina Marshes of the Northwestern Gulf of Mexico

Coastal wetlands worldwide are experiencing high rates of loss and degradation that may lead to a reduction in diversity in faunal populations. Since salt marsh habitats are subject to a multitude of stressors, evaluations of the genetic diversity, connectivity, and potential resilience of faunal communities within salt marsh habitats are relevant. This study characterizes mitochondrial DNA (mtDNA) diversity for three common faunal residents of salt marshes along the northern Gulf of Mexico. Gulf Killifish (Fundulus grandis) samples were characterized for 1077 bp of the concatenated nucleotide sequence corresponding to the Control Region and Nitrogen Dehydrogenase, Subunits 2 and 5. Daggerblade grass shrimp (Palaemon pugio) samples were characterized using 466 bp of 16sRNA sequence, and phloem-feeding planthoppers (Prokelisia marginata) were characterized using 372 bp of Cytochrome c Oxidase Subunit I (COI) sequence. For F. grandis, our data revealed high levels of haplotypic diversity, evidence of isolation by distance (IBD), and regional population structuring associated with the distribution of two distinct phylogroups and distinct historical demography signatures. P. pugio and P. marginata displayed low levels of haplotypic diversity and evidence of population structure, but both appear to contain only snapshots of the total potential diversity for these species in the Gulf of Mexico. Greater resolution of the patterns of historical demography of Gulf Killifish may be obtained in future studies by including localities from Florida and Mexico. For both P. pugio and planthoppers, future studies would benefit from the characterization of genetic markers with a higher degree of polymorphism. We conclude that despite these three species inhabiting the same habitats along the same stretch of coast, each is subject to a different combination of evolutionary forces, and this study was able to reconstruct differences in how the genetic variation in each of these species emerged, and how it is maintained.

THE FIRST RECORD OF CEPAEA NEMORALIS (LINNAEUS, 1758) (MOLLUSCA: GASTROPODA: HELICIDAE) IN THE DONBASS AND THE PHENETIC STRUCTURE OF THE FOUND COLONY

The phenetic composition of Cepaea nemoralis (Linnaeus, 1758) (Mollusca: Gastropoda: Helicidae) colony found in an area of continuous high-rise buildings in the center of Donetsk in 2022 is described. The area of the colony is 2.5 ha, the number reaches 15 ind./m2, the age is at least 10 years. Individuals with a pink ground colours (81.5%) and one central band in 64.7% of yellow-coloured and 76.8% of pink-coloured shells are clearly dominated. The absence of unbanded shells and shells with reduced 1, 2, 4 and 5 bands was a distinctive feature of the Donetsk colony. The trend of dominance of phenotypes with fused bands is traced for five-banded shells with the relative frequency of 69.7%. Morphs with fusion types of bands, namely F(45) - 13.2% and F(23) - 12.8% dominate. The colony is characterized by a relatively high degree of polymorphism (𝜇 = 4.78±0.039) and proportion of rare morphs (h±𝑆ℎ = 0.47±0.026) showing no similarities with the colonies in Eastern Europe available for meta-analysis. Cepaea nemoralis is likely to remain one of the elements in the urban fauna, not showing any tendency to expand into natural ecosystems.

Influence of allelic polymorphism in the 3’ untranslated region of the <i>StTCP23</i> gene on the tolerance of potato cultivars to the potato spindle tuber viroid

Background. It is known that the pathological phenotype of potato plants can be mediated by complementary interactions between the genomic RNA of PSTVd and mRNA of some regulatory genes, which consequently lead to RNA interference, the synthesis of small interfering RNAs (vd-sRNA PSTVd), and impaired morphogenesis. At the same time, symptoms caused by the viroid may vary in different potato cultivars. Here we predict the interactions between the 3’ UTRs of various alleles of the StTCP23 transcription factor gene and the complementary regions in PSTVd genomic RNA.Materials and methods. We selected eight commercial potato cultivars with different symptoms of viroid infection and disease. For each cultivar, six clones of each cDNA amplicon of StTCP23 with a 3’ UTR were identified, and the allelic compositions of the target regions within their 3’ UTRs were characterized.Results. In total, 11 types of alleles of the 3’ UTR StTCP23 segment complementary to the vd-sRNA PSTVd were identified. Cultivars with the A allele (‘Gala’, ‘Colomba’, ‘Favorit’, and ‘Fioletovy’) identical to the reference genome or a high dose of the C allele with a deletion of four nucleotides (cv. ‘Impala’) were characterized by high susceptibility already at the primary (firstyear) infection with the PSTVd. Cvs. ‘Krepysh’, ‘Labadia’ and ‘Riviera’, classified as tolerant during primary inoculation, on the contrary, were characterized by the absence of the A allele and the presence of cultivar-specific mutant alleles.Conclusion. A high degree of polymorphism in the target site (3’ UTR region) of StTCP23 indicates a possible selection pressure on this locus. It can be assumed that cultivars with shorter alleles, which have fewer bases complementary to vd-sRNA in hypothetical duplexes and therefore less likely to induce target gene silencing, are more tolerant to the PSTVd upon primary viroid infection.

Somatic Embryogenesis Induction and Genetic Stability Assessment of Plants Regenerated from Immature Seeds of Akebia trifoliate (Thunb.) Koidz

Akebia trifoliata is a perennial woody plant with considerable potential in nutrition, food, and health, and the production of seedlings with high quality is critical for its economic utilization. Plant regeneration through somatic embryogenesis is a powerful alternative for propagating many plant species. In this study, a simple and practicable protocol was developed for plant regeneration from immature seeds of A. trifoliata via somatic embryogenesis, and the genetic stability of regenerated plants was also assessed. In the somatic embryo (SE) induction stage, the highest frequency of somatic embryogenesis (35.2%) was observed on the WPM medium containing 1.0 mg L−1 of thidiazuron (TDZ) and 1.0 mg L−1 of 6-benzyladenine (6-BA). The concentration of 6-BA was optimized at 1.0 mg L−1 for the proliferation and maturation of the induced SEs, and the combination of 2.0 mg L−1 of indole-3-butyric acid (IBA) and 0.5 mg L−1 of TDZ was the most responsive for root development and plant growth. The leaf morphological characteristics greatly varied among the established plants, and they could be grouped into three plant types, namely the normal type, Type Ι, and Type ΙΙ. Remarkable differences in the number, size, shape, and color of the leaflets were observed among the three types, while their ploidy level was the same via flow cytometry analysis. The Type ΙΙ and the Type Ι plants had the highest and the lowest net photosynthesis rate, transpiration rate, and stomatal conductance among the three groups, respectively, and both had a smaller size of stomatal guard cells than the normal type. Simple sequence repeat (SSR) analysis detected that 41 bands (43.62%) were different from those observed in the wild, indicating a high degree of polymorphism between the regenerants and their donor parent. The obtained plants might hold potential for future genetic improvement and breeding in A. trifoliata, and the established regeneration protocol might serve as a foundation for in vitro propagation and germplasm preservation of this crop.

An alternative key for identifying three prostrate species of the genus Euphorbia

Plants are a valuable source of different folk and modern medicines. Therefore, correctly identifying the plant species is a prerequisite for using them in the health care system. Euphorbia species are of great medicinal importance but are difficult to distinguish due to the occurrence of ecotypes and a high degree of polymorphism within the species. Therefore, the present study was planned to explore the extent of variations in morphological characters among three Euphorbia species, namely, Euphorbia prostrata Aiton, Euphorbia serpens Kunth and Euphorbia thymifolia L. The edaphic ecotypes red and green forms of Euphorbia prostrata growing separately or adjacent to each other were reported during field surveys. To ensure identification accuracy, illustrated online key was prepared as a supportive tool for the traditional key.

ՀԱՅԱՍՏԱՆՈՒՄ ՏԱՐԱԾՎԱԾ ՃԱԿՆԴԵՂԻ ԳԵՆԵՏԻԿԱԿԱՆ ԲԱԶՄԱԶԱՆՈՒԹՅԱՆ ՈՒՍՈՒՄՆԱՍԻՐՈՒԹՅՈՒՆԸ ISSR ՄԱՐԿԵՐՆԵՐԻ ԿԻՐԱՌՄԱՄԲ

Genetic markers for the study of genetic diversity are divided into 4 groups: morphological or phenotypic, biochemical or protein, cytogenetic, and molecular or DNA. Currently, ISSR-DNA markers or Inter Simple Sequence Repeats is widely used for the genetic characterization of various crop varieties and wild species. In the present day, ISSR-DNA markers (Inter Simple Sequence Repeats) are widely used to characterize various crop varieties and wild species from their genetic makeup. It is used both for inter-species and intra-species genetic variability of populations and for genetic diversity, and species identification, as well as for genome mapping and marking of useful economic traits. The purpose of this research was to certify the wild species and population varieties of beet common in Armenia using DNA markers, to offer them as a selection starting material. The studied wild species and population varieties of beet are distinguished by a high degree of polymorphism: 33.3-88.9 %. According to the indicators of genetic diversity, most of the studied species and varieties are characterized as the main or typical gene pools with a minimum number and frequency of rare alleles. An exception is the Aparan population of table beet, which is distinguished by a high degree of originality. According to the indicators studied with ISSR DNA markers, it is possible to select the right parental forms for beet breeding activities.

Characterization of twenty-two fig cultivars (Ficus carica L.) collected in north-eastern Algeria

There is a significant lack of information and research about Algerian figs, whether for varietal characterization, preservation or enhancement of this genetic resource. The fig tree (Ficus carica L.) is one of the oldest cultivated fruits in the world. Algeria is among the first Mediterranean countries producing figs. The evaluation of the ITAFV collection will allow the establishment of a database for the identification of ecotypes of interest for the direct development of the species and its future use in variety breeding programs. Morphological characterization and pomological evaluation were studied on the basis of the methodology established by IPGRI and CIHEAM, 2003 during three seasons (2012, 2013 and 2014). The phenotypic variability of the twenty-two cultivars shows the richness of the fig heritage of the ITAFV collection, Skikda (Algeria). The hierarchical classification based on the morphological descriptors of the leaf and the fruit has clearly revealed the importance of these parameters in the discrimination of fig varieties. The morpho-pomological characters used for the leaves and fruits indicated a fairly significant genetic variability between the cultivars studied with a relatively high degree of polymorphism.

The first evidence of Heosminthus from North America and the phylogenetics of Sminthidae (Mammalia, Rodentia, Dipodoidea): biogeographical implications

The Eurasian fossil record is rich with the remains of sminthid rodents, a clade today represented by a single genus, Sicista. In contrast, the North American record of the family is sparse and exclusively composed of species of Plesiosminthus, an animal bearing a grooved incisor, during the Oligocene. This fossil record is the product of dispersal events from Eurasia. We here present the first occurrence of a smooth-incisor sminthid from North America. The new species, Heosminthus teredens from the Arikareean-aged Cabbage Patch beds of Montana, represents a rare occurrence of cranial material showing the clear association between incisors and cheek teeth, and the first postcranial remains for fossil sminthids. Our phylogenetic analysis, expanding on prior work, shows that Heosminthus teredens is most closely related to Heosminthus borrae, from Mongolia. In combination with published radioisotopic dating, our phylogenetic framework of sminthids provides evidence for an additional dispersal of sminthid rodents from Eurasia to North America 30 million years ago via Beringia. Our morphometric analysis of the tibia and metatarsal supports a terrestrial ecology for the new species; it is possible the animal had limited scansorial and/or saltatorial abilities. The large sample size of teeth available for the new taxon enables an analysis of morphological variation and indicates a high degree of polymorphism as well as a large size variation, even when controlling for stratigraphical and geographical range. https://zoobank.org/urn:lsid:zoobank.org:pub:B7D4245C-992F-4956-96AD-10D7CFA8E5C8

Genetic polymorphism and forensic application of 23 autosomal STR loci in the Han population of Panjin City, Liaoning Province, Northeastern China

Background Short tandem repeats (STRs) are consecutive repetition of a repeat motif and widely used in forensic medicine and human genetics because of their high polymorphism. Subjects and methods In the current study, 23 autosomal STR loci were genotyped from 1263 unrelated healthy individuals living in Panjin City, Liaoning Province, Northeastern China using the VeriFilerTM Express PCR Amplification Kit. The population comparison was performed between the Panjin Han population and the other relevant groups to further explore the structure of Panjin Han and its relationship with the other groups. Results The results found 316 alleles across the 23 STRs and the corresponding allelic frequencies ranged from 0.5198 to 0.0004. Except for D3S1358, TPOX, TH01, and D3S1358, all STR loci were highly polymorphic (PIC > 0.7), with the Penta E locus having the highest degree of polymorphism (0.9147). For population comparison, the exact test of population differentiation found that no significant difference was observed between the Panjin Han and the other Han populations, except for Guangdong Han and Jiangxi Han. Conclusion The Panjin Han population showed significant differences with the other ethnic groups in China (Bouyei, Dong, Hui, Miao, Tibetan, and Uygur) and the foreign ethnic groups.

The Total Synthesis of Glycolipids from Streptococcus pneumoniae and a Re-evaluation of Their Immunological Activity.

Invariant natural killer (iNK) T cells, Type I iNKTs, are responsible for the production of pro-inflammatory cytokines which induce a systemic immune response. They are distinctive in possessing an semi-invariant T-cell receptor that recognizes glycolipid antigens presented by CD1d, a protein closely related to the class I major histocompatibility complex, conserved across multiple mammalian species in a class of proteins well-renowned for their high degree of polymorphism. This receptor's first potent identified antigen is the α-galactosylceramide, KRN7000, a synthetic glycosphingolipid closely related to those isolated from bacteria that were found on a Japanese marine sponge. A corresponding terrestrial antigen remained unidentified until two specific diacylglycerol-containing glycolipids, reported to activate iNKT cells, were isolated from Streptococcus pneumoniae. We report the total synthesis and immunological re-evaluation of these two glycolipids. The compounds are unable to meaningfully activate iNKT cells. Computational modelling shows that these ligands, while being capable of interacting with the CD1d receptor, create a different surface for the binary complex that makes formation of the ternary complex with the iNKT T-cell receptor difficult. Together these results suggest that the reported activity might have been due to an impurity in the original isolated sample and highlights the importance of taking care when reporting biological activity from isolated natural products.

Abstract 1220: Accurately genotyping HLA and KIR alleles using cfDNA assay and k-mer based algorithm for immunotherapy

Abstract Background: HLA and KIR genotypes show great promise as emerging biomarkers for immune checkpoint inhibitors (ICIs) and understanding patient prognosis. Multiple studies have shown that HLA-I heterozygosity and high sequence divergence across alleles positively correlates with response to ICIs. However the high degree of polymorphism and allele sequence similarities in HLA and KIR present a challenge to accurate allele calling. To address these difficulties we developed kmerizer, a novel allele caller optimized for short fragments, such as reads from a cfDNA assay. Methods: We tested kmerizer on MHC class1 and class2 genes, and on KIR genes, on both simulated datasets and real samples (cell lines and plasma samples). To assess the capability of the algorithm to distinguish highly homologous allele pairs, we simulated cfDNA-like fragments with errors on randomly selected allele pairs, and on allele pairs with a high level of homology. Twelve plasma samples and 19 reference cell lines fragmented to cfDNA size were analyzed using a NGS cfDNA assay. For plasma samples, paired buffy coats were sent to an external vendor for HLA typing using multiplex PCR-based amplicon and sequenced by 300bp paired-end reads. Results: Of the 19 cell lines and 12 plasma samples for HLA typing (A, B, C, and DQB1 loci), kmerizer delivered 100% sensitivity with 98% specificity. On the simulated dataset, kmerizer achieved 99% sensitivity and specificity on all the MHC class1 and class 2 loci, and 90% sensitivity and specificity on all KIR loci, for both homozygous and heterozygous pairs. The novel allele caller kmerizer also demonstrated a lighter footprint on computational resource need: one deep-sequencing plasma sample on average can be processed in less than 2 minutes which is about 15 times faster than the most commonly used HLA typing tool HISAT21, which does not support KIR typing. Conclusions: As utilization of ICIs increases, the use of genetic and genomic information to accurately identify patients more likely to respond to ICIs will be critical. kmerizer is a fast and highly sensitive and specific allele caller, and it can effectively call alleles on both HLA and KIR.