Bio-mimicking DNA fingerprint profiling for HLS watermarking to counter hardware IP piracy.

The multifaceted, multivendor-based global design supply chain induces hardware threats of intellectual property (IP) piracy for modern computing and electronic systems. Current hardware watermarking techniques fall short either in terms of watermark strength (size of covert constraints generated) or number of security layers/variables involved in the security constraints generation process. This paper presents a novel approach for high level synthesis (HLS) watermarking by bio-mimicking DNA fingerprint profiling to counter hardware IP piracy. The proposed approach effectively captures the vital DNA fingerprint profiling phases such as DNA sequencing, DNA fragmentation, fragment replication, DNA ligase, etc. and bio-mimics them to generate a digital watermarking framework. The presented approach has been demonstrated on convolutional layer and JPEG compression-decompression (CODEC) algorithms that are widely used in several medical and machine learning applications. The proposed approach has been thoroughly compared with several state-of-the-art approaches. The proposed approach depicts superior security in the probability of coincidence of up to ~ 104 and tamper tolerance of up to ~ 10368 at 0% overhead as compared to the prior approaches.

Establishment and application of a SNP molecular identification system in Grifola frondosa.

Germplasm resources of edible mushrooms are essential for the breeding of varieties with improved traits. Analysis of the genetic diversity of Grifola frondosa germplasm resources and clarification of the genetic relationships among strains can provide valuable information for the selection of breeding parents. A total of 829,488 high-quality SNP loci were screened from 2,125,382 SNPs obtained by sequencing 60 G. frondose. Phylogenetic analysis, PCA, and population structure analysis based on the high-quality SNPs showed that the 60 strains could be divided into five subgroups, and the clustering results were consistent with the geographical distributions of these strains. Based on high-quality SNP loci, a core collection containing 18 representative germplasm resources was constructed, and 1,473 Kompetitive Allele-Specific PCR markers were obtained. A total of 722 SNP markers in the exonic regions were screened using KASP-genotyping experiments, and 50 candidate SNP markers and 12 core SNP markers were obtained. Genetic fingerprints of G. frondosa germplasm resources were constructed based on the selected SNP markers; these fingerprints provide an accurate, rapid, convenient, and efficient method for the identification of G. frondosa germplasm resources. The results of this study have important implications for the preservation and utilization of G. frondosa germplasm resources and the identification of varieties.

Antibiotyping, RAPD- and ERIC-PCR fingerprinting of Klebsiella pneumoniae clinical isolates at a tertiary reference hospital in Denpasar, Bali, Indonesia.

Background and Objectives: Klebsiella pneumoniae is a healthcare-associated infections agent and could be an extended spectrum ß-lactamase (ESBL) producer. Understanding the transmission of this bacterium in a hospital setting needs accurate typing methods. An antibiogram is used to detect the resistance pattern of the isolates. Random Amplified Polymorphic DNA (RAPD) and Enterobacterial Repetitive Intergenic Consensus (ERIC)-PCR are rapid, technically simple, and easy-to-interpret DNA typing methods. This study aimed to evaluate the use of antibiotyping, RAPD-, and ERIC-PCR to investigate the heterogeneity of K. pneumoniae isolated from clinical specimens. Materials and Methods: The antibiograms of 46 K. pneumoniae clinical isolates were determined by Vitek® 2 Compact. All isolates underwent RAPD-PCR using AP4 primer and ERIC-PCR using ERIC-2 primer. The dendrogram was generated using the GelJ software and analyzed to determine its similarity. The analysis of antibiogram and the molecular typing diversity index was calculated using the formula of the Simpson's diversity index. Results: About 71.7% of the isolates were ESBL-producers, and more than 80% of isolates were susceptible to amikacin, ertapenem, and meropenem. The antibiotyping produced 32 diverse types with DI = 0.964. In addition, the RAPD-PCR produced 47 different types with DI = 1, while ERIC-PCR was 46 (DI=0.999). Conclusion: Antibiotyping, RAPD- and ERIC-PCR showed powerful discrimination power among the isolates, supported the diversity of K. pneumoniae isolates in current study. These combination could be promising tools for clonal relationship determination, including in tracking the transmission of the outbreak's agent in hospital setting.

Exploring agro-morphological and fiber traits diversity in cotton (G. barbadense L.).

Cotton (Gossypium barbadense L.) is a leading fiber and oilseed crop globally, but genetic diversity among breeding materials is often limited. This study analyzed genetic variability in 14 cotton genotypes from Egypt and other countries, including both cultivated varieties and wild types, using agro-morphological traits and genomic SSR markers. Field experiments were conducted over two seasons to evaluate 12 key traits related to plant growth, yield components, and fiber quality. Molecular diversity analysis utilized 10 SSR primers to generate DNA profiles. The Molecular diversity analysis utilized 10 SSR primers to generate DNA profiles. Data showed wide variation for the morphological traits, with Egyptian genotypes generally exhibiting higher means for vegetative growth and yield parameters. The top-performing genotypes for yield were Giza 96, Giza 94, and Big Black Boll genotypes, while Giza 96, Giza 92, and Giza 70 ranked highest for fiber length, strength, and fineness. In contrast, molecular profiles were highly polymorphic across all genotypes, including 82.5% polymorphic bands out of 212. Polymorphism information content was high for the SSR markers, ranging from 0.76 to 0.86. Genetic similarity coefficients based on the SSR data varied extensively from 0.58 to 0.91, and cluster analysis separated genotypes into two major groups according to geographical origin. The cotton genotypes displayed high diversity in morphology and genetics, indicating sufficient variability in the germplasm. The combined use of physical traits and molecular markers gave a thorough understanding of the genetic diversity and relationships between Egyptian and global cotton varieties. The SSR markers effectively profiled the genotypes and can help select ideal parents for enhancing cotton through hybridization and marker-assisted breeding.

Development of eighteen novel microsatellite loci for Masked Bobwhite, Colinus virginianus ridgwayi.

BACKGROUND: Masked Bobwhite (Colinus virginianus ridgwayi) is a critically-endangered New World quail species endemic to Sonoran Desert grasslands of North America. It suffered severe population declines during the nineteenth and twentieth centuries, with its persistence now reliant upon a captive breeding program that requires careful genetic management to maintain extant genetic diversity. Although nuclear microsatellite DNA markers existed for the closely related Northern Bobwhite (C. virginianus), none were available for Masked Bobwhite to inform necessary management decisions. METHODS AND RESULTS: Paired-end Illumina© sequencing was conducted to screen the Masked Bobwhite genome for microsatellite loci. We identified 18 loci exhibiting high polymorphism and limited deviations from genetic equilibrium expectations. These loci were amplified in 78 individuals. Familial relationships were reconstructed via sibship methods and compared to manually-curated pedigree data. Thirteen of fifteen full-sibling groups in the pedigree were exactly reconstructed (86.6%). Three other full-sibling groups partially matched pedigree relationships with high statistical confidence, and likely represented pedigree inaccuracies. Four additional full-sibling pairs were identified with low statistical confidence and likely resulted from analytical artifacts. CONCLUSIONS: The novel microsatellite loci accurately reconstructed parent-offspring and sibling relationships. These loci will be useful for guiding genetic management decisions and identifying pedigree inaccuracies in the captive breeding program.

Assessment of co-contaminated soil amended by graphene oxide: Effects on pollutants, microbial communities and soil health.

Graphene oxide (GOx) is a nanomaterial with demonstrated capacity to remove metals from water. However, its effects on organic pollutants and metal(loid)s present in polluted soils when used for remediation purposes have not been extensively addressed. Likewise, few studies describe the effects of GOx on edaphic properties and soil biology. In this context, here we assessed the potential of GOx for remediating polluted soil focusing also on different unexplored effects of GOx in soil. To achieve this, we treated soil contaminated with concurrent inorganic (As and metals) and organic pollution (TPH and PAHs), using GOx alone and in combination with nutrients (N and P sources). In both cases increased availability of As and Zn was observed after 90 days, whereas Cu and Hg availability was reduced and the availability of Pb and the concentration of organic pollutants were not significantly affected. The application of GOx on the soil induced a significant and rapid change (within 1 week) in microbial populations, leading to a transient reduction in biodiversity, consistent with the alteration of several soil properties. Concurrently, the combination with nutrients exhibited a distinct behaviour, manifesting a more pronounced and persistent shift in microbial populations without a decrease in biodiversity. On the basis of these findings, GOx emerges as a versatile amendment for soil remediation approaches.

Effect of Carcinomas on Autosomal Trait Screening: A Review Article.

This review highlights the effect of carcinomas on the results of the examination of autosomal genetic traits for identification and paternity tests when carcinoid tissue is the only source and no other samples are available. In DNA typing or genetic fingerprinting, variable elements are isolated and identified within the base pair sequences that form the DNA. The person's probable identity can be determined by analysing nucleotide sequences in particular regions of DNA unique to everyone. Genetics plays an increasingly important role in the risk stratification and management of carcinoma patients. The available information from previous studies has indicated that in some incidents, including mass disasters and crimes such as terrorist incidents, biological evidence may not be available at the scene of the accident, except for some unknown human remains found in the form of undefined human tissues. If these tissues have cancerous tumours, it may affect the examination of the genetic traits derived from these samples, thereby resulting in a failure to identify the person. Pathology units, more often, verify the identity of the patients who were diagnosed with cancer in reference to their deceased tumorous relatives. Genetic fingerprinting (GF) is also used in paternity testing when the alleged parent disappeared or died and earlier was diagnosed and treated for cancer.

Comparative LC-MS/MS-based molecular networking, DNA fingerprinting, and in vitro anti-Helicobacter pylori activity of three Egyptian Ficus cultivars.

Ficus species (Moraceae) have been used for nutrition and traditional medicine, and plants from this family are phytochemically abundant and serve as a potential source of natural products. As a result of the inherent complexity of the plant metabolomes and the fact that these Ficus species chemical space has not yet been fully decoded, it is still difficult to characterize their phytochemistry. Therefore, this study, we suggest the use of the molecular networking to elucidate the chemical classes existing in leaves of three Ficus species (F. deltoidei Jack, F. drupacea Thunb and F. sycomorus L.) and highlight the importance of molecular networking in examining their chemotaxonomy . By using computational tools, 90 metabolites were annotated , including phenolic acids, flavonoids, furanocoumarins, fatty acids and terpenoids. Phenolic acids were detected as the main class present in the three studied species. Flavonoids-C-glycosides, flavonoids-O-glycosides and isoflavonoids were mainly present in F. drupacea and F. sycomorus, while furanocoumarins were proposed in F. sycomorus. Vomifoliol-based sesquiterpenes were proposed in F. deltoidei. The chemotaxonomic differentiation agreed with the DNA fingerprinting using SCOT and ISSR markers. F. deltoidei, in particular, had a divergent chemical fingerprint as well as a different genotype. Chemotype differentiation using chemical fingerprints, in conjunction with the proposed genetic markers, creates an effective identification tool for the quality control of the raw materials and products derived from those three Ficus species. As well, F. drupacea exploited the most potent inhibition of H. pylori with MIC of 7.81 µg/ mL compared with clarithromycin. Overall, molecular networking provides a promising approach for the exploration of the chemical space of plant metabolomes and the elucidation of chemotaxonomy.

Identification of grapevine clones via high-throughput amplicon sequencing: a proof-of-concept study.

Wine cultivars are available to growers in multiple clonal selections with agronomic and enological differences. Phenotypic differences between clones originated from somatic mutations that accrued over thousands of asexual propagation cycles. Genetic diversity between grape cultivars remains unexplored, and tools to discriminate unequivocally clones have been lacking. This study aimed to uncover genetic variations among a group of clonal selections of 4 important Vitis vinifera cultivars: Cabernet sauvignon, Sauvignon blanc, Chardonnay, and Merlot, and use this information to develop genetic markers to discriminate the clones of these cultivars. We sequenced with short-read sequencing technology the genomes of 18 clones, including biological replicates for a total of 46 genomes. Sequences were aligned to their respective cultivar's reference genome for variant calling. We used reference genomes of Cabernet sauvignon, Chardonnay, and Merlot and developed a de novo genome assembly of Sauvignon blanc using long-read sequencing. On average, 4 million variants were detected for each clone, with 74.2% being single nucleotide variants and 25.8% being small insertions or deletions (InDel). The frequency of these variants was consistent across all clones. From these variants, we validated 46 clonal markers using high-throughput amplicon sequencing for 77.7% of the evaluated clones, most of them small InDel. These results represent an advance in grapevine genotyping strategies and will benefit the viticulture industry for the characterization and identification of the plant material.

Geolocation prediction from STR genotyping: a pilot study in five geographically distinct global populations.

BACKGROUND: Traditional CE-based STR profiles are highly useful for the purpose of individualisation. However, they do not give any additional information without the presence of the reference sample for comparison. AIM: To assess the usability of STR-based genotypes for the prediction of an individual's geolocation. SUBJECTS AND METHODS: Genotype data from five geographically distinct populations, i.e. Caucasian, Hispanic, Asian, Estonian, and Bahrainian, were collected from the published literature. RESULTS: A significant difference (p < 0.05) in the observed genotypes was found between these populations. D1S1656 and SE33 showed substantial differences in their genotype frequencies across the tested populations. SE33, D12S391, D21S11, D19S433, D18S51, and D1S1656 were found to have the highest occurrence of "unique genotype's" in different populations. In addition, D12S391 and D13S317 exhibited distinct population-specific "most frequent genotypes." CONCLUSIONS: Three different prediction models have been proposed for genotype to geolocation prediction, i.e. (i) use of unique genotypes of a population, (ii) use of the most frequent genotype, and (iii) a combinatorial approach of unique and most frequent genotypes. These models could aid the investigating agencies in cases where no reference sample is available for comparison of the profile.

ISSR markers endorsed genetically stable plants regeneration of neem (Azadirachta indica A. Juss.) through indirect organogenesis using different explants.

BACKGROUND: Various parts of neem (Azadirachta indica) have high demand in several industries. However, the inadequate supply of sources hampers the commercialization of different neem products. In this scenario, the current research was undertaken to produce genetically stable plants through indirect organogenesis. METHODS AND RESULTS: Several explants like shoot tips, internodal segments, and leaves, were cultivated on MS media with different growth regulators. Maximum callus formation was achieved using 1.5 mg/L NAA, 0.5 mg/L 2,4-D and 0.2 mg/L both for Kn and BAP in combination with shoot tip (93.67%). These calli showed an organogenic potentiality on MS medium having coconut water (15%) without growth regulators. This medium along with 0.5 mg/L Kn and 0.1 mg/L both for BAP and NAA yielded the maximum adventitious shoot production with shoot tip-derived callus (95.24%). These calli further produced the most buds per shoot (6.38) and highest average shoot length (5.46 cm) with 0.5 mg/L both for BAP and Kn and 0.1 mg/L NAA in combination after the fifth subculture. The 1/3 strength of MS media was found to be best along with 0.5 mg/L IBA and 0.1 mg/L Kn in combination to generate maximum root response (92.86%), roots per shoot (5.86) and longest average root length (3.84 cm). The mean plant survival after initial hardening was 83.33% which increased to 89.47% after secondary hardening. The lack of variation in ISSR markers among the regenerated trees is evidence of clonal fidelity between hardened plants. CONCLUSIONS: This protocol will accelerate the propagation of neem for utilization of its sources.

DT-PICS: An Efficient and Cost-Effective SNP Selection Method for the Germplasm Identification of Arabidopsis.

Germplasm identification is essential for plant breeding and conservation. In this study, we developed a new method, DT-PICS, for efficient and cost-effective SNP selection in germplasm identification. The method, based on the decision tree concept, could efficiently select the most informative SNPs for germplasm identification by recursively partitioning the dataset based on their overall high PIC values, instead of considering individual SNP features. This method reduces redundancy in SNP selection and enhances the efficiency and automation of the selection process. DT-PICS demonstrated significant advantages in both the training and testing datasets and exhibited good performance on independent prediction, which validates its effectiveness. Thirteen simplified SNP sets were extracted from 749,636 SNPs in 1135 Arabidopsis varieties resequencing datasets, including a total of 769 DT-PICS SNPs, with an average of 59 SNPs per set. Each simplified SNP set could distinguish between the 1135 Arabidopsis varieties. Simulations demonstrated that using a combination of two simplified SNP sets for identification can effectively increase the fault tolerance in independent validation. In the testing dataset, two potentially mislabeled varieties (ICE169 and Star-8) were identified. For 68 same-named varieties, the identification process achieved 94.97% accuracy and only 30 shared markers on average; for 12 different-named varieties, the germplasm to be tested could be effectively distinguished from 1,134 other varieties while grouping extremely similar varieties (Col-0) together, reflecting their actual genetic relatedness. The results suggest that the DT-PICS provides an efficient and accurate approach to SNP selection in germplasm identification and management, offering strong support for future plant breeding and conservation efforts.

Novel strategy of multiple-locus variable number tandem repeats analysis for genetic fingerprinting of human.

BACKGROUND: The variable number of tandem repeat (VNTR) analyses are methods based on the detection of repeated sequences within the human genome. In order to perform DNA typing at the personal laboratory, it is necessary to improve the VNTR analysis. OBJECTIVE: The VNTR markers were difficult to popularize because PCR amplification was difficult due to its GC-rich and long nucleotide sequence. The aim of this study was to select the multiple VNTR markers that could only be identified by PCR amplification and electrophoresis. METHODS: We genotyped each of the 15 VNTR markers using genomic DNA from 260 unrelated individuals by PCR amplification. Differences in the fragment length of PCR products are visualized by agarose gel electrophoresis. To confirm their usefulness as a DNA fingerprint these 15 markers were simultaneously analyzed with the DNA of 213 individuals and verified the statistical significance. In addition, to investigate the usefulness of each of the 15 VNTR markers as paternity markers, Mendelian segregation by meiotic division within a family consisting of two or three generations was confirmed. RESULTS: Fifteen VNTR loci selected in this study could be easily amplified by PCR and analyzed by electrophoresis, and were newly named DTM1 ~ 15. The number of total alleles in each VNTR showed from 4 to 16, and 100 to 1600 bp in length, and their heterozygosity ranged from 0.2341 to 0.7915. In simultaneous analysis of 15 markers from 213 DNAs, the probability of chance appearing the same genotype in different individuals was less than 4.09E-12, indicating its usefulness as a DNA fingerprint. These loci were transmitted through meiosis by Mendelian inheritance in families. CONCLUSION: Fifteen VNTR markers have been found to be useful as DNA fingerprints for personal identification and kinship analysis that can be used at the personal laboratory level.

DNA marker identification of downy mildew resistance locus Rpv10 in grapevine genotypes.

One of the most common and harmful diseases of grapevine is downy mildew, caused by Plasmopara viticola. Cultivars of Vitis vinifera, the basis of high-quality viticulture, are mainly not resistant to downy mildew. Varieties with natural resistance to downy mildew belong to the vine species of North America and Asia (V. aestivalis, V. berlandieri, V. cinerea, V. labrusca, V. amurensis, etc.), as well as Muscadinia rotundifolia. The breeding of resistant cultivars is based on interspecific crossing. Currently, molecular genetic methods are increasingly used in pre-selection work and directly in breeding. One of the major loci of downy mildew resistance, Rpv10, was first identified in the variety Solaris and was originally inherited from wild V. amurensis. DNA markers that allow detecting Rpv10 in grapevine genotypes are known. We used PCR analysis to search for donors of resistance locus among 30 grape cultivars that, according to their pedigrees, could carry Rpv10. The work was performed using an automatic genetic analyzer, which allows obtaining high-precision data. Rpv10 locus allele, which determines resistance to the downy mildew pathogen, has been detected in 10 genotypes. Fingerprinting of grape cultivars with detected Rpv10 was performed at 6 reference SSR loci. DNA marker analysis revealed the presence of a resistance allele in the cultivar Korinka russkaya, which, according to publicly available data, is the offspring of the cultivar Zarya Severa and cannot carry Rpv10. Using the microsatellite loci polymorphism analysis and the data from VIVC database, it was found that Korinka russkaya is the progeny of the cultivar Severnyi, which is the donor of the resistance locus Rpv10. The pedigree of the grapevine cultivar Korinka russkaya was also clarified.

Genetic Diversity Analysis of Banana Cultivars (Musa sp.) in Saudi Arabia Based on AFLP Marker.

Banana plantation has been introduced recently to a temperate zone in the southeastern parts of Saudi Arabia (Fifa, Dhamadh, and Beesh, located in Jazan province). The introduced banana cultivars were of a clear origin without a recorded genetic background. In the current study, the genetic variability and structure of five common banana cultivars (i.e., Red, America, Indian, French, and Baladi) were analyzed using the fluorescently labeled AFLP technique. Nine different primer pairs combinations yielded 1468 loci with 88.96% polymorphism. Among all locations, high expected heterozygosity under the Hardy-Weinberg assumption was found (0.249 ± 0.003), where Dhamadh was the highest, followed by Fifa and Beesh, respectively. Based on the PCoA and Structure analysis, the samples were not clustered by location but in pairs in accordance with the cultivar's names. However, the Red banana cultivar was found to be a hybrid between the American and Indian cultivars. Based on ΦST, 162 molecular markers (i.e., loci under selection) were detected among cultivars. Identifying those loci using NGS techniques can reveal the genetic bases and molecular mechanisms involved in the domestication and selection indicators among banana cultivars.

Molecular Fingerprinting and Phytochemical Investigation of Syzygium cumini L. from Different Agro-Ecological Zones of India.

Syzygium cumini L. (ver Jamun; BlackBerry) is a native, evergreen multipurpose tree species of India. Besides being a fruit tree and for agroforestry in different regions, it is medicinally important too. This study aimed to determine genetic diversity using molecular and phytochemical markers in sixteen genotypes of Indian S. cumini from different agro-ecological zones. The present study used a combination of ISSR markers and the HPLC technique to explore these genotypes. The results showed a wide genetic diversity range based on the similarity coefficient values observed in S. cumini sixteen accessions from different sites. Four primary phenolic acids were discovered in all the accessions; caffeic acid (CA) was found in high concentrations. The intraspecific association between molecular and phytochemical characteristics was the primary goal of this investigation. By employing gene-specific markers for the route of secondary metabolites (polyphenols) production, it further investigated the progressive research of diversity analysis of polyphenol content in S. cumini accessions, which may also expand its nutraceutical and pharmaceutical utilization.

Tubulin-Based Polymorphism (TBP) in Plant Genotyping.

Tubulin-based polymorphism (TBP) is an intron length polymorphism (ILP) method widely applicable to any plant species and particularly suitable for a first and rapid classification of any plant genome. It is based on the selective, polymerase chain reaction (PCR)-based amplification of the two introns present at conserved positions within the coding sequences of plant ß-tubulin genes. Amplification releases a simple yet distinctive genomic profile.

Genetic polymorphism detection in brazilian perennial cottons (Gossypium spp.) using an ISSR marker system and its application for molecular interspecific differentiation.

BACKGROUND: The semi-domesticated Brazilian perennial cotton (Gossypium spp.) germplasm is considered a source of variability for creating modern upland cotton varieties. Here we used Inter-simple Sequence Repeat (ISSR) markers to detect intra and interspecific genetic polymorphism in Gossypium hirsutum L. r. marie-galante and Gossypium barbadense L. and to use molecular data to assessing genetic diversity and molecular discrimination of these species. METHODS AND RESULTS: The sets contained 12 G. barbadense genotypes and 16 G. hirsutum genotypes from a Brazilian collection. The 11 ISSR primers were used for genotyping yielded 101 bands (polymorphism = 47.5%) and were classified as moderately informative (PIC = 0.304). The ISSR markers exposed a greater diversity in G. hirsutum (P = 24.72%; HE =0.071 and I = 0.111) as compared to G. barbadense (P = 17.98%, HE = 0.043 and I = 0.070). The AMOVA analysis showed that 89.47% of the genetic variation was partitioned within species which is supported by Nei's genetic differentiation (Gst = 0.598) and gene flow (Nm = 0.338), suggesting that strong reproductive barriers between species. The UPGMA Cluster Analysis, Principal Coordinate Analysis and Bayesian Model-Based Structural Analysis divided the 28 genotypes into two main clades consistent with the taxonomical delimitation. CONCLUSION: The ISSR marker system offers a new approach to determining molecular differences between two cotton species (G. hirsutum L. r. marie-galante and G. barbadense L.). This study can expand the molecular marker resources for the identification and improvement of our knowledge about the genetic diversity and relationships between perennial cotton genotypes.

Establishment of a diverse head and neck squamous cancer cell bank using conditional reprogramming culture methods.

Most laboratory models of head and neck squamous cell cancer (HNSCC) rely on established immortalized cell lines, which carry inherent bias due to selection and clonality. We established a robust panel of HNSCC tumor cultures using a "conditional reprogramming" (CR) method, which utilizes a rho kinase inhibitor (Y-27632) and co-culture with irradiated fibroblast (J2 strain) feeder cells to support indefinite tumor cell survival. Sixteen CR cultures were successfully generated from 19 consecutively enrolled ethnically and racially diverse patients with HNSCC at a tertiary care center in the Bronx, NY. Of the 16 CR cultures, 9/16 were derived from the oral cavity, 4/16 were derived from the oropharynx, and 3/16 were from laryngeal carcinomas. Short tandem repeat (STR) profiling was used to validate culture against patient tumor tissue DNA. All CR cultures expressed ΔNp63 and cytokeratin 5/6, which are markers of squamous identity. Human papillomavirus (HPV) testing was assessed utilizing clinical p16 staining on primary tumors, reverse transcription polymerase chain reaction (RT-PCR) of HPV16/18-specific viral oncogenes E6 and E7 in RNA extracted from tumor samples, and HPV DNA sequencing. Three of four oropharyngeal tumors were p16 and HPV-positive and maintained HPV in culture. CR cultures were able to establish three-dimensional spheroid and murine flank and orthotopic tongue models. CR methods can be readily applied to all HNSCC tumors regardless of patient characteristics, disease site, and molecular background, providing a translational research model that properly includes patient and tumor diversity.

Epithelioid Trophoblastic Tumor Presenting as an Adnexal Mass: Report of a Diagnostically Challenging Case.

Epithelioid trophoblastic tumor (ETT) is a rare neoplasm derived from chorionic intermediate trophoblast cells, representing less than 2% of all gestational trophoblastic neoplasms. Classically, ETT presents as a uterine mass in women of reproductive age following a term pregnancy. The time from pregnancy to tumor development varies from months to several years. ETT most often arises in the endometrium, followed by the cervix. Extrauterine ETT are extremely infrequent, with few cases reported in the literature. We report a case of a 41-year-old woman, with history of three term pregnancies who presented with abdominal pain and elevated beta human chorionic gonadotropin (ß-hCG) level, ten years after her last pregnancy. Imaging reported a 3.5 cm adnexal mass, suspicious for ectopic pregnancy. Hysterectomy and mass resection revealed a 4.7 cm, tan-yellow, necrotic mass adjacent to the broad ligament. Histologic evaluation in conjunction with immunohistochemical stains revealed a tumor consistent with ETT. No connection to the endometrium was found grossly or microscopically. DNA fingerprinting analysis revealed the tumor to have two copies of paternal alleles, as seen in molar gestations. One of the primary differential diagnoses for ETT is squamous cell carcinoma due to similar morphologic features. In challenging cases, genetic analysis demonstrating paternally derived genes can establish the diagnosis. In this report, we discuss the challenges in the diagnosis of extrauterine ETT, due to its rarity and highly variable presentation, given that appropriate diagnosis is critical for correct patient management.