Profiling Methodology Research Articles

Comparison of Lysis and Amplification Methodologies for Optimal 16S rRNA Gene Profiling for Human and Mouse Microbiome Studies

When conducting sequence-based analysis of microbiome samples, it is important to accurately represent the bacterial communities present. The aim of this study was to compare two commercially available DNA isolation and PCR amplification approaches to determine their impact on the taxonomic composition of microbiome samples following 16S rRNA gene sequencing. A well-established 16S rRNA gene profiling approach, which was widely used in the Human Microbiome Project (HMP), was compared with a novel alkaline degenerative technique that utilizes alkaline cell lysis in combination with a degenerate pool of primers for nucleic acid extraction and PCR amplification. When comparing these different approaches for the microbiome profiling of human and mouse fecal samples, we found that the alkaline-based method was able to detect greater taxonomic diversity. An in silico analysis of predicted primer binding against a curated 16S rRNA gene reference database further suggested that this novel approach had the potential to reduce population bias found with traditional methods, thereby offering opportunities for improved microbial community profiling.

Integrative transcriptome profiling for identifying ALS potential treatment using the drug repurposing approach

BackgroundAmyotrophic lateral sclerosis (ALS), an alarming neurodegenerative disorder, induces muscle atrophy and motor deterioration. The current treatments exhibit limited improvement in survival rates. Thus, we here attempted to identify crucial genetic biomarkers through transcriptome profiling and systems biology methodologies to advance our knowledge of the diagnosis and pathogenesis of ALS. Following this, a drug repurposing approach was employed to introduce possible treatments for ALS.ResultsAfter analyzing differentially expressed genes (DEG) using different in silico approaches, 43 DEGs (23 upregulated and 20 downregulated) were identified, which were abnormally expressed in ALS patients compared to healthy individuals. Two proteins CMPK2 and IFI44L were identified as ALS biomarkers and selected for molecular docking. Then, molecular docking was performed to repurpose drugs that have the potential to suppress upregulated proteins. Accordingly, three drugs including ketoprofen, thalitone, and cromolyn have been repurposed against CMPK2 and IFI44L proteins.ConclusionsCMPK2 and IFI44L serve as potential biomarkers for ALS and may be applied in the diagnostic assessment of this disorder within the bloodstream of affected individuals. Furthermore, three drugs were proposed as potential therapeutic candidates for ALS with the help of transcriptomics profiling. We advocate for the implementation of these identified pharmacotherapies in animal models of ALS to validate their therapeutic efficacy. This approach also would help narrow down the options to more suitable targets and economically viable treatments.

Clinical evaluation of long-read sequencing-based episignature detection in developmental disorders

BackgroundA subset of developmental disorders (DD) is characterized by disease-specific genome-wide methylation changes. These episignatures inform on the underlying pathogenic mechanisms and can be used to assess the pathogenicity of genomic variants as well as confirm clinical diagnoses. Currently, the detection of these episignature requires the use of indirect methylation profiling methodologies. We hypothesized that long-read whole genome sequencing would not only enable the detection of single nucleotide variants and structural variants but also episignatures.MethodsGenome-wide nanopore sequencing was performed in 40 controls and 20 patients with confirmed or suspected episignature-associated DD, representing 13 distinct diseases. Following genomic variant and methylome calling, hierarchical clustering and dimensional reduction were used to determine the compatibility with microarray-based episignatures. Subsequently, we developed a support vector machine (SVM) for the detection of each DD.ResultsNanopore sequencing-based methylome patterns were concordant with microarray-based episignatures. Our SVM-based classifier identified the episignatures in 17/19 patients with a (likely) pathogenic variant and none of the controls. The remaining patients in which no episignature was identified were also classified as controls by a commercial microarray assay. In addition, we identified all underlying pathogenic single nucleotide and structural variants and showed haplotype-aware skewed X-inactivation evaluation directs clinical interpretation.ConclusionThis proof-of-concept study demonstrates nanopore sequencing enables episignature detection. In addition, concurrent haplotyped genomic and epigenomic analyses leverage simultaneous detection of single nucleotide/structural variants, X-inactivation, and imprinting, consolidating a multi-step sequential process into a single diagnostic assay.

Methodologies for Mitochondrial Omic Profiling During Spaceflight.

To be able to understand how spaceflight can affect human biology, there is a need for maximizing the amount of information that can be obtained from experiments flown to space. Recently there has been an influx of data obtained from astronauts through multi-omics approaches based on both governmental and commercial spaceflight missions. In addition to data from humans, mitochondrial specific data is gathered for other experiments from rodents and other organisms that are flown in space. This data has started to universally demonstrate that mitochondrial dysfunction is the key regulator associated with increasing health risks associated with spaceflight. This mitochondrial dysfunction can have influence downstream on immune suppression, inflammation, circadian rhythm issues, and more. Due to the space environment, standard methodologies have to be altered for performing mitochondrial specific analysis and in general sample collection for omics. To perform mitochondrial specific analysis and data collection from samples flown to space we will outline the current sample collection methods, processing of the samples, and specific analysis. Specifically we will highlight the different mitochondrial methodologies and challenges involved with research associated with spaceflight.

Microarray-Based Methodology for Lipid Profiling, Enzymatic Activity, And Binding Assays in Printed Lipid Raft Membranes from Astrocytes and Neurons.

Lipid rafts are liquid-ordered domains in which specific enzymes and receptors are located. These membrane platforms play crucial roles in a variety of signaling pathways. Alterations in the lipid environment, such as those elicited by oxidative stress, can lead to important functional disruptions in membrane proteins. Cell membrane microarrays have emerged in the past decade as a powerful methodology for the study of both lipids and membrane proteins at large scales. Based on that technology and the importance of liquid-ordered subdomains, we have developed a new printed lipid raft technology with a preserved native protein structure and lipid environment. To validate this technology and evaluate its potential for different aims, raft membrane microarrays (RMMAs) containing two different cell types (astrocytes and neurons) and three different conditions (astrocytes in control situation, metabolic stress, and oxidative stress) were developed. To study differences in lipid profiles between raft domains, the MALDI-MS assay was performed on RMMAs. To evaluate the preservation of native protein activities (enzymatic activity and ligand binding) in the printed raft domains, differences in NADH oxidoreductase, GAPDH, cholinesterase activities, and sigma-1 and sigma-2 binding assays were performed. We demonstrate the performance of this new microarray technology, adapted to membrane subdomains, as valid to explore changes in lipid composition and protein activities in raft domains from brain cell lines under different stress conditions relevant for neuropathology.

Root microbiome of Panax ginseng in comparison with three other medicinal plants in the families of Araliaceae and Apiaceae

The intricate interplay between endophytic microorganisms and plants in the upkeep of biodiversity, the stability of communities, and the operation of ecosystems needs to be more adequately extensive. Although root-associated microbial communities of plants have been revealed for the last decade, the understanding of bacterial and fungal communities associated with the roots of medicinal plants remains elusive. To highlight the importance of Panax ginseng Meyer (PG) in our research, we investigated the root endophytic bacterial and fungal communities of Panax ginseng Meyer (PG), alongside Aralia cordata (AC), Angelica gigas (AG), and Peucedanum japonicum (PJ), utilizing amplicon-based community profiling and advanced bioinformatic methodologies. The study aimed to investigate the root-endophytic microbiota of ginseng and three other medicinal plants and identify similarities in microbiome composition across different plant species and families. Results revealed that root-endophytic bacterial and fungal communities were influenced by plant species and phylogenetic differences at the family level. Differential abundance tests and random forest models showed microbial features within the same plant family. PG had a distinctive microbial profile with significant B1653_o_Enterobacterales and F8_o_Helotiales. PG had a core microbiome, B10_Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium, and a more evenly distributed microbial network compared to AG, PJ, and AC. Our research reveals the intricate endophytic microbial communities within the roots of medicinal plants, pinpointing specific taxa that may be pivotal to their medicinal qualities and overall plant health. These insights carry notable implications for future studies, particularly those focused on the endophytes of PG and their secondary metabolites, as they deepen our understanding of plant-microbe interactions and their role in enhancing the plants’ therapeutic potential.

Distinct substrate specificities of the three catalytic subunits of the Trichomonas vaginalis proteasome.

The protozoan parasite Trichomonas vaginalis (Tv) causes trichomoniasis, the most common non-viral sexually transmitted infection in the world. Although Tv has been linked to significant health complications, only two closely related 5-nitroimidazole drugs are approved for its treatment. The emergence of resistance to these drugs and lack of alternative treatment options poses an increasing threat to public health, making development of novel anti-Trichomonas compounds an urgent need. The proteasome, a critical enzyme complex found in all eukaryotes has three catalytic subunits, β1, β2, and β5 and has been validated as a drug target to treat trichomoniasis. With the goal of developing tools to study the Tv proteasome, we isolated the enzyme complex and identified inhibitors that preferentially inactivate either one or two of the three catalytic subunits. Using a mass spectrometry-based peptide digestion assay, these inhibitors were used to define the substrate preferences of the β1, β2 and β5 subunits. Subsequently, three model fluorogenic substrates were designed, each specific for one of the catalytic subunits. This novel substrate profiling methodology will allow for individual subunit characterization of other proteasomes of interest. Using the new substrates, we screened a library of 284 peptide epoxyketone inhibitors against Tv and determined the subunits targeted by the most active compounds. The data show that inhibition of the Tv β5 subunit alone is toxic to the parasite. Taken together, the optimized proteasome subunit substrates will be instrumental for understanding the molecular determinants of proteasome specificity and for accelerating drug development against trichomoniasis.

Abstract A062: Insightful analysis: Harnessing aqueous humor ctDNA for retinoblastoma stratification

Abstract Retinoblastoma (RB) the most common intraocular malignancy in children poses a significant challenge as traditional tissue biopsies are not feasible. Due to its critical location within the eye and the high risk of tumor seeding, clinicians are limited in performing molecular analysis-based risk stratification prior to enucleation. In this study, we explored the potential of aqueous humor (AH) as a source for minimally invasive liquid biopsies, aiming to establish a molecular profiling methodology for circulating tumor DNA (ctDNA) for risk stratification. We analyzed AH samples and corresponding tumor tissues from 19 enucleated eyes of patients diagnosed with RB. The extracted ctDNA from AH underwent low-coverage whole-genome sequencing (lcWGS) and methylation profiling. These data were then compared with the genomic and methylation landscapes of the matched tumor DNA. Additionally, we assessed the ability of AH- derived ctDNA to predict retinoblastoma methylation cluster. Our findings demonstrate a high concordance between ctDNA from AH and the primary tumor DNA in both methylation patterns and genomic alterations detected by lcWGS allowing the correct prediction of the retinoblastoma molecular group. The shared aberrations underscore the potential of AH as a reliable surrogate for tumor tissue in retinoblastoma. Additionally, the methylation profiles revealed consistent epigenetic modifications, further supporting the accuracy of AH-derived ctDNA in reflecting the tumor’s molecular characteristics. This study highlights the feasibility and effectiveness of using AH for molecular ctDNA genetic analysis in retinoblastoma. The overall high concordance between ctDNA profiles with primary tumor DNA and the ability to accurately predict RB methylation classes, highlights the superiority of the liquid biopsy approach overcoming the limitations of traditional biopsies and transforming the potential for early molecular analysis and risk stratification. This revolutionary technique could redefine pre-enucleation diagnostics, offering unprecedented insights and paving the way for tailored, precision medicine in retinoblastoma care. Citation Format: Sophia H Montigel, Tatsiana Ryl, Stefanie Volz, Elena Afanasyeva, Tatjana Wedig, Nathalie Schwarz, Stefan M Pfister, Kristian W Pajtler, Petra Ketteler, Kendra K Maaß. Insightful analysis: Harnessing aqueous humor ctDNA for retinoblastoma stratification [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A062.

EPCO-48. RESOLVING INTRATUMORAL TRANSCRIPTIONAL HETEROGENEITY ACROSS THE CORE-EDGE SPATIAL AXIS IN GLIOBLASTOMA

Abstract Intratumoral heterogeneity promotes tumor progression, treatment resistance, and recurrence in glioblastoma (GBM), and the cellular tumor core and infiltrating edge represent transcriptionally distinct cellular ecosystems. In this study, we characterize and validate the molecular differences across the core-edge spatial axis in GBM by applying whole-transcriptome GeoMx Digital Spatial Profiling (DSP) to 5 IDH-wildtype tumors. Three 3-micron tissue cores were extracted from each tumor and arranged into a tissue microarray (TMA). Core and edge regions of interest (ROIs) were chosen through coregistration of H&E and immunofluorescent TMA scans. Following DSP, data for 44 ROIs (30 core and 14 edge) and 8530 RNA targets were bioinformatically analyzed. Single-sample gene set enrichment analysis aligned the DSP core and edge regional signatures to those of publicly annotated datasets, substantiating our profiling methodology. Unsupervised analyses revealed ROI clusters unique to each tumor specimen for both pre- and post-normalized data. To mitigate a potential patient-level effect in subsequent differential gene expression and pathway analyses, we leveraged a linear mixed-effect model using core versus edge as a fixed effect and tumor specimen as a random effect. Our analyses showed upregulation of epigenomic markers in core ROIs and neuronal markers in edge ROIs. Further, core and edge signatures were incongruent with the canonical GBM subtypes (Mesenchymal, Classical, Proneural) and malignant cell states (OPC-like, NPC-like, AC-like, MES-like). Cellular deconvolution analysis highlighted differential enrichment of neoplastic cells in core ROIs and neuroglia (astrocytes and oligodendrocytes) in edge ROIs. In summary, we leverage spatial profiling to further validate the geographic molecular heterogeneity of GBM, which is characterized by divergent neuronal programs within the infiltrating edge.

Highly reliable LC-MS lipidomics database for efficient human plasma profiling based on NIST SRM 1950

Liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS)-based methods have become the gold standard methodology for the comprehensive profiling of the human plasma lipidome. However, both the complexity of lipid chemistry and LC-HRMS-associated data pose challenges to the characterization of this biological matrix. In accordance with the current consensus of quality requirements for LC-HRMS lipidomics data, we aimed to characterize the NIST® Standard Reference Material for Human Plasma (SRM 1950) using an LC-ESI(+/−)-MS method compatible with high-throughput lipidome profiling. We generated a highly curated lipid database with increased coverage, quality, and consistency, including additional quality assurance procedures involving adduct formation, within-method m/z evaluation, retention behavior of species within lipid chain isomers, and expert-driven resolution of isomeric and isobaric interferences. As a proof-of-concept, we showed the utility of our in-house LC-MS lipidomic database—consisting of 592 lipid entries—for the fast, comprehensive, and reliable lipidomic profiling of the human plasma from healthy human volunteers. We are confident that the implementation of this robust resource and methodology will have a significant impact by reducing data redundancy and the current delays and bottlenecks in untargeted plasma lipidomic studies.

Measles Population Immunity Profiles: Updated Methods and Tools.

Measles is a highly contagious disease and remains a major cause of child mortality worldwide. While measles vaccine is highly effective, high levels of population immunity are needed to prevent outbreaks. Simple but accurate tools are needed to estimate the profile of population measles immunity by age to identify and fill immunity gaps caused by low levels of vaccination coverage. The measles immunity profile estimates and visualizes the percentage of each birth cohort immune or susceptible to measles based on measles vaccination coverage. Several tools that employed this approach have been developed in the past, including informal unpublished versions. However, these tools used varying assumptions and produced inconsistent results. We updated the measles population immunity profile methodology to standardize and better document the assumptions and methods; provide timely estimates of measles population immunity; and facilitate prompt actions to close immunity gaps and prevent outbreaks. We recommend assuming that the second dose of the measles-containing vaccine (MCV2) and doses given during supplementary immunization activities (SIAs) first reach children who have been previously vaccinated against measles, so that previously unvaccinated children are reached only when the coverage of MCV2 or SIA is higher than the coverage achieved by all previous measles vaccination opportunities. This updated method provides a conservative estimate of immunization program impact to assess measles outbreak risk and to facilitate early planning of timely preventive SIAs to close population immunity gaps.

Editing and genome-wide analysis upstream open reading frames contributes to enhancing salt tolerance in tomato.

The salinization of soil constitutes a substantial hindrance to the advancement of sustainable agriculture. Our research seeks to elucidate the role of a Rab GTPase-activating protein (RabGAP) family member, SlRabGAP22, in salt tolerance and its translational regulation under salt stress in tomatoes, employing gene-editing techniques and ribosome profiling methodologies. Findings demonstrate that SlRabGAP22 acts as a positive regulator of tomato salt tolerance, with four predicted upstream open reading frames (uORFs) classified into three categories. Functional uORFs were found to be negative regulation. Editing these uORFs along with altering their classifications and characteristics mitigated the inhibitory effects on primary ORFs and fine-tuned gene expression. Enhanced tomato salt tolerance was attributed to improved scavenging of reactive oxygen species, reduced toxicity Na+, and diminished osmotic stress effects. Furthermore, we conducted genome-wide analysis of ORFs to lay the foundation for further research on uORFs in tomatoes. In summary, our findings offer novel perspectives and important data for the enhancement of genetic traits via uORF-based strategies and translational regulation against the backdrop of salt stress.

Quantifying Membrane Alterations with Tailored Fluorescent Dyes: A Rapid Antibiotic Resistance Profiling Methodology.

Accurate detection of bacterial antibiotic sensitivity is crucial for theranostics and the containment of antibiotic-resistant infections. However, the intricate task of detecting and quantifying the antibiotic-induced changes in the bacterial cytoplasmic membrane, and their correlation with other metabolic pathways leading to antibiotic resistance, poses significant challenges. Using a novel class of 4-aminophthalimide (4AP)-based fluorescent dyes with precisely tailored alkyl chains, namely 4AP-C9 and 4AP-C13, we quantify stress-mediated alterations in E. coli membranes. Leveraging the unique depth-dependent positioning and environment-sensitive fluorescence properties of these dyes, we detect antibiotic-induced membrane damage through single-cell imaging and monitoring the fluorescence peak maxima difference ratio (PMDR) of the dyes within the bacterial membrane, complemented by other methods. The correlation between the ROS-induced cytoplasmic membrane damage and the PMDR of dyes quantifies sensitivity against bactericidal antibiotics, which correlates to antibiotic-induced lipid peroxidation. Significantly, our findings largely extend to clinical isolates of E. coli and other ESKAPE pathogens like K. pneumoniae and Enterobacter subspecies. Our data reveal that 4AP-Cn probes can potentially act as precise scales to detect antibiotic-induced membrane damage ("thinning") occurring at a subnanometer scale through the quantification of dyes' PMDR, making them promising membrane dyes for rapid detection of bacterial antibiotic resistance, distinguishing sensitive and resistant infections with high specificity in a clinical setup.

Atypical applications of transverse diffusion of laminar flow profiles methodology for in-capillary reactions in capillary electrophoresis.

Capillary electrophoresis (CE) is a powerful separation technique offering quick and efficient analyses in various fields of bioanalytical chemistry. It is characterized by many well-known advantages, but one, which is perhaps the most important for this application field, is somewhat overlooked. It is the possibility to perform chemical and biochemical reactions at the nL scale inside the separation capillary. There are two basic formats applicable for this purpose, heterogeneous and homogeneous. In the former, one reactant is immobilized onto a particle or monolithic support or directly on the capillary wall, and the other is injected. In the latter, the reactant mixing inside a capillary is based on electromigration or diffusion. One of the diffusion-based methodologies, termed Transverse Diffusion of Laminar Flow Profiles, is the subject of this review. Since most studies utilizing in-capillary reactions in CE focus on enzymes, which are being continuously and exhaustively reviewed, this review covers the atypical applications of this methodology, but still in the bioanalytical field. As can be seen from the demonstrated applications, they are not limited to reactions, but can also be utilized for other biochemical systems.

Cytokine Signatures in Inflamed Mucosa of IBD Patients: State-of-the-Art

The process of development, recurrence, and exacerbation of the inflammatory process depends on the cytokine levels in IBD. For that reason, many cytokine therapies have been developed for treating IBD patients. Researchers employ various techniques and methodologies for cytokine profiling to identify cytokine signatures in inflamed mucosa. These include enzyme-linked immunosorbent assays (ELISA), multiplex immunoassays, flow cytometry, and gene expression analysis techniques (i.e., microarray, RNA-seq, single-cell RNA-seq (scRNA-seq), mass cytometry (CyTOF), Luminex). Research knowledge so far can give us some insights into the cytokine milieu associated with mucosal inflammation by quantifying cytokine levels in mucosal tissues or biological fluids such as serum or stool. The review is aimed at presenting state-of-the-art techniques for cytokine profiling and the various biomarkers for follow-up and treatment.

Evaluation of comparative chemical profiling and bioactivities of medicinal and non-medicinal parts of Ampelopsis delavayana

Ampelopsis delavayana, a distinctive Yi medicine, utilized the roots as an essential medicinal substance for trauma treatment of the “Yunnan Hong Yao”. A. delavayana, however, cannot be cultivated artificially presently, and it has been described with a phenomenon of mixed utilization of roots and stems, impeding pharmaceutical quality control. In response to resource scarcity and standardization issues, the research comprehensively compares the material basis and efficacy of medicinal (roots) and non-medicinal (stems) parts by using chemical profiling and pharmacological methodologies. Chemical disparity between two parts was compared by TLC and HPLC. Analgesia and anti-inflammatory capabilities of both parts were comprehensively evaluated through acetic acid writhing test, hot plate test, and xylene-induced mouse ear swelling test. Additionally, all the extracts were evaluated for anti-inflammatory activities by monitoring regulation of the levels of TNF-α, IL-1β, IL-6, and IgE in ear tissue. Consequently, the findings of TLC and HPLC revealed substantial similarity in the material basis of the medicinal and non-medicinal parts of A. delavayana, and pharmacological activities of anti-inflammatory and analgesic between two parts were consistent. Different extracts remarkably reduced the levels of TNF-α, IL-1β, IL-6, and IgE, demonstrating no discernible differences. Collectively, the comprehensive exploitation indicated that the medicinal and non-medicinal parts of A. delavayana exhibited identical chemical profiling and bioactivities, providing a theoretical rationale and scientific evidence for using stems as a therapeutic part, thereby holding considerable potential for ameliorating the current status of its medicinal reserves.

Automated myeloma cell selection using machine learning and artificial intelligence.

7565 Background: Precision of sampling is critical to achieve accurate results relevant to diagnostics and prognostics. Sampling errors may compromise the sensitivity and specificity of otherwise credible diagnostics technologies. Our 3- dimensional (3D) telomeres profiling methodology is conducted on individual target cells selected from the patient sample processed using 3D immuno-FISH. In our traditional workflow target cells are manually selected by highly trained personnel based on morphological characteristics and/ or immunophenotypic characteristics, namely cells positive for CD138 and CD56. However, the variability among trained operators required rounds of verification by a second and third operator and an approval by a Hematopathologist or the Lab Director to confirm the selected cells in some cases. Therefore, automated detection of myeloma cells is of paramount importance. Methods: In this study we employed machine learning and artificial intelligence (AI) tools to develop an automated algorithm capable of streamlining target cell (i.e. myeloma) selection and minimize or eliminate operator introduced variability. Training libraries were built using positive and negative cells. Over 5000 cells were used in the algorithm training process. We employed iterative rounds of validation and refinement to achieve a high precision automated tool. AI then identified myeloma cells in an automated fashion and the results of AI identification were confirmed by a Hematopathologist. Results: We report the results of a comparative analysis done on 20 myeloma patients at different stages of the disease including MGUS, smouldering myeloma and active myeloma. Also, the sample type included bone marrow smears, purified mono-nucleated plasma cells and bone marrow biopsies. 50 verified manually selected target cells and 50 target cells selected by the automated cell selection tool, approved by a skilled operator, from the same sample of each patient were analyzed using the TeloView platform. The TeloView platform quantifies 6 telomeric molecular and structural parameters. We conducted statistical analysis to calculate Coefficient of Variation (CV) between the results of each patient. We set the acceptable variability to < 15%. We achieved CVs of less than 10% among all patients included in the analysis. Conclusions: The automated cell selection tool streamlined this critical sampling process in our workflow minimizing operator-introduced variability, reducing the processing time to a fraction of the time required for manual cell selection and verification which enables high throughput for the 3D telomeres profiling while maintaining precision.

Assessing clinical pathological characteristics and gene expression patterns associated with hepatoid adenocarcinoma of the stomach.

The objective of this study is to assess the clinical pathological attributes of Hepatoid Adenocarcinoma of the Stomach (HAS) and to delineate the differential diagnostic considerations about it. The investigation involved analyzing 31 HAS cases using histomorphological assessment, immunohistochemical profiling, and relevant gene detection methodologies. Among the 31 HAS cases, 9 (29.0%) were of trabecular hepatoid adenocarcinoma of the stomach, 7 (22.6%) were of glandular hepatoid adenocarcinoma of the stomach, 4 (12.9%) were of nesting hepatoid adenocarcinoma of the stomach, 3 (9.7%) were of clear cell hepatoid adenocarcinoma of the stomach, and 8 (25.8%) were of diverse hepatoid adenocarcinoma of the stomach. Of these 31 cases, 24 were male, accounting for 77.4% of the cases. Serum alpha-fetoprotein (AFP) levels were notably elevated, with radioimmunoassay results reaching 1240ng/ml; 28 out of 31 cases had AFP levels below 25µg/l, accounting for 90.3%. Related genes: HER2 protein indicated positive expression on the cell membrane in 35.5% (11/31) of the cases; HER2 gene amplification detected by the FISH technique was 12.9% (4/31). Tumoral stromal lymphocytes exhibited a PD-1 positive expression rate of 58.1% (18/31). In gastric cancer tissues, the PD-L1 positive rate was 45.1% (14/31). HAS represents a distinctive subtype of gastric cancer with a propensity for mimicking other forms of tumors, underscoring the significance of discerning its unique histopathological attributes for accurate differential diagnosis and tailored therapeutic interventions.

Adaptation of Wind Drag Coefficient Parameterization: Improvement of Hydrodynamic Modeling by a Wave‐Dependent Cd in Large Shallow Lakes

AbstractWind is a critical driving force in hydrodynamic and water quality modeling of large shallow lakes, and is characterized by the wind drag coefficient Cd, representing the momentum transfer at the air‐water interface. Contemporary empirical formulae for Cd estimation were derived over oceans and some of which are solely wind velocity U10 dependent. These formulae were previously found to be inadequate in inland lake models often resulting in the water velocity underestimation. To address this problem, a physical scale experiment was designed, in which Cd was measured using a wind profile and eddy covariance methodology. A new wind‐induced wave‐dependent Cd parameterization was also established and validated in two lake studies. The driving force was modified by the wave‐dependent Cd formula in a hydrodynamic model of the shallow Upper Klamath Lake (UKL), OR, USA. The experimental Cd was negatively correlated to the wind velocity up until the critical U10 = 1.6 m s−1 which was 1.0~3.1 times previous empirical extrapolations at light winds. The variation partitioning results showed that wave parameters contributed to more than 30% of Cd variation combined with wind parameters. The modified wind stress field was spatially heterogeneous and the modeled water velocity was closer to the observations at two sites. Significant main circulation and outer bank circulation were modeled accompanied by higher surface vorticity, compared to the original UKL model. Overall, the wave‐dependent Cd formula provided an improvement of the surface flow field in the UKL model and will improve the management of the lake ecosystems.

Sensory profile and chemical quality of Merlot aged in barrels of different wood species

The cultivation of Vitis vinifera varieties in Brazil was established in 1970. With the opening of the market to imported labels and a greater variety of wines, viticulture became more competitive, and consumers became more demanding. The aging of wine is a system that involves several reactions based on the extraction of molecules from the wood and also on the micro-oxygenation of beverages. The objective of this study was to investigate the sensorial and chemical evolution of wines aged in barrels of different woods: French oak (Quercus petraea), Amburana (Amburana cearensis), and Jequitibá Rosa (Cariniana legalis). The comparison of the sensorial and chemical characteristics of the aged wine with the non-aged one allowed us to know the modifications resulting from the maturation time. Their distinct evolution among the different species was identified through chromatographic analysis of maturation congeners. Perceived sensory characteristics and acceptance of Brazilian woods were measured using the Napping and UltraFlash Profile methodologies. Tropical woods stood out in terms of acceptance by tasters, and the sensory attributes listed by the panel differ between both.