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Evolution and Expression Divergence of Legume PAL Genes Suggest Associations with Drought Response and Root Nodule Development.

Comparative genomic analyses provide insight into the mechanisms underlying gene-family evolution and crop adaptation. Here, we used the legume phenylalanine ammonia-lyase (PAL) gene family as a model and integrated pan-genomic, phylogenetic, molecular evolutionary, duplication-mode, and transcriptomic analyses, while developing GFtool for gene family identification. Across 45 genomes, we identified 302 PAL genes and classified them into five Groups. Groups 1-3 represented ancient lineages shared with outgroups, whereas Groups 4 and 5 were legume-specific. Molecular-clock analyses placed the divergence of Group 2 near the Paleocene-Eocene transition, while Groups 4 and 5 diversified from the middle Eocene to the early Oligocene. WGD/segmental duplication broadly contributed to PAL copy-number expansion, whereas tandem duplication was enriched in Group 5 of Papilionoideae. Group 2 genes showed drought-induced expression, whereas Group 5 genes were associated with early root nodule development. GFtool provides a scalable framework for gene-family studies.

Fabaceae

Inhibition of 3'-methyl-4-dimethylaminoazobenezene-induced hepatocarcinogenesis by portacaval shunt.

Adult male Sprague Dalwey rats on which end-to-side portacaval shunt (PCS) operation was performed did not hyperplastic nodules and hepatoms when they were fed 3'-methyl-4-dimethylaminoazobenzene in semisynthetic basal diet for periods of up to 169 days. In contrast, all the intact rats fed the same diet for only 75 days, developed hyperplastic nodules in the liver. Transferred to normal pellet for another 25 days, hepatomas developed in 100% of these animals. The amount of protein-bond 3'-Me-DAB was found to be much smaller in operated rats than in intact animals. The glutathione (GSH) level in PCS-operated rats was lower than in intact controsl. A single large dose of 3'-Me-DAB led to the increase of only about 30% in the concentration of GSH during the period of 24-48 h, compared to the increase of 50-100% in non-operated rats. No clear tendency to a gradual increase in the activity of gamma-glutamyl transpeptidase was noted in PCS-operated rats during the period of 5 1/2 months of 3'-Me-DAB feeding. The increase in GT-ase activity never exceeded 30% above the level of GT-ase in the livers of PCS-operated rats fed basal diet without the carcinogen. This striking inhibibiton of GT-ase increase induced by 3'Me-DAB in PCS-operated rats contrasted with an increase of GI-ase activity by 5,000% found in livers of non-operated rats with hyperplastic nodules after 75 days of 3'-Me-DAB feeding and the increase by up to 10,000% in developed hepatomas. These effects and the inhibition of 3'Me-DAB-induced hepatocarcinogenesis, manifested by lack preneoplastic morphologic changes and the absecnce of hepatomas in rats after PCS, can best be explained by functional deficiency of the liver to metabolize the procarcinogen 3'-Me-DAB into an activated carcinogen.

Animals

Endoscopic ultrasound-guided biopsy of left and right adrenal metastases enabling pathological diagnosis and genomic profiling after nondiagnostic conventional biopsies: two case reports.

Obtaining adequate tissue for histologic diagnoses and genomic testing can be challenging in metastatic lung cancer, particularly when conventional biopsy approaches are nondiagnostic. The study reports an effective salvage strategy using endoscopic ultrasound (EUS)-guided adrenal tissue acquisition in two cases. A 66-year-old woman (case 1) developed recurrent lung adenocarcinoma with progressive metastases to the left adrenal, liver, and lungs following multiple lines of systemic therapy. A percutaneous biopsy of a suspected liver metastasis proved nondiagnostic. Subsequently, transgastric EUS-guided biopsy was performed, which confirmed metastatic adenocarcinoma originating in the lung. Oncomine-based genomic testing detected a human epidermal growth factor receptor 2 exon 20 insertion. Trastuzumab deruxtecan was subsequently introduced, resulting in disease stabilization for 6 months. A 75-year-old man (case 2) developed bilateral pulmonary nodules and a right adrenal mass detected on positron emission tomography. Bronchoscopy failed to yield diagnostic tissue. Following careful review of cross-sectional anatomy, EUS-guided biopsy of the right adrenal gland was safely performed via the duodenal bulb, confirming metastatic squamous cell carcinoma and yielding adequate tissue for genomic testing. EUS-guided adrenal biopsy, including transduodenal sampling of the right adrenal gland, may provide tissue for histopathologic and precision oncology testing, facilitating definitive diagnoses.

Adrenal metastasis

Glutathione and gamma glutamyl transpeptidase in rat liver during chemical carcinogenesis.

Continued administration of several hepatocarcinogens led to an increase in the concentration of glutathione (GSH) in the livers of intact, but not of hypophysectomized or adrenalectomized rats. The concentration of GSH remained high untill the development of hyperplastic nodules. Subsequently, the concentration of GSH dropped to the normal level or below. A single dose of 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) produced an increase of GSH which, within a certain range, depended upon the amount of the carcinogen. In well differentiated, slowly growing hepatomas, the concentration of GSH approached the level in normal adult rat liver. On the other hand, in nondifferentiated and rapidly growing hepatomas, GSH was only 30-40% of that in normal liver. The activity of gamma-glutamyl transpeptidase (GTase) increased within 24-48 hours after a single large dose of 3'-Me-DAB. Continued feeding of 3'-Me-DAB led to an exponential increase of GTase. During hepatocarcinogenesis, the level of GTase activity corresponded to the degree and size of pathologic changes produced in rat liver. Chloramphenicol partially inhibited the increase of GTase induced by 2-acetylaminofluorene. Pretreatment with 3-methylcholanthrene partially inhibited the increase of GTase that had been induced by a single dose of 3'-Me-DAB. Puromycin partially inhibited the increase of GTase induced by several doses of dimethylnitrosamine. These observations indicated a close connection between the activation of GTase and chemical carcinogenesis in rat liver. Measurements of GTase activity in 12 Morris hepatomas supported this conclusion; their GTase levels were greatly elevated compared with that in normal adult rat liver.

2-Acetylaminofluorene

Nissolia brasiliensis as a nonnodulating model legume.

The nitrogen-fixing root nodule symbiosis is specifically formed by 4 orders of angiosperms. The largest of these 4 orders includes the legume family, the Fabaceae. Among legumes, historical model species have emerged, such as the root nodule symbiosis-forming Medicago truncatula and Lotus japonicus or, more recently, Aeschynomene evenia. By contrast, legume species that have lost root nodule symbiosis have been largely ignored. Here, we describe the first near chromosome-level assembly for a non-root nodule symbiosis-forming legume, the tropical Papilionoideae Nissolia brasiliensis. We compared its genome to closely related legumes and identified genes associated with root nodule symbiosis. Finally, we developed a stable transformation protocol that can be deployed in the future to reevolve root nodule symbiosis in legumes, a first step toward the goal of engineering root nodule symbiosis in nonlegume crops.

Fabaceae

Kinetic model of a determinate legume root nodule reveals plant metabolic characteristics for more efficient nitrogen fixation symbiosis.

While nitrogen fertilizers are widely used in agricultural production, their application incurs significant environmental and energetic costs. In contrast, some crops are less dependent on these fertilizers because they engage in symbioses with rhizobia, nitrogen-fixing bacteria that provide ammonium to the plant in exchange for carbon. However, the carbon cost associated with nitrogen fixation can negatively impact crop yields. Improving the efficiency of this metabolic process could alleviate this impact on crop productivity. Mathematical models can help us quantitatively explore metabolic behavior and identify potential targets for metabolic engineering. In this work, we developed a kinetic model of determinate root nodule metabolism, where this symbiotic exchange of carbon from the plant and nitrogen from the bacteria occurs. We used this model to evaluate how the predicted metabolic behavior differs between inefficient and efficient nodules, and to identify potential engineering targets for improving nitrogen fixation efficiency and rate. We show that the enzymes phosphoenolpyruvate carboxylase and pyruvate kinase have significant influence on the predicted rate and efficiency of nitrogen fixation, especially when their expression is varied in combination with oxidative Pentose Phosphate Pathway enzymes like glucose-6-phosphate dehydrogenase and 6-phosphogluconolactonase. The model predicts that pairing a 3-fold decrease in glucose-6-phosphate dehydrogenase activity along with either a 3-fold increase in phosphoenolpyruvate carboxylase activity or decrease in pyruvate kinase activity could increase nitrogen fixation rate by 8.82% while improving nitrogen fixation efficiency by 10.99%.

Enzyme kinetics

Genomic and Molecular Interaction Analysis of NodD1 in a Novel Bradyrhizobium yuanmingense sp. B64 Isolate for Nodulation and Symbiosis of Legume Plants.

Rhizobial bacteria are known for their ability to fix nitrogen for leguminous plants and their essential function for sustainable agriculture. This study characterizes the taxonomic status and functional potential of the Bradyrhizobium B64 isolate using integrated genomic and molecular approaches. The whole genome of the B64 isolate was sequenced via Illumina paired-end technology. Species delimitation was performed using average nucleotide identity (ANI) and digital DNA-DNA Hybridization (dDDH). The NodD1 protein structure was modeled using AlphaFold3 and validated by Ramachandran plot analysis. Molecular docking was then conducted to evaluate interactions between NodD1 and four signaling flavonoids: Apigenin, Daidzein, Genistein, and Naringenin. Genomic analysis revealed a maximum ANI of 94.4% and dDDH values between 51.4 and 62.4%. Since these values fall below the standard prokaryotic thresholds (ANI&#x2009;<&#x2009;95%; dDDH&#x2009;<&#x2009;70%), the B64 isolate is identified as a novel species. Physiological assays confirmed nitrogen fixation (1.97 ppm), IAA production (3.67 ppm), and phosphate solubilization (26.10 ppm). Structural validation showed 100% of NodD1 residues in allowed regions, ensuring high model reliability. Docking simulations demonstrated strong binding affinities across all flavonoids, with binding free energies ranging from -&#x2009;8.8 to -&#x2009;9.0&#xa0;kcal/mol. Daidzein exhibited the highest thermodynamic stability (-&#x2009;9.0&#xa0;kcal/mol), whereas apigenin showed the most extensive residue interaction network. The B64 isolate is a novel Bradyrhizobium species with a high symbiotic capacity. The stable NodD1-flavonoid interactions provide a molecular basis for efficient nodulation, positioning B64 as a promising candidate for developing lipo-chitooligosaccharide (LCO)-based biofertilizers.

Bradyrhizobium

Calcification of the collagenous axial skeleton of Veretillum cynomorium pall. (Cnidaria: Pennatulacea).

The axial skeletal rod of Veretillium cynomorium consists of a fibrillar collagenous matrix calcified with calcite. The present paper describes ultrastructural and crystallographic details of its organization and deposition. At the inferior end of the rod is a calcification gradient between the noncalcified tip and the rest of the axis. Initial mineral deposits, which are sometimes associated with cell debris, give rise to calcitic nodules which enlarge by the radical growth of several lobes. These nodules fuse and form the core of the axis. Subsequent increase in diameter of the rod involves the radial development of irregular columns of calcite which arise from the peripheral nodules. Mineral surfaces exhibit a distinctive microarchitecture which can be related to the predominantly c-axis parallel growth of the calcite. Particular attention is paid to the relationship between mineral and matrix. The collagen fibrils, embedded in the calcite but never impregnated with it, are not responsible for the initial nucleation of mineral. The crystallographic orientation of the calcite also appears to be independent of these fibrils.

Animals

Postpubertal untreated cryptorchidism. Anatomical relationship between Leydig cells and tubular sclerosis.

A histological study of the postpubertal untreated cryptorchid testis establishes a direct anatomical relationship between Leydig cells and tubular sclerosis, an age-related process ranging from mild fibrosis of the tubular tunica propria to complete tubular hyalinization. Two separate processes are involved. Mild or moderate tunical fibrosis is related to pretubular hyperplastic Leydig cell nodules, while severe tubular sclerosis and hyalinization depends on the development of intratunical Leydig cells, a finding hitherto undescribed. More probing studies are needed to define the mechanisms whereby these abnormal Leydig cells cause tubular tunical fibrosis.

Adolescent

Nodules-associated Klebsiella oxytoca complex: genomic insights into plant growth promotion and health risk assessment.

The swift emergence of antibiotic resistance genes (ARGs) across interconnected One Health compartments poses a significant global threat. Although plant growth-promoting (PGP) bacteria possess numerous attributes beneficial to host plants, many of these bacteria also harbor ARGs, necessitating a focused assessment of their negative implications. In this context, here we performed whole genome sequencing of 14 PGP endophytic strains isolated from root nodules of faba beans, belonging to three Klebsiella oxytoca species complex (KoSC): K. grimontii (n&#x2009;=&#x2009;5), K. michiganensis (n&#x2009;=&#x2009;5), and K. pasteurii (n&#x2009;=&#x2009;4). We performed comparative genomics, molecular typing, and pangenome analyses on these strains. We identified significant diversity within the KoSC population, classifying the strains into five sequence types (STs), three of which are novel to this study (ST-542, ST-569, and ST-629). Phylogenomic analysis revealed that the bacterial strains clustered more closely by ST than by their source of isolation. Annotation of gene clusters indicated that all assembled genomes are enriched with genes involved in PGP activities, alongside a robust array of genes conferring tolerance to abiotic stresses. Importantly, our findings disclosed that the 14 assembled genomes harbored multiple ARGs, conferring resistance to various antibiotic classes, with 71% of the population classified as multidrug-resistant based on the in vitro antibiotic susceptibility assay. Furthermore, all genomes contained an array of virulence factors critical for survival, pathogenesis, biofilm formation, and root colonization. In conclusion, this study substantiates the hypothesis that certain PGP bacteria may serve as potential reservoirs of multidrug resistance, posing significant public health risks. Thus, the future advancement of bacteria-based biofertilizers should integrate environmental considerations and monitor their impact on antibiotic resistance dissemination in soil ecosystems.

Klebsiella oxytoca

Trade-off between photosynthetic promotion and nitrogen fixation suppression induced by chloroplast-targeted Mo nanoparticles in soybean.

Organelle-targeted nanomaterials offer opportunities to improve crop photosynthesis, yet their unintended effects on symbiotic nitrogen fixation remain poorly understood. Here, we developed chloroplast-targeted molybdenum nanoparticles (Chl-Mo) and compared their effects with those of ionic Mo (IonMo) and non-targeted Mo nanoparticles in soybean. Chl-Mo preferentially accumulated in chloroplasts, enhancing photosynthetic carbon assimilation, thylakoid development, PSII performance, sucrose transport, and biomass accumulation. However, this growth promotion was accompanied by suppressed nodule nitrogenase activity, reduced nif gene expression, inhibited GS/GOGAT-mediated nitrogen assimilation, and disrupted microoxic and ROS homeostasis in nodules. Integrated nodule proteomics and metabolomics showed downregulation of sucrose transport, glycolysis, pyruvate metabolism, and amino acid biosynthesis, indicating a decoupling between enhanced carbon input and nitrogen utilization. Root transcriptomics further revealed oxidative stress, impaired nitrate assimilation, and attenuated early symbiotic signaling. These findings demonstrate that chloroplast-targeted Mo delivery can enhance photosynthesis while compromising symbiotic nitrogen fixation, highlighting the need to evaluate belowground symbiotic functions when developing organelle-targeted nanotechnologies for sustainable agriculture.

Chloroplast-targeted Mo

Isolation of rhizobia from Ontario soils that are effective at fixing nitrogen with common bean (Phaseolus vulgaris).

UNLABELLED: Common bean (Phaseolus vulgaris) is an important crop in Canada and globally. Like other legumes, common bean establishes symbiotic interactions with nitrogen-fixing bacteria called rhizobia. However, nitrogen fixation by rhizobia in association with common bean is often suboptimal, constraining its productivity and necessitating the application of nitrogen fertilizer. To support the development of high-performing, locally adapted rhizobial inoculants for Ontario common bean growers, we isolated 216 common bean-nodulating rhizobia from southern Ontario soils using a nodule trapping approach with four common bean cultivars. Whole genome sequencing followed by phylogenomic analyses of all rhizobial isolates revealed substantial diversity, assigning them to 11 Rhizobium species, including two novel species. Nearly all isolates belong to the symbiovar phaseoli, spanning the nodC &#x3b3;-a, &#x3b3;-b, and &#x3b1; alleles, with four isolates belonging to the symbiovar gallica. Soil origin had a significant impact on the species-level community composition recovered during the nodule trapping experiments. In contrast, host trapping cultivar had only a minor influence on the recovered Rhizobium population. Greenhouse assays demonstrated that one of the novel Rhizobium species exhibited the highest average symbiotic effectiveness, although high-quality isolates were found across multiple species. Together, these results revealed a diverse and genomically variable Rhizobium community capable of forming effective symbioses with common bean in southern Ontario soils. Importantly, our genome-sequenced Rhizobium collection will serve as a valuable resource for identifying competitive and high-quality strains for the development of inoculants tailored to Ontario common bean production. IMPORTANCE: Common bean is a globally important food crop, yet its productivity is often limited by suboptimal nitrogen fixation, forcing growers to rely on synthetic fertilizers. Consequently, identifying high&#x2011;performing, locally adapted inoculant strains is essential for reducing dependence on synthetic nitrogen fertilizers and improving the sustainability of temperate agroecosystems. Our study provides a genome&#x2011;sequenced collection of common bean-nodulating Rhizobium from southern Ontario, revealing substantial species and genomic diversity across sampling locations. Greenhouse studies allowed us to identify multiple isolates that consistently fix nitrogen with, and enhance the growth of, common bean plants. Our findings highlight strong biogeographical structuring of the effective and competitive subpopulations of rhizobial communities and demonstrate that Ontario soils already harbor strains with high symbiotic potential. In addition, our Rhizobium collection represents a foundational resource to support future inoculant development and enables future work on the ecology, evolution, and applied optimization of legume-rhizobium symbioses.

Nanopore

Incidence and risk factors for malignancy in patients with incidental solitary pulmonary nodules: a systematic review and meta-analysis.

BACKGROUND: The increasing use of chest imaging has led to a higher detection rate of incidental solitary pulmonary nodules (SPNs), often causing patient anxiety. Determining the malignancy rate and associated risk factors is crucial for developing appropriate follow-up strategies to prevent overdiagnosis, overtreatment, or missed diagnoses. This meta-analysis aims to investigate the malignancy rate and risk factors in patients with incidental SPNs. METHODS: A systematic search of PubMed, Embase, Web of Science, and the Cochrane Library was conducted up to June 30, 2025. Data on malignancy rates and potential risk factors were extracted from eligible studies. All pooled analyses were performed using a random-effects model. RESULTS: Fifty-four studies involving 19,985 patients were included. The pooled malignancy rate for incidental SPNs was 56.7% (95% CI: 51.5-62.0), with significant between-study heterogeneity (I2 = 98.5%, p&#x2009;<&#x2009;0.001). The pooled effect size showed a minimal change after adjustment for potential publication bias using the non-parametric Trim-and-Fill method (54.7%; 95%CI: 50.9-58.8). Risk factor analysis identified that older age, history of cancer, cigarette smoker, larger nodule diameter, spiculation, upper lobe location, lobulation, pleural indentation, vascular convergence, solid nodules, family history of cancer, and irregular or ill-defined margins were significantly associated with an increased risk of malignancy. Conversely, male sex, presence of calcification, and clear borders were significantly associated with a reduced risk of malignancy. CONCLUSION: This meta-analysis provides a comprehensive assessment of malignancy rates and risk factors in incidental SPNs. The high pooled malignancy rate should be interpreted considering the significant heterogeneity and the inclusion of a high proportion of retrospective studies and populations from high-risk regions. Nonetheless, these findings offer essential evidence for clinical risk stratification, supporting optimized follow-up and informed decision-making.

Humans

A multi-analyte cfDNA-based blood test for early detection of hepatocellular carcinoma.

BACKGROUND & AIMS: Patients at high risk for hepatocellular carcinoma (HCC) are recommended to undergo biannual abdominal ultrasound surveillance; however, ultrasound has low sensitivity for small HCC nodules and is associated with poor adherence. To address these limitations, the multianalyte HelioLiver Dx blood test was developed to aid in the detection of HCC in patients with cirrhosis who are at high risk for HCC. METHODS: The performance of the HelioLiver Dx test and ultrasound for the detection of HCC in adults with cirrhosis was evaluated in a cross-sectional, prospective, blinded, multicenter validation study. All participants provided blood specimens for the HelioLiver Dx test and underwent ultrasound. All participants also underwent multiphasic MRI, which served as the reference standard for determining HCC status. RESULTS: Of 1,268 evaluable participants, 46 (3.6%) were considered to have HCC as determined by MRI, with many (46%) having small HCC lesions &#x2264;2 cm in diameter. The HelioLiver Dx test had a sensitivity of 47.8% (95% CI 32.9-63.1) for all HCC lesions and 28.6% (95% CI 11.3-52.2) for HCC lesions &#x2264;2 cm. In contrast, ultrasound demonstrated a lower sensitivity of 28.3% (95% CI 16.0-43.5) for all HCC lesions and failed to detect any (0%; 95% CI 0.0-16.1) HCC lesions &#x2264;2 cm. The specificity of HelioLiver Dx and ultrasound were 87.6% (95% CI 85.6-89.4) and 93.9% (95% CI 92.5-95.2), respectively. The HelioLiver Dx test met prespecified co-primary endpoints for superior sensitivity and non-inferior specificity compared to ultrasound. CONCLUSION: Compared with ultrasound and alpha-fetoprotein, the HelioLiver Dx test identified more HCC lesions overall, including more small lesions. A convenient and accurate blood-based test may improve HCC surveillance by facilitating earlier detection and reducing patient barriers to testing. GOV IDENTIFIER: NCT03694600 IMPACT AND IMPLICATIONS: There is a significant, unmet clinical need for more sensitive and accessible methods for the early detection of hepatocellular carcinoma (HCC) in high-risk patient populations. The current study is the first blinded, multicenter, prospective study to evaluate the performance of a multianalyte blood test compared to abdominal ultrasound for the detection of HCC among patients with cirrhosis. The multianalyte HelioLiver Dx test met prespecified co-primary endpoints for superior sensitivity and non-inferior specificity compared to ultrasound for detection of HCC lesions. The availability of a more accessible, convenient and sensitive blood test to aid in the detection of HCC may improve utilization and consequently clinical outcomes for high-risk patients via reduction of care barriers and improved early HCC detection. CLINICALTRIALS: gov identifier: NCT03694600.

Humans

Development of hematopoietic spleen colonies in nonirradiated genetically normal mice.

The question as to whether prior irradiation or injection of cytotoxic drugs is essential for the development of spleen colonies was examined in genetically normal mice. Mixtures of lymph node and bone marrow cells from C57BL mice were injected into (C57BL X CBA-T6T6) F1 hybrid mice without pretreatment. Hematopoietic nodules were observed in the spleens of F1 hybrid mice killed 18 days after injection. The average number of nodules increased linearly with increased numbers of injected bone marrow cells. Hematopoietic stem cells (CFU-S) and dividing cells in the nodules were shown to be of C57BL origin. Histologic examination showed that erythroid cell colonies predominated over granulocytic cell colonies. These results suggest that any kind of treatment that causes the depletion of CFU-S in the spleen of hosts would provide a suitable environment for the production of colonies by transplanted CFU-S.

Animals

A germline PDGFRB splice site variant associated with infantile myofibromatosis and resistance to imatinib.

PURPOSE: Infantile myofibromatosis is characterized by the development of myofibroblastic tumors in young children. In most cases, the disease is caused by somatic gain-of-function variants in platelet-derived growth factor (PDGF) receptor beta (PDGFRB). Here, we reported a novel germline intronic PDGFRB variant, c.2905-8G>A, in 6 unrelated infants with multifocal myofibromatosis and their relatives. METHODS: We performed constitutional and tumor DNA and RNA sequencing to identify novel variants, which were subsequently characterized in cellular assays. RESULTS: All patients had multiple skin nodules, 4 had bone lesions, and 2 had aggressive disease with bowel obstruction. The c.2905-8G>A substitution creates an alternative acceptor splice site in intron 21, inserting 2 codons in the PDGFRB transcript. Functional studies revealed that the splice change induced a partial loss of function, contrasting with previously described variants. In 4 tumor samples, we identified a second somatic hit at position Asp850 in PDGFRB exon 18, triggering constitutive receptor activation and resistance to imatinib. In addition to vinblastine and methotrexate, 2 patients received imatinib without objective response. One of them switched to dasatinib with concomitant improvement. CONCLUSION: This splice-site PDGFRB variant favors the development of myofibroma, featuring an acquired oncogenic variant in the same gene and resistance to targeted therapy.

Humans

Biomarker identification through spatial proteomics for the characterization of indeterminate thyroid nodules.

PURPOSE: The identification of novel molecular biomarkers may assist in the characterization of indeterminate thyroid nodules, which pose significant diagnostic challenges. Here, we aimed to explore the potential of proteomic analyses to support biomarker discovery in challenging thyroid lesions. METHODS: Linear Discriminant Analysis (LDA) was applied to Matrix-Assisted Laser Desorption Ionization Mass Spectrometry Imaging (MALDI-MSI) data from 44 thyroid neoplasms to select the most impactful molecular features for the classification of different tumor histologies, as well as for the distinction between NRAS-mutant (mNRAS) and NRAS-wild-type (wtNRAS) tumors. Relevant peaks were subsequently identified through nanoscale liquid chromatography electrospray ionization tandem mass spectrometry (nLC-ESI-MS/MS). RESULTS: The LDA selected nine relevant molecular markers distinguishing noninvasive follicular thyroid neoplasms with papillary-like nuclear features (NIFTPs) from other tumor histologies (balanced accuracy = 73%), as well as 19 relevant markers able to identify mNRAS cases (balanced accuracy = 84%). Nine differentially expressed proteins were putatively identified: among them, ATP-dependent RNA helicase DDX42 showed a similar distribution between NIFTPs and papillary thyroid carcinomas (PTCs) / follicular variant PTCs (FVPTCs), while the distribution of the Histone H4 signal was similar between NIFTPs and follicular adenomas (FAs). In addition, Protein disulfide-isomerase A1 and Complement C4-B were overexpressed in wtNRAS compared to mNRAS cases, regardless of histology. CONCLUSION: The LDA-selected features enable to distinguish NIFTPs from morphologically similar lesions and to discriminate between mNRAS and wtNRAS cases. The identified markers might complement genetic analyses and provide insights into the distinct pathogenic drivers behind the development of mNRAS compared to wtNRAS lesions.

Humans

Genetic structuring and estimation of reproductive adults in Onchocerca volvulus: A genome-wide analysis across hosts and regions.

Genomic analysis of parasites can deepen our understanding of their transmission, population structure, and important biological characteristics. Onchocerciasis (river blindness), caused by the parasitic nematode Onchocerca volvulus, involves adult worms residing in subcutaneous nodules that produce larval-stage microfilariae (mf), which are routinely detected in the skin for diagnosis. Whole-genome studies of mf are limited; most analyses have focused on the mitochondrial genome. We conducted a genome-wide analysis with 94% median nuclear genome coverage, analyzing 171, 37, and 98 mf from 16, 3, and 5 individuals from Ghana, Liberia, and the Democratic Republic of Congo, respectively. These data were used to investigate population differentiation, estimate the number of reproductive adult worms, and analyze genetic variation across chromosomes. Population genetic analyses across hosts and countries showed that nuclear genome diversity can reveal fine-scale genetic structure, even between geographically close countries, providing more resolution than mitochondrial haplotype data. By reconstructing maternal and paternal sibships, we estimated the number of reproductively active adult filariae. Comparisons between adult worm estimates from genetic data and nodule observations showed that genetics-based estimates were higher or equal to observed worm counts in 8 out of 9 hosts for female worms and 7 out of 9 hosts for male worms. Our analysis also revealed lower-than-expected X chromosome diversity, consistent with neo-X chromosome fusions in filarial species. This study represents an important step in using nuclear genome data from mf to support onchocerciasis elimination efforts and in developing genetic tools that could inform mass drug administration programs.

Onchocerca volvulus