A Cuproptosis-Related gene Signature as a Prognostic Biomarker in Thyroid Cancer Based on Transcriptomics.

Thyroid cancer (THCA) is the most prevalent endocrine tumor, and its incidence continues to increase every year. However, the processes underlying the aggressive progression of thyroid cancer are unknown. We concentrated on the prognostic and biological importance of thyroid cancer cuproptosis-related genes in this investigation. Genomic and clinical data were obtained from the UCSC XENA website, and cuproptosis-related genes were obtained from the FerrDb website. We performed differential expression analysis and Cox regression analysis to identify possible predictive targets associated with thyroid cancer prognosis. To assess the role of CDKN2A in thyroid cancer and the ability to predict prognosis on the basis of the CDKN2A expression level, we performed immunohistochemical staining, survival analysis, immunological analysis, functional analysis, and clinical analysis with respect to CDKN2A gene expression. CDKN2A expression levels were found to be inversely correlated with thyroid cancer prognosis. Higher levels of CDKN2A expression were associated with higher T, N, and clinicopathological stage and more residual tumor cells. Through univariate and multivariate Cox regression analyses, the CDKN2A expression level was shown to be linked with thyroid cancer patients' overall survival (OS). Moreover, we discovered that CDKN2A expression was linked to a dysfunctional tumor immune microenvironment. The study shows that CDKN2A, a cuproptosis-related gene, can be used as a prognostic marker for thyroid cancer.

Ubiquitin-specific peptidase 10 attenuates the ferroptosis to promote thyroid cancer malignancy by facilitating GPX4 via elevating SIRT6.

PURPOSE: Ubiquitin-specific peptidase 10 (USP10) has been found to have oncogenic activity in several human tumors. This study first revealed the exact function of USP10 on the progression of thyroid cancer (THCA) by researching its effect on the ferroptosis. METHODS: USP10 expression in THCA patients was analyzed by online data analysis and in 75 THCA cases was scrutinized by real-time quantitative reverse transcription-polymerase chain reaction and Western blot. Influence of USP10 on the viability, colony formation, migration and invasion of THCA cells was demonstrated by cell counting kit-8, colony formation, wound healing and Transwell invasion assays. Effect of USP10 on the Erastin-induced ferroptosis in THCA cells was evaluated by detecting the ferroptosis-related indicators. Intrinsic mechanism of USP10, glutathione peroxidase 4 (GPX4) and sirtuin 6 (SIRT6) in regulating THCA progression was identified. In vivo xenograft experiment was implemented. RESULTS: USP10 was abundantly expressed in THCA patients, linking to poor outcome. USP10 overexpression enhanced the viability, colony formation, migration and invasion of THCA cells. USP10 mitigated the Erastin-induced ferroptosis in THCA cells, decreased the levels of iron, Fe2+ , malondialdehyde, lipid reactive oxygen species, reduced mitochondrial superoxide level, and increased mitochondrial membrane potential. USP10 facilitated the expression of ferroptosis suppressor GPX4 by elevating SIRT6. Loss of USP10 repressed the in vivo growth of THCA cells. CONCLUSION: USP10 might attenuate the ferroptosis to promote thyroid cancer malignancy by facilitating GPX4 via elevating SIRT6. It might be novel target for the treatment of THCA.

The Cannabinoids, CBDA and THCA, Rescue Memory Deficits and Reduce Amyloid-Beta and Tau Pathology in an Alzheimer's Disease-like Mouse Model.

Most studies related to hemp are focused on Cannabidiol (CBD) and Tetrahydrocannabinol (THC); however, up to 120 types of phytocannabinoids are present in hemp. Hemp leaves contain large amounts of Cannabidiolic acid (CBDA) and Tetrahydrocannabinolic acid (THCA), which are acidic variants of CBD and THC and account for the largest proportion of CBDA. In recent studies, CBDA exhibited anti-hyperalgesia and anti-inflammatory effects. THCA also showed anti-inflammatory and neuroprotective effects that may be beneficial for treating neurodegenerative diseases. CBDA and THCA can penetrate the blood-brain barrier (BBB) and affect the central nervous system. The purpose of this study was to determine whether CBDA and THCA ameliorate Alzheimer's disease (AD)-like features in vitro and in vivo. The effect of CBDA and THCA was evaluated in the Aß1-42-treated mouse model. We observed that Aß1-42-treated mice had more hippocampal Aß and p-tau levels, pathological markers of AD, and loss of cognitive function compared with PBS-treated mice. However, CBDA- and THCA-treated mice showed decreased hippocampal Aß and p-tau and superior cognitive function compared with Aß1-42-treated mice. In addition, CBDA and THCA lowered Aß and p-tau levels, alleviated calcium dyshomeostasis, and exhibited neuroprotective effects in primary neurons. Our results suggest that CBDA and THCA have anti-AD effects and mitigate memory loss and resilience to increased hippocampal Ca2+, Aß, and p-tau levels. Together, CBDA and THCA may be useful therapeutic agents for treating AD.

An immune-related prognostic signature for thyroid carcinoma to predict survival and response to immune checkpoint inhibitors.

Thyroid carcinoma (THCA) is the most common endocrine malignancy, and its incidence is increasing worldwide. Several studies have explored whether the tumor immune microenvironment and immune-related genes (IRGs) influence the prognosis of patients with THCA and can be used to predict the response to immune checkpoint inhibitors (ICIs). We developed an IRG prognostic/risk signature using a bioinformatics method, and its predictive capacity was validated in patients in the test set and the total set. Subsequently, we analyzed the correlation between this IRG prognostic signature and tumor-infiltrating immune cells, tumor mutation burden (TMB), and immune checkpoint protein expression in patients with THCA. With a multivariate analysis, the IRG prognostic signature, which comprised eight IRGs, was identified as an independent prognostic factor. High-risk patients had poor overall survival compared with low-risk patients. Plasma cells, monocytes, and dendritic cells infiltrated differently according to the IRG prognostic signature. The low-risk group had a higher TMB and immunophenoscore (IPS), which indicated a better response to ICIs. The qRT-PCR validated eight IRGs with differential expression in thyroid cancer and normal tissues. We conclude that the IRG prognostic signature may be a useful tool to predict survival and response to ICIs. However, further testing is required to assess the predictive capacity of this IRG prognostic signature.

Evaluate the immune-related eRNA models and signature score to predict the response to immunotherapy in thyroid carcinoma.

BACKGROUND: The functional alterations of eRNAs have been reported to be correlated with tumorigenesis. However, the roles of eRNAs in thyroid cancer (THCA) remain still unclear. This study aimed to construct an immune-related eRNA prognostic signature that could effectively predict the survival and prognosis for THCA. METHODS: The Weighted Gene Co-Expression Network Analysis (WGCNA) was performed to identify THCA-specific immune-related hub genes and immune-related eRNAs were obtained using Pearson correlation analysis. Univariate and least absolute shrinkage and selection operator (LASSO) Cox regression were conducted to construct an immune-related eRNA prognostic signature in training cohort, and the predictive capability was verified in test cohort and entire cohort. Kaplan-Meier analysis, principal component analysis (PCA), receiver operating characteristic (ROC) curves, and nomogram were used to validate the risk signature. Furthermore, CIBERSORT, ESTIMATE and ssGSEA were analyzed to explore the tumor immune microenvironment (TIME) of the risk signature, and the response of potential immunotherapeutic were also discussed. RESULTS: A total of 125 immune-related eRNAs were obtained and 16 immune-related eRNAs were significantly correlated with overall survival (OS). A 9-immune-related eRNA prognostic signature was constructed, and the risk score was identified as an independent predictor. High-risk groups were associated with a poorer OS. Immune microenvironment analysis indicated that low risk score was correlated with higher immuneScore, high immune cell infiltration, and the better response of immunotherapy. Additionally, we also detected 9 immune-related eRNA expression levels in sixty-two matched tumorous and non-tumorous tissues using qRT-PCR analysis. CONCLUSION: Our immune-related eRNA risk signature that was an independent prognostic factor was strongly correlated with the immune microenvironment and may be promising for the clinical prediction of prognosis and immunotherapeutic responses in THCA patients.

Evolution of intra-tumoral heterogeneity across different pathological stages in papillary thyroid carcinoma.

BACKGROUND: Intra-tumor heterogeneity (ITH) results from the continuous accumulation of mutations during disease progression, thus impacting patients' clinical outcome. How the ITH evolves across papillary thyroid carcinoma (PTC) different tumor stages is lacking. METHODS: We used the whole-exome sequencing data from The Cancer Genome Atlas Thyroid Cancer (TCGA-THCA) cohort to track the ITH and assessed its relationship with clinical features through different stages of the PTC progression. We further assayed the expression levels of the specific genes in papillary thyroid cancer cell lines compared to an immortalized normal thyroid epithelial cell line by qRT-PCR. RESULTS: We revealed the timing of mutational processes and the dynamics of the temporal acquisition of somatic events during the lifetime of the PTC. ITH significantly influences the PTC patient's survival rate and, as genetic heterogeneity increases, the prognosis gets worse in advanced tumor stages. ITH also affects the mutational architecture of each clinical stage which is subject to periodic fluctuations. Different mutational processes may cooperate to shape a stage-specific mutational spectrum during the progression from early to advanced tumor stages. Moreover, different evolutionary paths characterize PTC progression across pathological stages due to both mutations recurrently occurring in all stages in hotspot positions and distinct codon changes dominating in different stages. A different expression level of specific genes also exists in different thyroid cancer cell lines. CONCLUSIONS: Our findings suggest ITH as a potential unfavorable prognostic factor in PTC and highlight the dynamic changes in different clinical stages of PTC, providing some clues for the precision medicine and suggesting different diagnostic decisions depending on the clinical stages of patients. Finally, complete clear guidelines to define risk stratification of PTC patients are lacking; thus, this work could contribute to defining patients who need more aggressive treatments and, in turn, could reduce the social burden of this cancer.

Three novel transcription factors involved in cannabinoid biosynthesis in Cannabis sativa L.

KEY MESSAGE: Three novel transcription factors were successfully identified and shown to interact with the trichome-specific THCAS promoter regulatory region. Cannabinoids are important secondary metabolites present in Cannabis sativa L. (cannabis). One cannabinoid that has received considerable attention, 9-tetrahydrocannabinol (THC), is derived from Delta-9-Tetrahydrocannabinolic acid (THCA) and responsible for the mood-altering and pain-relieving effects of cannabis. A detailed understanding of transcriptional control of THCA synthase (THCAS) is currently lacking. The primary site of cannabinoid biosynthesis is the glandular trichomes that form on female flowers. Transcription factors (TFs) have been shown to play an important role in secondary-metabolite biosynthesis and glandular trichome formation in Artemisia annua, Solanum lycopersicum and Humulus lupulus. However, analogous information is not available for cannabis. Here, we characterize a 548 bp fragment of the THCAS promoter and regulatory region that drives trichome-specific expression. Using this promoter fragment in a yeast-one-hybrid screen, we identified 3 novel TFs (CsAP2L1, CsWRKY1 and CsMYB1) and provided evidence that these 3 TFs regulate the THCAS promoter in planta. The O-Box element within the proximal region of the THCAS promoter is necessary for CsAP2L1-induced transcriptional activation of THCAS promoter. Similar to THCAS, the genes for all three TFs have trichome-specific expression, and subcellular localization of the TFs indicates that all three proteins are in the nucleus. CsAP2L1 and THCAS exhibit a similar temporal, spatial and strain-specific gene expression profiles, while those expression patterns of CsWRKY1 and CsMYB1 are opposite from THCAS. Our results identify CsAP2L1 playing a positive role in the regulation of THCAS expression, while CsWRKY1 and CsMYB1 may serve as negative regulators of THCAS expression.

Identification and validation of a pyroptosis-related prognostic signature for thyroid cancer.

BACKGROUND: Pyroptosis is a form of programmed cell death triggered by inflammasomes. However, the roles of pyroptosis-related genes in thyroid cancer (THCA) remain still unclear. OBJECTIVE: This study aimed to construct a pyroptosis-related signature that could effectively predict THCA prognosis and survival. METHODS: A LASSO Cox regression analysis was performed to build a prognostic model based on the expression profile of each pyroptosis-related gene. The predictive value of the prognostic model was validated in the internal cohort. RESULTS: A pyroptosis-related signature consisting of four genes was constructed to predict THCA prognosis and all patients were classified into high- and low-risk groups. Patients with a high-risk score had a poorer overall survival (OS) than those in the low-risk group. The area under the curve (AUC) of the receiver operator characteristic (ROC) curves assessed and verified the predictive performance of this signature. Multivariate analysis showed the risk score was an independent prognostic factor. Tumor immune cell infiltration and immune status were significantly higher in low-risk groups, which indicated a better response to immune checkpoint inhibitors (ICIs). Of the four pyroptosis-related genes in the prognostic signature, qRT-PCR detected three of them with significantly differential expression in THCA tissues. CONCLUSION: In summary, our pyroptosis-related risk signature may have an effective predictive and prognostic capability in THCA. Our results provide a potential foundation for future studies of the relationship between pyroptosis and the immunotherapy response.

Similarities and differences upon binding of naturally occurring Δ9-tetrahydrocannabinol-derivatives to cannabinoid CB1 and CB2 receptors.

We have here assessed, using Δ9-tetrahydrocannabinol (Δ9-THC) for comparison, the effect of Δ9-tetrahydrocannabinolic acid (Δ9-THCA) and of Δ9-tetrahydrocannabivarin (Δ9-THCV) that is mediated by human versions of CB1, CB2, and CB1-CB2 receptor functional units, expressed in a heterologous system. Binding to the CB1 and CB2 receptors was addressed in living cells by means of a homogeneous assay. A biphasic competition curve for the binding to the CB2 receptor, was obtained for Δ9-THCV in cells expressing the two receptors. Signaling studies included cAMP level determination, activation of the mitogen-activated protein kinase pathway and ß-arrestin recruitment were performed. The signaling triggered by Δ9-THCA and Δ9-THCV via individual receptors or receptor heteromers disclosed differential bias, i.e. the bias observed using a given phytocannabinoid depended on the receptor (CB1, CB2 or CB1-CB2) and on the compound used as reference to calculate the bias factor (Δ9-THC, a selective agonist or a non-selective agonist). These results are consistent with different binding modes leading to differential functional selectivity depending on the agonist structure, and the state (monomeric or heteromeric) of the cannabinoid receptor. In addition, on studying Gi-coupling we showed that Δ9-THCV and Δ9-THCA and Δ9-THCV were able to revert the effect of a selective CB2 receptor agonist, but only Δ9-THCV, and not Δ9-THCA, reverted the effect of arachidonyl-2'-chloroethylamide (ACEA 100 nM) a selective agonist of the CB1 receptor. Overall, these results indicate that cannabinoids may have a variety of binding modes that results in qualitatively different effects depending on the signaling pathway that is engaged upon cannabinoid receptor activation.

Good or not good: Role of miR-18a in cancer biology.

miR-18a is a member of primary transcript called miR-17-92a (C13orf25 or MIR17HG) which also contains five other miRNAs: miR-17, miR-19a, miR-20a, miR-19b and miR-92a. This cluster as a whole shows specific characteristics, where miR-18a seems to be unique. In contrast to the other members, the expression of miR-18a is additionally controlled and probably functions as its own internal controller of the cluster. miR-18a regulates many genes involved in proliferation, cell cycle, apoptosis, response to different kinds of stress, autophagy and differentiation. The disturbances of miR-18a expression are observed in cancer as well as in different diseases or pathological states. The miR-17-92a cluster is commonly described as oncogenic and it is known as 'oncomiR-1', but this statement is a simplification because miR-18a can act both as an oncogene and a suppressor. In this review we summarize the current knowledge about miR-18a focusing on its regulation, role in cancer biology and utility as a potential biomarker.

Identification of candidate aberrantly methylated and differentially expressed genes in thyroid cancer.

Aberrant methylation of DNA sequences plays a criticle role in finding novel aberrantly methylated genes and pathways in thyroid cancer (THCA). This study aimed to integrate three cohorts profile datasets to find novel aberrantly methylated genes and pathways in THCA. Data of gene expression profiling microarrays (GSE33630 and GSE65144) and gene methylation profiling microarrays (GSE51090) were downloaded from the Gene Expression Omnibus database. Aberrantly methylated and differentially expressed genes were sorted and pathways were analyzed. Functional and enrichment analyses of selected genes were performed using the String database. A protein-protein interaction network was constructed using the Cytoscape software, and module analysis was performed using Molecular Complex detection. In total, we identified 12 hypomethylation/high-expression genes and 30 hypermethylation/low-expression genes at the screening step and, finally, found 6 mostly changed hub genes including PPARGC1A, CREBBP, EP300, CD44, SPP1, and MMP9. Pathway analysis showed that aberrantly methylated differentially expressed genes were mainly associated with the thyroid hormone signaling pathway, AMP-activated protein kinase (AMPK) signaling pathway, and cell cycle process in THCA. After validation in the Cancer Genome Atlas database, the methylation and expression status of hub genes was significantly altered and the same with our results. Taken together, we identified novel aberrantly methylated genes and pathways in THCA, which could improve our understanding of the cause and underlying molecular events, and these candidate genes could serve as aberrant methylation-based biomarkers for precise diagnosis and treatment of THCA.

Chemical fingerprinting of single glandular trichomes of Cannabis sativa by Coherent anti-Stokes Raman scattering (CARS) microscopy.

BACKGROUND: Cannabis possesses a rich spectrum of phytochemicals i.e. cannabinoids, terpenes and phenolic compounds of industrial and medicinal interests. Most of these high-value plant products are synthesised in the disk cells and stored in the secretory cavity in glandular trichomes. Conventional trichome analysis was so far based on optical microscopy, electron microscopy or extraction based methods that are either limited to spatial or chemical information. Here we combine both information to obtain the spatial distribution of distinct secondary metabolites on a single-trichome level by applying Coherent anti-Stokes Raman scattering (CARS), a microspectroscopic technique, to trichomes derived from sepals of a drug- and a fibre-type. RESULTS: Hyperspectral CARS imaging in combination with a nonlinear unmixing method allows to identify and localise Δ9-tetrahydrocannabinolic acid (THCA) in the secretory cavity of drug-type trichomes and cannabidiolic acid (CBDA)/myrcene in the secretory cavity of fibre-type trichomes, thus enabling an easy discrimination between high-THCA and high-CBDA producers. A unique spectral fingerprint is found in the disk cells of drug-type trichomes, which is most similar to cannabigerolic acid (CBGA) and is not found in fibre-type trichomes. Furthermore, we differentiate between different cell types by a combination of CARS with simultaneously acquired two-photon fluorescence (TPF) of chlorophyll a from chloroplasts and organic fluorescence mainly arising from cell walls enabling 3D visualisation of the essential oil distribution and cellular structures. CONCLUSION: Here we demonstrate a label-free and non-destructive method to analyse the distribution of secondary metabolites and distinguish between different cell and chemo-types with high spatial resolution on a single trichome. The record of chemical fingerprints of single trichomes offers the possibility to optimise growth conditions as well as guarantee a direct process control for industrially cultivated medicinal Cannabis plants. Moreover, this method is not limited to Cannabis related issues but can be widely implemented for optimising and monitoring all kinds of natural or biotechnological production processes with simultaneous spatial and chemical information.

Identification of Cannabis sativa L. using the 1-kbTHCA synthase-fluorescence in situ hybridization probe.

This study reports a successful application of fluorescence in situ hybridization (FISH) technique in the identification of Cannabis sativa L. cells recovered from fresh and dried powdered plant materials. Two biotin-16-dUTP-labeled FISH probes were designed from the Cannabis-specific tetrahydrocannabinolic acid synthase (THCAS) gene and the ITS region of the 45S rRNA gene. Specificity of probe-target hybridization was tested against the target and 4 non-target plant species, i.e., Humulus lupulus, Mitragyna speciosa, Papaver sp., and Nicotiana tabacum. The 1-kb THCA synthase hybridization probe gave Cannabis-specific hybridization signals, unlike the 700-bp Cannabis-ITS hybridization probe. Probe-target hybridization was also confirmed against 20 individual Cannabis plant samples. The 1-kb THCA synthase and 700-bp Cannabis-ITS hybridization probes clearly showed 2 hybridization signals per cell with reproducibility. The 1-kb THCA synthase probe did not give any FISH signal when tested against H. lupulus, its closely related member of the Canabaceae family. It was also showed that 1-kb THCA synthase FISH probe can be applied to identify small amount of dried powdered Cannabis material with an addition of rehydration step prior to the experimental process. This study provided an alternative identification method for Cannabis trace.

Anoikis-related gene CDKN2A predicts prognosis and immune response and mediates proliferation and migration in thyroid carcinoma.

Thyroid carcinoma (THCA) is a tumor commonly occurring in the endocrine system, and its incidence rate is increasing yearly. Anoikis is a type of cell death involved in the carcinogenesis process. This study aimed to investigate the prognosis and immune correlations of anoikis in THCA. Our study used several bioinformatics algorithms (co-expression analysis, univariate and multivariate Cox analysis) to screen anoikis-related genes (ARGs) to construct risk models. Through receiver operating characteristic (ROC) curve, nomogram, and independent prognostic analysis found that the constructed model had ideal predictive value for THCA. The consensus clustering method was used to divide ARG patterns into three subgroups, and there were significant differences in survival among the three subgroups. The CIBERSORT algorithm demonstrated strong correlations among immune infiltrating cells, prognostic genes, and risk scores. Univariate and multivariate Cox analysis showed that CDKN2A is an independent prognostic gene. Basic experiments (immunohistochemistry, qRT-PCR, etc.) showed that the expression levels of CDKN2A mRNA and protein were highly expressed in THCA, which was consistent with the results of bioinformatics analysis. In vitro, the knockdown of CDKN2A significantly inhibited the proliferation and migration of THCA cells. In summary, our study utilized eight ARGs to construct an accurate risk model. ARGs, especially CDKN2A, play a crucial role in the occurrence and development of THCA and can become potential targets for treating THCA patients.

NOLC1 was identified as a tumor suppressor gene in thyroid cancer and correlated with prognosis by bioinformatics.

Thyroid cancer (THCA) is the most common endocrine malignancy, mainly affecting women's unilateral glandular lobes. However, for relapsed and distant metastasis of THCA patients, the existing early diagnosis and treatment methods were still insufficient, and a new method was urgently needed to diagnose and treat them. Nucleolar and coiled-body phosphoprotein 1 (NOLC1) was one of the most phosphorylated proteins in the cell, which was located mainly in the nucleolus. In addition, more and more studies have confirmed that NOLC1 plays a crucial role in various pathological processes, such as the occurrence and progression of cancer and viral infection. A previous study showed that NOLC1, as a member of RNA-binding protein, was significantly correlated with the prognosis of THCA patients. However, further exploration of NOLC1 in THCA is limited. To further explore the role of NOLC1 in THCA, we conducted expression and survival prognosis analysis of NOLC1 using multiple databases. We also evaluated the correlation between NOLC1 gene expression and clinical characteristics of THCA patients. Furthermore, we analyzed the relationship between NOLC1 and other genes, followed by enrichment analysis to investigate its metabolic pathways and molecular metabolism processes. Additionally, we examined the association between immune cell infiltration in tumor microenvironment and NOLC1. Notably, through vitro experiments, we confirmed the tumor suppressive effect of NOLC1 on the proliferation and migration of human THCA cells, providing evidence for clinical diagnosis of THCA. Furthermore, we confirmed the tumor suppressive effect of NOLC1 in vivo xenograft assay. To sum up, our results suggest that NOLC1 is a tumor suppressor gene for THCA.

Does the Quantification of Δ9-Tetrahydrocannabinolic Acid A in Serum/Plasma Provide Any Additional Information About Consumption Pattern from Drivers Under the Influence of Cannabis?

Introduction: Δ9-tetrahydrocannabinolic acid A (THCA-A) is one of the main ingredients of cannabis plants and is converted to the psychoactive substance Δ9-tetrahydrocannabinol (THC) by decarboxylation during heating above ∼90°C. During the consumption of cannabis, a varying proportion of THCA-A is absorbed into the body. Therefore, the quantification of THCA-A in serum/plasma might provide additional information on consumption behavior in driving under the influence of cannabis cases. Materials and Methods: In this study, an already established gas-chromatography mass-spectrometry (GC-MS) method for the quantification of THC, 11-OH-THC, and THC-COOH in serum and plasma samples was extended to include THCA-A. This validated method was then applied to 1228 routinely achieved serum/plasma samples from drivers suspected of cannabis consumption in Western Saxony. Two different grouping systems for chronic/occasional consumption, one system for acute/subacute consumption, Huestis formulas, and the cannabis influence factor (CIF) were used for evaluation. Results: Method validation showed appropriate results for forensic toxicological routine analysis. Limit of detection and lower limit of quantification (LLOQ) for THCA-A were 0.3 and 1.0 ng/mL, respectively. Reproducibility was <11% and accuracy ranged between 104% and 107%. THCA-A was stable in native samples at least for 2 weeks at room temperature or 4°C as well as 1 month at -20°C. Freeze-thaw stability for three cycles and processed sample stability over 3 days was proven. A total of 865 cases with a THC concentration above the German analytical cutoff of 1 ng/mL as well as the analytical LLOQs of 0.9 and 2.5 ng/mL for 11-OH-THC and THC-COOH, respectively, were included in further statistical analysis. In 407 (47.1%) of these samples, THCA-A was quantifiable. Different statistical analyses indicated a correlation between THCA-A and THC concentrations in cases of chronic and acute consumption. In addition, an increase of chronic and acute cases with increasing THCA-A concentrations was observed. However, no correlation between THCA-A and CIF was found. Discussion: These data show that THCA-A might be an additional indicative marker to provide information about consumption frequency and acuteness. Additional studies with known consumption frequencies and times are required to verify these findings.

Quantification of sixteen cannabinoids in hemp seed edible oils and the influence of thermal processing on cannabinoid contents and profiles.

To investigate cannabinoid content and profiles, 16 cannabinoids were quantified in 30 commercial hemp seed edible oils. In addition, one hemp seed oil was subjected to thermal processing up to 200 °C for up to 60 min. UHPLC-MS/MS was used for analysis. The content of cannabinoids in the samples ranged from 9 to 279 mg kg-1 (sum) and for Δ9-tetrahydrocannabinol (Δ9-THC) from 0.2 to 6.7 mg kg-1. Three samples exceeded the EU Δ9-THC equivalent maximum levels of 7.5 mg kg-1 for hemp seed oils. Cannabinoid profiles can provide indications of different product characteristics (e.g. degree of processing, variety of plant material). Furthermore, intense thermal processing (200 °C, 60 min) led to 38% decrease in sum cannabinoid content (sum of all analysed cannabinoids in this study), 99% decrease in cannabinoid acids, and 22% increase in Δ9-THC.

Delta-9-Tetrahydrocannabinolic Acid B: A Mechanism for its Formation in Cannabis.

There appears to be consensus among Cannabis biologists that delta-9-tetrahydrocannabinolic acid A (THCA-A) is the exclusive product of the enzyme THCA synthase. This then leaves an open question for formation of the THCA-A structural isomer, delta-9-tetrahydrocannabinolic acid B (THCA-B), discovered as a minor product in Cannabis in 1969. With no reasonable biochemical pathway to explain the presence of THCA-B in Cannabis, a synthetic route was next considered. Using established literature precedent, a photochemical mechanism has been proposed for the conversion of THCA-A to cannabidiolic acid (CBDA), followed by conversion of CBDA to THCA-B employing bond-breaking and bond-forming reactions.

NKD2 Trigger NF-κB Signaling Pathway and Facilitates Thyroid Cancer Cell Proliferation.

NKD inhibitor of WNT signaling pathway 2 (NKD2) is an emerging player in cancer onset and progression. Here, it was confirmed that THCA patients have robustly expressed NKD2, which was linked to an advanced pathologic stage. The prognosis was worse for those with high NKD2 levels. Functionally, ectopically produced NKD2 promotes THCA cell proliferation, whereas NKD2 knockdown impairs the ability of THCA cells to proliferate. Mechanically, ectopically expressed NKD2 activated NF-κB transcriptional activity, whereas NKD2-deficient THCA cells showed lower NF-κB transcriptional activity. As a result, NKD2 activates the NF-κB signaling pathway, encouraging the growth of THCA cells.

The prognostic and immune significance of C15orf48 in pan-cancer and its relationship with proliferation and apoptosis of thyroid carcinoma.

Background: C15orf48 was recently identified as an inflammatory response-related gene; however there is limited information on its function in tumors. In this study, we aimed to elucidate the function and potential mechanism of action of C15orf48 in cancer. Methods: We evaluated the pan-cancer expression, methylation, and mutation data of C15orf48 to analyze its clinical prognostic value. In addition, we explored the pan-cancer immunological characteristics of C15orf48, especially in thyroid cancer (THCA), by correlation analysis. Additionally, we conducted a THCA subtype analysis of C15orf48 to determine its subtype-specific expression and immunological characteristics. Lastly, we evaluated the effects of C15orf48 knockdown on the THCA cell line, BHT101, by in vitro experimentation. Results: The results of our study revealed that C15orf48 is differentially expressed in different cancer types and that it can serve as an independent prognostic factor for glioma. Additionally, we found that the epigenetic alterations of C15orf48 are highly heterogeneous in several cancers and that its aberrant methylation and copy number variation are associated with poor prognosis in multiple cancers. Immunoassays elucidated that C15orf48 was significantly associated with macrophage immune infiltration and multiple immune checkpoints in THCA, and was a potential biomarker for PTC. In addition, cell experiments showed that the knockdown of C15orf48 could reduce the proliferation, migration, and apoptosis abilities of THCA cells. Conclusions: The results of this study indicate that C15orf48 is a potential tumor prognostic biomarker and immunotherapy target, and plays an essential role in the proliferation, migration, and apoptosis of THCA cells.