Extraction Protocol Research Articles

Preservation of scRNA-Seq Libraries Using Existing Inactivation Protocols.

Single-cell RNA sequencing has soared in popularity in recent years. The ability to deeply profile the states of individual cells during the course of disease or infection has helped to expand our knowledge of coordinated responses. However, significant challenges arise when performing this analysis in high containment settings such as biosafety level 3 (BSL-3), BSL-3+ and BSL-4. Working in containment is necessary for many important pathogens, such as Ebola virus, Marburg virus, Lassa virus, Nipah and Hendra viruses. Since standard operating procedures (SOPs) for inactivation are extensive and may compromise sample integrity, we tested whether the removal of single-cell sequencing libraries from containment laboratories using existing inactivation protocols for nucleic acid extraction (Trizol, RLT buffer, or AVL buffer) was feasible. We have demonstrated that the inactivation does not affect sample quality and can work with existing methods for inactivation.

Biochemical, Molecular Characterization, Antioxidant, And Cytotoxicity Of Dicliptera Bupleuroides With Potential Drug Discovery: In Vitro And In Silico Analysis

AbstractDicliptera is a genus belonging to family Acanthaceae. Dicliptera bupleuroides is a small perennial herb commonly known as roxburg's foldwing. It is locally used in folkloric medicines for the treatment of different diseases and as a general tonic for wound healing. The current investigational study was to optimize solvent extraction protocol, to identify quantitative and qualitative biochemical composition, antioxidant and cytotoxic activities of D. bupleuroides and to characterize the gene associated with biosynthesis of flavonol. The plant of D. bupleuroides was collected from district Abbottabad and separated into its four parts including leaf, stem, flower, and root. To extract the biochemical compounds, five different solvents were used such as methanol, acidified methanol, 80 % methanol, ethyl acetate, and n‐butanol. Total phenolic, flavonoids, flavonol, flavanol (catechin), and anthocyanin contents were determined and antioxidant potential of the extracts was evaluated using various antioxidant assays. The methanolic extracts of D. bupleuroides showed promising antioxidant potential towards DPPH, ABTS, H2O2, ferric, phosphomolybdate and hydroxyl radical scavenging capabilities.The extracts obtained from the solvents mentioned above were tested for cytotoxicity using the 3T3 cell line by using mouse fibroblast cells. The results indicated that none of the extracts demonstrated any toxic effects on the cells, suggesting that they are non‐toxic. The RNA was extracted and cDNA was synthesized using reverse transcriptase enzyme. Degenerate primers for dihydroflavonol reductase (DFR) gene were synthesized to amplify the desired product. The high quality of cDNA amplification showed the presence of the DFR gene in the Dicliptera bupleuroides leaf for the first time with a product size of 419 bp respectively. The DFR gene isolated from Dicliptera bupleuroides is involved in flavonol biosynthesis regulation. In addition, Glide molecular docking results proposed that the constituents within the n‐hexanic extract of Dicliptera bupleuroides not only furnish an updated approach to better understand the antioxidant potential of its various constituents to interact with an antioxidant target protein (PDB ID:3MNG) but also ensure promise in addressing these potential therapeutic modalities. The active compounds of D. bupleuroides could be suitable for pharmaceutical industries to develop stress related drugs.

Enhanced molecular release from elderly bone samples using collagenase I: insights into fatty acid metabolism alterations

BackgroundBone is a metabolically active tissue containing different cell types acting as endocrine targets and effectors. Further, bone is a dynamic depot for calcium, phosphorous and other essential minerals. The tissue matrix is subjected to a constant turnover in response to mechanical/endocrine stimuli. Bone turnover demands high energy levels, making fatty acids a crucial source for the bone cells. However, the current understanding of bone cell metabolism is poor. This is partly due to bone matrix complexity and difficulty in small molecules extraction from bone samples. This study aimed to evaluate the effect of metabolite sequestering from a protein-dominated matrix to increase the quality and amount of metabolomics data in discovering small molecule patterns in pathological conditions.MethodsHuman bone samples were collected from 65 to 85 years old (the elderly age span) patients who underwent hip replacement surgery. Separated cortical and trabecular bone powders were treated with decalcifying, enzymatic (collagenase I and proteinase K) and solvent-based metabolite extraction protocols. The extracted mixtures were analyzed with the high-resolution mass spectrometry (HRMS). Data analysis was performed with XCMS and MetaboAnalystR packages.ResultsFast enzymatic treatment of bone samples before solvent addition led to a significantly higher yield of metabolite extraction. Collagenase I and proteinase K rapid digestion showed more effectiveness in cortical and trabecular bone samples, with a significantly higher rate (2.2 folds) for collagenase I. Further analysis showed significant enrichment in pathways like de novo fatty acid biosynthesis, glycosphingolipid metabolism and fatty acid oxidation-peroxisome.ConclusionThis work presents a novel approach for bone sample preparation for HRMS metabolomics. The disruption of bone matrix conformation at the molecular level helps the molecular release into the extracting solvent and, therefore, can lead to higher quality results and trustable biomarker discovery. Our results showed β-oxidation alteration in the aged bone sample. Future work covering more patients is worthy to identify the effective therapeutics to achieve healthy aging.

Extraction and selection of high-molecular-weight DNA for long-read sequencing from Chlamydomonas reinhardtii.

Recent advances in long-read sequencing technologies have enabled the complete assembly of eukaryotic genomes from telomere to telomere by allowing repeated regions to be fully sequenced and assembled, thus filling the gaps left by previous short-read sequencing methods. Furthermore, long-read sequencing can also help characterizing structural variants, with applications in the fields of genome evolution or cancer genomics. For many organisms, the main bottleneck to sequence long reads remains the lack of robust methods to obtain high-molecular-weight (HMW) DNA. For this purpose, we developed an optimized protocol to extract DNA suitable for long-read sequencing from the unicellular green alga Chlamydomonas reinhardtii, based on CTAB/phenol extraction followed by a size selection step for long DNA molecules. We provide validation results for the extraction protocol, as well as statistics obtained with Oxford Nanopore Technologies sequencing.

Comparing the Extraction Performance in Mouse Plasma of Different Biphasic Methods for Polar and Nonpolar Compounds.

Many metabolomic studies are interested in both polar and nonpolar analyses. However, the available sample volume often precludes multiple separate extractions. Therefore, there are major advantages in performing a biphasic extraction and retaining both phases for subsequent separate analyses. To be successful, such approaches require the method to be robust and repeatable for both phases. Hence, we determined the performance of three extraction protocols, plus two variant versions, using 25 μL of commercially available mouse plasma. The preferred option for nonpolar lipids was a modified diluted version of a method employing methyl tert-butyl ether (MTBE) suggested by Matyash and colleagues due to its high repeatability for nonpolar compounds. For polar compounds, the Bligh-Dyer method performs best for sensitivity but with consequentially poorer lipid performance. Overall, the scaled-down version of the MTBE method gave the best overall performance, with high sensitivity for both polar and nonpolar compounds and good repeatability for polar compounds in particular.

Extraction and Profiling of Antifungal Compounds Produced by Azotobacter Species

Food loss and wastage occur in large quantities globally every year and it occurs in the entire supply chain right from the production to the processing stage. The loss of food is due to various factors like adopting traditional cultivation practices, low investment in the food sector, and more loss from poor pests and disease management of agricultural crops. The most important and major cause is due to microbial spoilage; fungi are most harmful to the consumers and also to the agriculture sector. Synthetic chemical strategies can prevent fungal growth and may reduce wastage but still causes accumulation of chemical substances in the environment and food chain in a long run. For these reasons, the use of bio-control technologies can be a great solution to agriculture and food sector as well. In view of this, the present study has been conducted using an efficient Azotobacter species, which belongs to the PGPR group. In this study, antifungal compounds produced by Azotobacter have been extracted by following solvent extraction protocols and identified using GCMS methods. The antifungal compounds were tested against the major fungal pathogens viz., Aspergillus, Fusarium, and Penicillium species. The metabolites produced by Azotobacter species were efficient in controlling the growth of the fungal species. These compounds can be used as a potential bio-preservative in the food sector instead of synthetic chemicals. Thus, these compounds can further be analyzed and tested on the food sample, having a great scope in the future to replace the chemical preservatives.

Improving the efficiency of DNA extraction from iron incrustations and oilfield-produced water

The quantity and quality of DNA isolated from environmental samples are crucial for getting robust high-throughput sequencing data commonly used for microbial community analysis. The differences in the nature and physicochemical properties of environmental samples impact DNA yields, and therefore, an optimisation of the protocols is always recommended. For instance, samples collected from corroded areas contain high concentrations of metals, salts, and hydrocarbons that can interfere with several steps of the DNA extraction protocols, thereby reducing yield and quality. In this study, we compared the efficiency of commercially available DNA extraction kits and laboratory-adopted methods for microbial community analysis of iron incrustations and oilfield-produced water samples. Modifications to the kits manufacturers’ protocols were included to maximise the yield and quality. For iron incrustations, the modified protocol for FastDNA Spin Kit for Soil yielded higher DNA and resulted in higher diversity, including the recovery of low-abundant and rare taxa in the samples, compared to DNeasy PowerSoil Pro Kit. The DNA extracted with modified phenol–chloroform methods yielded higher DNA but failed to pass quality control PCR for 16S sequencing with and without purification. The protocols mentioned here can be used to maximise DNA recovery from iron incrustations and oilfield-produced water samples.

LC-HRMS-Driven Computational Toolbox to Assess Extraction Protocols Dedicated to Untargeted Analysis: How to Ease Analyzing Pesticide-Contaminated Soils?

Metabolomics is a powerful approach that allows for high throughput analysis and the acquisition of large biochemical data. Nonetheless, it still faces several challenging requirements, such as the development of optimal extraction and analytical methods able to respond to its high qualitative and quantitative requisites. Hence, the objective of the present article is to suggest a LC-HRMS-based untargeted profiling approach aiming to provide performant tools that help assess the performance and the quality of extraction methods. It is applied in a herbicide-contaminated soil metabolomics context. The trifactorial experimental design consists of 150 samples issued from five different extraction protocols, two types of soils, and three contamination conditions (contaminated soils with two different formulated herbicides against uncontaminated soils). Four performance and quality criteria are investigated using adapted LC-HRMS-driven computational tools. First, 861 metabolic features are annotated, and then the width of metabolome coverage and quantitative performance of the five different extraction protocols are assessed in all samples using various optimized configurations of heatmaps as well as van Krevelen diagrams. Then, the reproducibility of LC-HRMS profiles issued from the five extractions is studied by two different approaches: Euclidean distances and relative standard deviations. The two methods are examined and compared. Their advantages and limitations are thus discussed. After, the capacity of the different extractions to discriminate between contaminated and uncontaminated soils will be evaluated using orthogonal projections to latent structures-discriminant analysis. Different data scaling parameters are tested, and the results are explored and discussed. All of the suggested computational and visualization tools are performed using public-access platforms or open-source software. They can be readapted by metabolomics developers and users according to their study contexts and fields of application.

Chestnut Episperm as a Promising Natural Source of Phenolics from Agri-Food Processing by-Products: Optimisation of a Sustainable Extraction Protocol by Ultrasounds

Chestnut processing has increasingly grown in recent years. All the processes involved in the chestnut supply chain are characterized by the production of high levels of by-products that cause several environmental and disposal issues. The Castanea spp. fruit production is related to a high number of chestnut episperm. This underutilized agricultural by-product may be evaluated as a good resource for the extraction of health-promoting natural molecules, such as phenolics. This preliminary study aimed to develop and optimize, using a multivariate statistical approach, a sustainable protocol for the ultrasound-assisted extraction (UAE) of the main phenolics from chestnut episperm (cv Marsol, C. sativa × C. crenata). A design of experiment (DoE) approach was employed. This approach focused on the two quantitative UAE process factors: the extraction time (X1), within a timeframe ranging from 10 to 30 min, and the sample-to-solvent (w/v) ratio (X2), ranging from 1/30 to 1/10. These variables were investigated to determine their impact on phenol extraction yield. Exploratory analysis, in particular principal component analysis (PCA) and multiple linear regression (MLR), were carried out on the studied responses. The phenolic characterization of ten different extracts was also performed using high-performance liquid chromatography (HPLC), both to define the levels of specific phenolics selected for their health-promoting properties and to evaluate some important features, such as the total antioxidant capacity. The values of total polyphenolic content (TPC) obtained in the different experiments ranged between 97 (extract 4) and 142 (extract 6) mg GAE/g of dried weight (DW). Moreover, results from the ferric reducing antioxidant power (FRAP) test confirmed the high TPC values, highlighting that all the ultrasound extracts contained excellent levels of molecules with good antioxidant properties. In particular, extracts 2 and 3 showed the highest AOC values (about 490–505 mmol Fe2+/Kg of dried weight). The proposed optimized protocol allowed for obtaining formulations characterized by high levels of tannins, phenolic acids, and catechins. Indeed, episperm extracts contained high levels of chlorogenic acid (15–25 mg/100 g DW), ferulic acid (80–120 mg/100 g DW), castalagin (20–80 mg/100 g DW), and vescalagin (40–75 mg/100 g). Finally, in this research study, the potential of chestnut episperm as a source of polyphenolic molecules to be extracted by green technologies and used in several food and/or pharmaceutical applications was evaluated to valorize a sustainable reuse strategy of agri-food processing by-products, also reducing the environmental impact of this waste derived from chestnut processing.

Detection of Chikungunya Virus RNA in Oral Fluid and Urine: An Alternative Approach to Diagnosis?

To evaluate whether oral fluids (OF) and urine can serve as alternative, non-invasive samples to diagnose chikungunya virus (CHIKV) infection via RT-qPCR, we employed the same RNA extraction and RT-qPCR protocols on paired serum, OF and urine samples collected from 51 patients with chikungunya during the acute phase of the illness. Chikungunya patients were confirmed through RT-qPCR in acute-phase sera (N = 19), IgM seroconversion between acute- and convalescent-phase sera (N = 12), or IgM detection in acute-phase sera (N = 20). The controls included paired serum, OF and urine samples from patients with non-arbovirus acute febrile illness (N = 28) and RT-PCR-confirmed dengue (N = 16). Nine (47%) of the patients with positive RT-qPCR for CHIKV in sera and two (17%) of those with CHIKV infection confirmed solely via IgM seroconversion had OF positive for CHIKV in RT-qPCR. One (5%) patient with CHIKV infection confirmed via serum RT-qPCR was positive in the RT-qPCR performed on urine. None of the negative control group samples were positive. Although OF may serve as an alternative sample for diagnosing acute chikungunya in specific settings, a negative result cannot rule out an infection. Further research is needed to investigate whether OF and urine collected later in the disease course when serum becomes RT-qPCR-negative may be helpful in CHIKV diagnosis and surveillance, as well as to determine whether urine and OF pose any risk of CHIKV transmission.

A cost-effective RNA extraction and RT-qPCR approach to detect California serogroup viruses from pooled mosquito samples

Mosquito-borne diseases pose ongoing global health concerns, demanding more cost-efficient methods to detect pathogens to support enhanced surveillance efforts. This study introduces an adapted TRIzol-based high-throughput RNA extraction protocol, tailored for the detection of California serogroup viruses in pooled mosquito samples in a rapid and cost-effective manner. This approach provided consistent RNA yields and sensitive viral detection relative to two commercial extraction kits (QIAGEN RNeasy Mini Kit and MACHEREY–NAGEL NucleoSpin RNA Plus Kit). The incorporation of a user-friendly and non-spiking-based RT-qPCR internal control designed for the 18S rRNA gene in mosquitoes minimizes potential false positives/negatives, improving the fidelity of viral detection outcomes. Effective RNA yields, purity, and successful target amplification across 25 mosquito species and varied pool sizes (1–50 mosquitoes per tube) affirm the reliability of our approach. The extraction method is cost-effective, with an incurred cost of $0.58 CAD per sample, in contrast to the $5.25 CAD cost per sample of the two kits, rendering it promising for mosquito-borne disease surveillance initiatives.

3D-printed extraction devices fabricated from silica particles suspended in acrylate resin

In recent years, there has been an increasing worldwide interest in the use of alternative sample preparation methods. Digital light processing (DLP) is a 3D printing technique based on using UV light to form photo-curable resin layer upon layer, which results in a printed shape. This study explores the application of this technique for the development of novel drug extraction devices in analytical chemistry. A composite material consisting of a photocurable resin and C18-modified silica particles was employed as a sorbent device, demonstrating its effectiveness in pharmaceutical analysis. Apart from estimating optimal printing parameters, microscopic examination of the material surface, and sorbent powder to resin ratio, the extraction procedure was also optimised. Optimisation included the type and amount of sample matrix additives, desorption solvent, sorption and desorption times, and proper number of sorbent devices needed in extraction protocol. To demonstrate this method's applicability for sample analysis, the solid-phase extraction followed by gas chromatography coupled with mass spectrometry (SPE-GC–MS) method was validated for its ability to quantify benzodiazepine-type drugs. This evaluation confirmed good linearity in the concentration range of 50–1000 ng/mL, with R2 values being 0.9932 and 0.9952 for medazepam and diazepam, respectively. Validation parameters proved that the presented method is precise (with values ranging in-between 2.98 %-7.40 %), and accurate (88.81 % to 110.80 %). A negative control was also performed to investigate possible sorption properties of the resin itself, proving that the addition of C18-modified silica particles significantly increases the extraction efficiency and repeatability.The cost-effectiveness of this approach makes it particularly advantageous for single-use scenarios, eliminating the need for time-consuming sorbent-cleaning procedures, common in traditional solid-phase extraction techniques. Future optimisation opportunities include refining sorbent size, shape, and geometry to achieve lower limits of quantification. As a result of these findings, 3D-printed extraction devices can serve as a viable alternative to commercially available SPE or solid-phase microextraction (SPME) protocols for studying new sample preparation approaches.

Evaluation of SP3 for antibody-free quantification of tau in CSF mimic and brain by mass spectrometry.

Tubulin-associated unit (tau) has an important role in the pathogenesis and the diagnosis of Alzheimer's disease (AD) and other tauopathies. In view of the diversity of tau proteoforms, antibody-free methods represent a good approach for unbiased quantification. We adapted and evaluated the single-pot, solid-phase-enhanced sample-preparation (SP3) protocol for antibody-free extraction of the tau protein in cerebro-spinal fluid (CSF) mimic and in human brain. A total of 13 non-modified peptides were quantified by high-resolution mass spectrometry (HRMS) after digestion of tau by trypsin. We significantly improved the basic SP3 protocol by carefully optimizing the organic solvents and incubation time for tau binding, as well as the digestion step for the release directly from the SP3 beads of the 13 tau peptides. These optimizations proved to be primarily beneficial for the most hydrophilic tau peptides, increasing the sequence coverage of recombinant tau. Mean recovery in CSF mimic of the 13 non-modified peptides was of 53%, with LODs ranging from 0.75 to 10 ng/mL. Next, we tested the optimized SP3 protocol on pathological tau extracted from the soluble fraction from an AD brain sample (middle frontal gyrus). We could successfully identify and quantify biologically relevant tau peptides including representative peptides of two isoforms and two phospho-peptides (pTau217 and pTau181).

Comprehensive multiresidue chromatographic methods for monitoring pesticides in agricultural areas and corresponding plant protection zones

This article reports a comprehensive analytical method for the identification and quantification of a broad range of pesticides in green plant crops. The sample preparation method for pesticides involved an optimization of the QuEChERS-based extraction protocol, with sample mass, volume of added water, and the type of cleanup sorbent as variables. A sorbent combination based on ENVI-Carb and ChloroFiltr was examined. A highly efficient method was developed for the purification of plant extracts with 900 mg MgSO4, 150 mg PSA, and 15 mg ENVI-Carb at the d-SPE stage, combined with gas chromatography and liquid tandem mass spectrometry for the determination of 197 pesticides in crop plants containing chlorophyll. The method was validated in accordance with the requirements of international guidelines SANTE/11312/2021. The method was applied to quantify pesticide residues in 29 pairs of green crop plants and plants from the corresponding crop protection zone to verify whether the zones are effective barriers to prevent pesticides from penetrating outside agricultural areas. The number and types of agrochemical preparations were chosen by farmers. In total, more than 60 one- and several-component pesticide formulations were applied to the crops included in the study. The pesticide residues were detected in 21 crop samples and 3 samples from protection zones. Epoxiconazole, an active substance that was banned for use in 2021, was found in a spring barley sample. Based on the conducted research, the effectiveness of the protection zones has been clearly demonstrated, and it has been proven that environmental migration of pesticides and unauthorized agricultural practices pose a risk to ecosystems.

Ultrasound assisted extraction of amino acids and nucleobases from clay minerals and astrobiological samples

The study of organic molecules in meteorite and return samples allows for the understanding of the chemistry that undergoes in our Solar System. The present work aims at studying ultrasound assisted extraction technique as effective extraction method for these molecules in extraterrestrial samples and analogs. Optimal conditions were selected from the investigation of ultrasonic frequency, irradiation duration and solvent effects on amino acids, nucleobases and dipeptides extraction yields from a model clay-rich mineral matrix. Optimal ultrasound-assisted extraction parameters were frequency of 20 kHz within 20 min irradiation time and methanol/water solvent ratio of 1. We then validated this protocol on Mukundpura and Tarda meteorite fragments and compared it to the reference extraction protocol used in astrobiology and based on 24 h extraction time at 100 °C in water We obtained similar quantitative results without any racemization with both methodologies.

An innovative optimized protocol for high-quality genomic DNA extraction from recalcitrant Shea tree (Vitellaria paradoxa, C.F. Gaertn) plant and its suitability for downstream applications.

It is not always easy to find a universal protocol for the extraction of genomic DNA (gDNA) from plants. Extraction of gDNA from plants such as shea with a lot of polysaccharides in their leaves is done in two steps: a first step to remove the polysaccharides and a second step for the extraction of the gDNA. In this work, we designed a protocol for extracting high-quality gDNA from shea tree and demonstrate its suitability for downstream molecular applications. Fifty milligrams of leaf and root tissues were used to test the efficiency of our protocol. The quantity of gDNA was measured with the NanoDrop spectrometer and the quality was checked on agarose gel. Its suitability for use in downstream applications was tested with restriction enzymes, SSRs and RAPD polymerase chain reactions and Sanger sequencing. The average yield of gDNA was 5.17; 3.96; 2.71 and 2.41µg for dry leaves, dry roots, fresh leaves and fresh roots respectively per 100mg of tissue. Variance analysis of the yield showed significant difference between all tissue types. Leaf gDNA quality was better compared to root gDNA at the absorbance ratio A260/280 and A260/230. The minimum amplifiable concentration of leaf gDNA was 1pg/µl while root gDNA remained amplifiable at 10pg/µl. Genomic DNA obtained was also suitable for sequencing. This protocol provides an efficient, convenient and cost effective DNA extraction method suitable for use in various vitellaria paradoxa genomic studies.

Unambiguous Analysis and Systematic Mapping of Oxygenated Aromatic Compounds in Atmospheric Aerosols Using Ultrahigh-Resolution Mass Spectrometry.

Compositional analysis of organic aerosols (OAs) at the molecular level has been a long-standing challenge in field and laboratory studies. In this work, we applied different extraction protocols to aerosol samples collected from the ambient atmosphere and biomass burning sources, followed by Orbitrap mass spectrometric analysis with a soft electrospray ionization source operating in both positive and negative ionization modes. To systematically map the distribution of mono- and dioxygenated aromatic compounds (referred to as aromatic CHO1 and CHO2 formulas) in OA, we developed a unique two-dimensional Kendrick mass defect (2D KMD) framework. Our analysis unveiled a total of (76, 64, 70) aromatic CHO1 formulas and (103, 110, 106) CHO2 formulas, corresponding to samples obtained from ambient air, rice straw burning, and sugarcane leaf burning, respectively. These results reveal a significant number of additional distinct formulas exclusively present in ambient samples, suggesting a significant chemical transformation of OAs in the atmosphere. The analytical approach can be further extended to incorporate multiple layers of 2D KMD, enabling systematic mapping of the unexplored chemical space for complex environmental samples.

FTA-LAMP based biosensor for a rapid in-field detection of Globodera pallida-the pale potato cyst nematode.

The combination of a sensitive and specific magnetoresistive sensing device with an easy DNA extraction method and a rapid isothermal amplification is presented here targeting the on-site detection of Globodera pallida, a potato endoparasitic nematode. FTA-cards were used for DNA extraction, LAMP was the method developed for DNA amplification and a nanoparticle functionalized magnetic-biosensor was used for the detection. The combinatorial effect of these three emerging technologies has the capacity to detect G. pallida with a detection limit of one juvenile, even when mixed with other related species. This combined system is far more interesting than what a single technology can provide. Magnetic biosensors can be combined with any DNA extraction protocol and LAMP forming a new solution to target G. pallida. The probe designed in this study consistently distinguished G. pallida (∆Vac binding/Vac sensor above 1%) from other cyst nematodes (∆Vac binding/Vac sensor below 1%). It was confirmed that DNA either extracted with FTA-cards or Lab extraction Kit was of enough quantity and quality to detect G. pallida whenever present (alone or in mixed samples), ensuring probe specificity and sensitivity. This work provides insights for a new strategy to construct advanced devices for pathogens in-field diagnostics. LAMP runs separately but can be easily integrated into a single device.

Wide-spread dispersal in a deep-sea brooding polychaete: the role of natural history collections in assessing the distribution in quill worms (Onuphidae, Annelida)

BackgroundModern integrative taxonomy-based annelid species descriptions are detailed combining morphological data and, since the last decades, also molecular information. Historic species descriptions are often comparatively brief lacking such detail. Adoptions of species names from western literature in the past led to the assumption of cosmopolitan ranges for many species, which, in many cases, were later found to include cryptic or pseudocryptic lineages with subtle morphological differences. Natural history collections and databases can aid in assessing the geographic ranges of species but depend on correct species identification. Obtaining DNA sequence information from wet-collection museum specimens of marine annelids is often impeded by the use of formaldehyde and/or long-term storage in ethanol resulting in DNA degradation and cross-linking.ResultsThe application of ancient DNA extraction methodology in combination with single-stranded DNA library preparation and target gene capture resulted in successful sequencing of a 110-year-old collection specimen of quill worms. Furthermore, a 40-year-old specimen of quill worms was successfully sequenced using a standard extraction protocol for modern samples, PCR and Sanger sequencing. Our study presents the first molecular analysis of Hyalinoecia species including the previously known species Hyalinoecia robusta, H. tubicloa, H. artifex, and H. longibranchiata, and a potentially undescribed species from equatorial western Africa morphologically indistinguishable from H. tubicola. The study also investigates the distribution of these five Hyalinoecia species. Reassessing the distribution of H. robusta reveals a geographical range covering both the Atlantic and the Indian Oceans as indicated by molecular data obtained from recent and historical specimens.ConclusionOur results represent an example of a very wide geographical distribution of a brooding deep-sea annelid with a complex reproduction strategy and seemingly very limited dispersal capacity of its offspring, and highlights the importance of molecular information from museum specimens for integrative annelid taxonomy and biogeography.

Abstract A068: Proteomic analysis of keratin 17 positive cells from laser-capture microdissected pancreatic ductal adenocarcinoma

Abstract Introduction. Although patients with pancreatic ductal adenocarcinoma (PDAC) have one of the lowest survival of all carcinomas, there is considerable variation in survival and disease progression, even when accounting for factors such as age and pathologic stage. Our group identified keratin 17 (K17) as a negative prognostic and predictive biomarker in PDAC. Patients with high K17 expression in their tumors have shorter overall survival as well as decreased response to chemotherapeutic agents, including gemcitabine. K17 has also emerged as an established biomarker of the basal molecular subtype of PDAC. In this study, we aim to identify proteins which correlate, directly or indirectly, with K17 expression via a combined approach using immunohistochemistry (IHC) followed by laser capture microdissection (LCM) and mass spectrometry. Methods. We selected 5 PDAC cases of low K17 expression and 5 PDAC cases of high K17 expression from the archival collections from the Stony Brook Cancer Center Biobank. A modified indirect immunoperoxidase approach was performed on sections mounted on LCM-compatible membrane slides. Carcinoma cells that were either K17 positive or negative by IHC were precisely dissected using LMD7 microscope (Leica) from 10-µM thick sections, followed by a modified tissue digestion and protein extraction protocol. Collision-induced fragment mass information and peptide abundance values were obtained by a liquid chromatography-electrospray ionization tandem mass spectrometry (LC-MS/MS). Spectral cutoffs were analyzed at both <5% and 1% false discovery rates. The human UniProt dataset was used for data alignment. Results. Mass spectrometry yielded over 1600 detected proteins with approximately 50 of which correlated strongly with K17 expression. Our preliminary results show that keratin 5, a known marker of basal-like PDAC, is positively correlated with keratin 17. Genes whose increased expression contribute to the Moffitt classification of basal molecular subtype of PDAC, such as S100A2, keratin 6A and keratin 7, also showed positive correlation with K17 in our study. The remainder of proteins associated with K17 expression include those involved with cell adhesion, cell-extracellular matrix interaction and smooth muscle contraction pathways, with specific types and classes being identified, pending in-depth analysis. Proteins with inverse relationship to K17 expression included those involved in intracellular organelle trafficking and apoptosis pathways. Conclusion. Our findings demonstrate the utility of mass spectrometry-based spatial proteomic analysis of microdissected cell populations from formalin-fixed, paraffin embedded samples. These methods have the potential to uncover complementary biomarkers to K17 that could enhance prognostic accuracy or serve as therapeutic targets and provide mechanistic insight into the mechanisms through which K17 drives tumor aggression. Citation Format: Ji Dong K. Bai, John D. Haley, Natalia Marchenko, Luisa F. Escobar-Hoyos, Kenneth R. Shroyer. Proteomic analysis of keratin 17 positive cells from laser-capture microdissected pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A068.