Sex-Specific Expression of Histone Lysine Demethylases (KDMs) in Thyroid Cancer.

BACKGROUND: The incidence of thyroid cancer in women is 3-4-fold higher than in men. To characterize sex-specific molecular alterations in thyroid cancer, we examined the expression of sex-biased genes in normal thyroids and thyroid tumors. METHODS: Ingenuity pathways analysis was used to define sex-biased gene networks using data from the Cancer Genome Atlas (TCGA). Confirmatory studies were performed through the analysis of histone lysine demethylases (KDMs) expression by real-time PCR and immunostaining. RESULTS: In normal thyroids, 44 sex-biased genes were comparatively upregulated in male and 28 in female patients. The expressions of 37/72 (51%) sex-biased genes were affected in cancer tissues compared with normal thyroids. Gene network analyses revealed sex-specific patterns in the expressions of KDM5C, KDM5D, and KDM6A. In confirmatory studies, KDM5D mRNA and protein were detected only in males, whereas KDM5C and KDM6A were detected in samples from male and female patients. Nuclear staining with anti-KDMs was found in normal thyroids, but a loss of nuclear expression with a concomitant gain of cytoplasmic staining was observed in cancer tissues. CONCLUSIONS: Normal thyroids have a sex-specific molecular signature, and the development of thyroid cancer is associated with a differential expression of sex-biased genes. The sex-specific expression of KDMs, coupled with cancer-related alterations in their intracellular localization, may contribute to mechanisms underlying sex differences in thyroid tumorigenesis.

FixNCut: single-cell genomics through reversible tissue fixation and dissociation.

The use of single-cell technologies for clinical applications requires disconnecting sampling from downstream processing steps. Early sample preservation can further increase robustness and reproducibility by avoiding artifacts introduced during specimen handling. We present FixNCut, a methodology for the reversible fixation of tissue followed by dissociation that overcomes current limitations. We applied FixNCut to human and mouse tissues to demonstrate the preservation of RNA integrity, sequencing library complexity, and cellular composition, while diminishing stress-related artifacts. Besides single-cell RNA sequencing, FixNCut is compatible with multiple single-cell and spatial technologies, making it a versatile tool for robust and flexible study designs.

Novel Approach for Pancreas Transcriptomics Reveals the Cellular Landscape in Homeostasis and Acute Pancreatitis.

BACKGROUND & AIMS: Acinar cells produce digestive enzymes that impede transcriptomic characterization of the exocrine pancreas. Thus, single-cell RNA-sequencing studies of the pancreas underrepresent acinar cells relative to histological expectations, and a robust approach to capture pancreatic cell responses in disease states is needed. We sought to innovate a method that overcomes these challenges to accelerate study of the pancreas in health and disease. METHODS: We leverage FixNCut, a single-cell RNA-sequencing approach in which tissue is reversibly fixed with dithiobis(succinimidyl propionate) before dissociation and single-cell preparation. We apply FixNCut to an established mouse model of acute pancreatitis, validate findings using GeoMx whole transcriptome atlas profiling, and integrate our data with prior studies to compare our method in both mouse and human pancreas datasets. RESULTS: FixNCut achieves unprecedented definition of challenging pancreatic cells, including acinar and immune populations in homeostasis and acute pancreatitis, and identifies changes in all major cell types during injury and recovery. We define the acinar transcriptome during homeostasis and acinar-to-ductal metaplasia and establish a unique gene set to measure deviation from normal acinar identity. We characterize pancreatic immune cells, and analysis of T-cell subsets reveals a polarization of the homeostatic pancreas toward type-2 immunity. We report immune responses during acute pancreatitis and recovery, including early neutrophil infiltration, expansion of dendritic cell subsets, and a substantial shift in the transcriptome of macrophages due to both resident macrophage activation and monocyte infiltration. CONCLUSIONS: FixNCut preserves pancreatic transcriptomes to uncover novel cell states during homeostasis and following pancreatitis, establishing a broadly applicable approach and reference atlas for study of pancreas biology and disease.

CHEK2 Founder Variants and Thyroid Cancer Risk.

Background: Germline pathogenic variants in CHEK2 are associated with a moderate increase in the lifetime risk for breast cancer. Increased risk for other cancers, including non-medullary thyroid cancer (NMTC), has also been suggested. To date, data implicating CHEK2 variants in NMTC predisposition primarily derive from studies within Poland, driven by a splice site variant (c.444 + 1G>A) that is uncommon in other populations. In contrast, the predominant CHEK2 variants in non-Polish populations are c.1100del and c.470T>C/p.I157T, representing 61.1% and 63.8%, respectively, of all CHEK2 pathogenic variants in two large U.S.-based commercial laboratory datasets. To further delineate the impact of common CHEK2 variants on thyroid cancer, we aimed to investigate the association of three CHEK2 founder variants (c.444 + 1G>A, c.1100del, and c.470T>C/p.Ile157Thr) on NMTC susceptibility in three groups of unselected NMTC patients. Methods: The presence of three CHEK2 founder variants was assessed within three groups: (1) 1544 NMTC patients (and 1593 controls) from previously published genome-wide association study (GWAS) analyses, (2) 789 NMTC patients with germline exome sequencing (Oncology Research Information Exchange Network [ORIEN] Avatar), and (3) 499 NMTC patients with germline sequence data available in The Cancer Genome Atlas (TCGA). A case-control study design was utilized with odds ratios (ORs) calculated by comparison of all three groups with the Ohio State University GWAS control group. Results: The predominant Polish variant (c.444 + 1G>A) was present in only one case. The proportion of patients with c.1100del was 0.92% in the GWAS group, 1.65% in the ORIEN Avatar group, and 0.80% in the TCGA group. The ORs (with 95% confidence intervals [CIs]) for NMTC associated with c.1100del were 1.71 (0.73-4.29), 2.64 (0.95-7.63), and 2.5 (0.63-8.46), respectively. The proportion of patients with c.470T>C/p.I157T was 0.91% in the GWAS group, 0.76% in the ORIEN Avatar group, and 0.80% in the TCGA group, respectively. The ORs (with CIs) for NMTC associated with c.470T>C/p.I157T were 1.75 (0.74-4.39), 1.52 (0.42-4.96), and 2.31 (0.58-7.90), respectively. Conclusions: Our analyses of unselected patients with NMTC suggest that CHEK2 variants c.1100del and c.470T>C/p.I157T have only a modest impact on thyroid cancer risk. These results provide important information for providers regarding the relatively low magnitude of thyroid cancer risk associated with these CHEK2 variants.

Variation in CD8 T cell IFNγ differentiation to strains of Toxoplasma gondii is characterized by small effect QTLs with contribution from ROP16.

Introduction: Toxoplasma gondii induces a strong CD8 T cell response characterized by the secretion of IFNγ that promotes host survival during infection. The initiation of CD8 T cell IFNγ responses in vitro differs widely between clonal lineage strains of T. gondii, in which type I strains are low inducers, while types II and III strains are high inducers. We hypothesized this phenotype is due to a polymorphic "Regulator Of CD8 T cell Response" (ROCTR). Methods: Therefore, we screened F1 progeny from genetic crosses between the clonal lineage strains to identify ROCTR. Naïve antigen-specific CD8 T cells (T57) isolated from transnuclear mice, which are specific for the endogenous and vacuolar TGD057 antigen, were measured for their ability to become activated, transcribe Ifng and produce IFNγ in response to T. gondii infected macrophages. Results: Genetic mapping returned four non-interacting quantitative trait loci (QTL) with small effect on T. gondii chromosomes (chr) VIIb-VIII, X and XII. These loci encompass multiple gene candidates highlighted by ROP16 (chrVIIb-VIII), GRA35 (chrX), TgNSM (chrX), and a pair of uncharacterized NTPases (chrXII), whose locus we report to be significantly truncated in the type I RH background. Although none of the chromosome X and XII candidates bore evidence for regulating CD8 T cell IFNγ responses, type I variants of ROP16 lowered Ifng transcription early after T cell activation. During our search for ROCTR, we also noted the parasitophorous vacuole membrane (PVM) targeting factor for dense granules (GRAs), GRA43, repressed the response suggesting PVM-associated GRAs are important for CD8 T cell activation. Furthermore, RIPK3 expression in macrophages was an absolute requirement for CD8 T cell IFNγ differentiation implicating the necroptosis pathway in T cell immunity to T. gondii. Discussion: Collectively, our data suggest that while CD8 T cell IFNγ production to T. gondii strains vary dramatically, it is not controlled by a single polymorphism with strong effect. However, early in the differentiation process, polymorphisms in ROP16 can regulate commitment of responding CD8 T cells to IFNγ production which may have bearing on immunity to T. gondii.

Both maternal IFNγ exposure and acute prenatal infection with Toxoplasma gondii activate fetal hematopoietic stem cells.

Infection directly influences adult hematopoietic stem cell (HSC) function and differentiation, but the fetal hematopoietic response to infection during pregnancy is not well-studied. Here, we investigated the fetal hematopoietic response to maternal infection with Toxoplasma gondii (T. gondii), an intracellular parasite that elicits Type II IFNγ-mediated maternal immunity. While it is known that maternal infection without direct pathogen transmission can affect fetal immune development, the effects of maternal IFNγ on developing HSCs and the signals that mediate these interactions have not been investigated. Our investigation reveals that the fetal HSCs respond to T. gondii infection with virulence-dependent changes in proliferation, self-renewal potential, and lineage output. Furthermore, maternal IFNγ crosses the fetal-maternal interface, where it is perceived by fetal HSCs. By comparing the effects of maternal IFNγ injection with maternal T. gondii infection, we reveal that the effects of IFNγ treatment mimic some aspects of the fetal HSC response to infection. Moreover, our findings illuminate that the fetal HSC response to prenatal infection is distinct from the adult HSC response to IFNγ-induced inflammation. Altogether, our data disentangle the role of infection-induced inflammatory cytokines in driving the expansion of downstream hematopoietic progenitors.

T cell exhaustion dynamics in systemic autoimmune disease.

Unlike in infection and cancer, T cell exhaustion in autoimmune disease has not been clearly defined. Here we set out to understand inhibitory protein (PD-1, Tim3, CTLA4, Lag3) expression in CXCR5- and CXCR5+ CD8 and CD4 T cells in systemic lupus erythematosus. CXCR5+ CD8 and CD4 T cells express PD-1 and engage B cells in germinal center reactions, leading to autoantibody formation in autoimmunity. We hypothesized that CXCR5+ CD8 T cells develop an exhausted phenotype as SLE autoimmunity expands from initial to chronic, self-perpetuating disease due to chronic self-antigen exposure. Our results indicate that there is no exhaustion frequency differences between sexes, although disease kinetics vary by sex. CXCR5+ CD8 T cells express primarily IFNγ, known to promote autoimmune disease development, whereas CXCR5-CD8 T cells express TNFα and IFNγ as disease progresses from 2-6 months. Tim3 is the highest expressed inhibitory marker for all CD4 and CD8 T cell populations demonstrating potential for terminally exhausted populations. CTLA4 expression on CD4 T cells suggests potential tolerance induction in these cells. We identified exhaustion phenotypes within autoimmune disease that progress with increasing lupus erythematosus severity and possibly provide a feedback mechanism for immunological tolerance. Highlights: CXCR5- and CXCR5+ CD8 T cells expand with rate of disease in SLE mouse model.CXCR5+ CD8 T cells are low contributors to TNFα disease progression unlike CXCR5-CD8 T cells but may increase disease mechanisms through high IFNγ production.Inhibitory markers upregulate in frequency with the highest amounts seen in Tim3+ populations. Tim3+Lag3+ expression may be an indicator of terminal differentiation for all populations.Inhibitory marker expression frequency was unrelated to sex.

Inadvertent Radiation Exposures in Combat Zones: Risk of Contamination and Radiobiologic Consequences.

On February 24, 2022, Russia began a military invasion of Ukraine. Missile and air strikes were reported throughout the country, shortly followed by a large ground invasion from multiple directions. Four major theaters developed: the Kyiv offensive, the Northeastern Ukraine offensive, the Eastern Ukraine offensive, and the Southern Ukraine offensive, with continued missile and air strikes far into Western Ukraine. Advancing Russian military units launched an attack and captured the Chernobyl nuclear station. Russian troops dug trenches into the area commonly known as the "Red Forest," violating the established radiation safety measures and threatening security within the Chernobyl Exclusion Zone. The placement of military units in such close proximity to the station also sparked concerns of possible damage occurring to the containment vessel constructed around the station's wrecked fourth reactor. There are 15 operating nuclear reactors in Ukraine. Each is vulnerable to an attack or sabotage that could precipitate a malfunction and possible release of radioactive isotopes. In this short commentary, we will discuss radiobiologic data obtained after the analysis of historical nuclear power plant (NPP) accidents and emphasize new challenges for nuclear security when NPPs are found and are possible targets within a conflict zone.

Forward Genetics in Apicomplexa Biology: The Host Side of the Story.

Forward genetic approaches have been widely used in parasitology and have proven their power to reveal the complexities of host-parasite interactions in an unbiased fashion. Many aspects of the parasite's biology, including the identification of virulence factors, replication determinants, antibiotic resistance genes, and other factors required for parasitic life, have been discovered using such strategies. Forward genetic approaches have also been employed to understand host resistance mechanisms to parasitic infection. Here, we will introduce and review all forward genetic approaches that have been used to identify host factors involved with Apicomplexa infections, which include classical genetic screens and QTL mapping, GWAS, ENU mutagenesis, overexpression, RNAi and CRISPR-Cas9 library screens. Collectively, these screens have improved our understanding of host resistance mechanisms, immune regulation, vaccine and drug designs for Apicomplexa parasites. We will also discuss how recent advances in molecular genetics give present opportunities to further explore host-parasite relationships.

Impaired Glucose Metabolism, Anti-Diabetes Medications, and Risk of Thyroid Cancer.

The prevalence of obesity is progressively increasing along with the potential high risk for insulin resistance and development of type 2 diabetes mellitus. Obesity is associated with increased risk of many malignancies, and hyperinsulinemia has been proposed to be a link between obesity and cancer development. The incidence of thyroid cancer is also increasing, making this cancer the most common endocrine malignancy. There is some evidence of associations between obesity, insulin resistance and/or diabetes with thyroid proliferative disorders, including thyroid cancer. However, the etiology of such an association has not been fully elucidated. The goal of the present work is to review the current knowledge on crosstalk between thyroid and glucose metabolic pathways and the effects of obesity, insulin resistance, diabetes, and anti-hyperglycemic medications on the risk of thyroid cancer development.

Fast pesticide analysis using low-pressure gas chromatography paired with a triple quadrupole mass spectrometer equipped with short collision cell technology.

RATIONALE: A proof of concept showing GC-MS/MS analysis time for pesticides can be dramatically reduced while maintaining a similar separation efficiency by combining a low-pressure gas chromatography (LPGC) column with the enhanced selected reaction monitoring (SRM) switching speed of the short collision cell of a JEOL JMS-TQ4000GC. METHODS: Triple-quadrupole tandem mass spectrometry (standard EI + at 70 eV) was used to measure pesticides eluting from a low-pressure gas chromatograph capillary column. Three transitions for each of 244 pesticide compounds were measured within an 11-min analysis time, and the data were checked to confirm the method's reproducibility and ability to distinguish all three transitions for each pesticide. RESULTS: All three transitions for all 244 pesticides were detected in the standard mixture at 1X concentration within the 11-min analysis time. Relative standard deviation (RSD) of peak areas was less than 15% for 242 pesticides, and I/Q RSDs were less than 10% for 242 compounds. Retention time RSD over 15 replicates was less than 0.1%. CONCLUSIONS: Results show that analysis time can be markedly decreased using an LPGC column, and that the ability of the short collision cell to distinguish a large number of coeluting peaks makes the two technologies a natural pairing. The effective measurement of pesticides within a short time could benefit any scientists doing pesticide analysis work.

Eukaryotic elongation factor 2 kinase regulates foam cell formation via translation of CD36.

Eukaryotic elongation factor 2 kinase (eEF2K) is an atypical protein kinase that controls protein synthesis in cells under stress. Although well studied in cancer, less is known about its roles in chronic inflammatory diseases. Here, we examined its regulation of macrophage cholesterol handling in the context of atherosclerosis. eEF2K mRNA expression and protein activity were upregulated in murine bone marrow-derived macrophages (BMDMs) exposed to oxidized low-density lipoprotein cholesterol (oxLDL). When incubated with oxLDL, BMDMs from eEF2K knockout (Eef2k-/- ) mice formed fewer Oil Red O+ foam cells than Eef2k+/+ BMDMs (12.5% ± 2.3% vs. 32.3% ± 2.0%, p < .01). Treatment with a selective eEF2K inhibitor, JAN-384, also decreased foam cell formation for C57BL/6J BMDMs and human monocyte-derived macrophages. Disabling eEF2K selectively decreased protein expression of the CD36 cholesterol uptake receptor, mediated by a reduction in the proportion of translationally active Cd36 mRNA. Eef2k-/- mice bred onto the Ldlr-/- background developed aortic sinus atherosclerotic plaques that were 30% smaller than Eef2k+/+ -Ldlr-/- mice after 16 weeks of high cholesterol diet (p < .05). Although accompanied by a reduction in plaque CD36+ staining (p < .05) and lower CD36 expression in circulating monocytes (p < .01), this was not associated with reduced lipid content in plaques as measured by oil red O staining. Finally, EEF2K and CD36 mRNA levels were higher in blood mononuclear cells from patients with coronary artery disease and recent myocardial infarction compared to healthy controls without coronary artery disease. These results reveal a new role for eEF2K in translationally regulating CD36 expression and foam cell formation in macrophages. Further studies are required to explore therapeutic targeting of eEF2K in atherosclerosis.

Eviction notice served on Toxoplasma.

The gene RARRES3 uses an unexpected strategy to eliminate the parasite Toxoplasma gondii from human cells.

Genetic mapping reveals Nfkbid as a central regulator of humoral immunity to Toxoplasma gondii.

Protective immunity to parasitic infections has been difficult to elicit by vaccines. Among parasites that evade vaccine-induced immunity is Toxoplasma gondii, which causes lethal secondary infections in chronically infected mice. Here we report that unlike susceptible C57BL/6J mice, A/J mice were highly resistant to secondary infection. To identify correlates of immunity, we utilized forward genetics to identify Nfkbid, a nuclear regulator of NF-κB that is required for B cell activation and B-1 cell development. Nfkbid-null mice ("bumble") did not generate parasite-specific IgM and lacked robust parasite-specific IgG, which correlated with defects in B-2 cell maturation and class-switch recombination. Though high-affinity antibodies were B-2 derived, transfer of B-1 cells partially rescued the immunity defects observed in bumble mice and were required for 100% vaccine efficacy in bone marrow chimeric mice. Immunity in resistant mice correlated with robust isotype class-switching in both B cell lineages, which can be fine-tuned by Nfkbid gene expression. We propose a model whereby humoral immunity to T. gondii is regulated by Nfkbid and requires B-1 and B-2 cells for full protection.

capCLIP: a new tool to probe translational control in human cells through capture and identification of the eIF4E-mRNA interactome.

Translation of eukaryotic mRNAs begins with binding of their m7G cap to eIF4E, followed by recruitment of other translation initiation factor proteins. We describe capCLIP, a novel method to comprehensively capture and quantify the eIF4E (eukaryotic initiation factor 4E) 'cap-ome' and apply it to examine the biological consequences of eIF4E-cap binding in distinct cellular contexts. First, we use capCLIP to identify the eIF4E cap-omes in human cells with/without the mTORC1 (mechanistic target of rapamycin, complex 1) inhibitor rapamycin, there being an emerging consensus that rapamycin inhibits translation of TOP (terminal oligopyrimidine) mRNAs by displacing eIF4E from their caps. capCLIP reveals that the representation of TOP mRNAs in the cap-ome is indeed systematically reduced by rapamycin, thus validating our new methodology. capCLIP also refines the requirements for a functional TOP sequence. Second, we apply capCLIP to probe the consequences of phosphorylation of eIF4E. We show eIF4E phosphorylation reduces overall eIF4E-mRNA association and, strikingly, causes preferential dissociation of mRNAs with short 5'-UTRs. capCLIP is a valuable new tool to probe the function of eIF4E and of other cap-binding proteins such as eIF4E2/eIF4E3.

Do Molecular Profiles of Primary Versus Metastatic Radioiodine Refractory Differentiated Thyroid Cancer Differ?

Management of metastatic radioiodine refractory differentiated thyroid cancer (DTC) can be a therapeutic challenge. Generally, little is known about the paired molecular profile of the primary tumor and the metastases and whether they harbor the same genetic abnormalities. The present study compared the molecular profile of paired tumor specimens (primary tumor/metastatic sites) from patients with radioiodine refractory DTC in order to gain insight into a possible basis for resistance to radioiodine. Twelve patients with radioiodine refractory metastases were studied; median age at diagnosis of 61 years (range, 25-82). Nine patients had papillary TC (PTC), one had follicular TC (FTC), and two had Hürthle cell TC (HTC). Distant metastases were present in the lungs (n = 10), bones (n = 4), and liver (n = 1). The molecular profiling of paired tumors was performed with a panel of 592 genes for Next Generation Sequencing, RNA-sequencing, and immunohistochemistry. Digital microfluidic PCR was used to investigate TERT promoter mutations. The genetic landscape of all paired sites comprised BRAF, NRAS, HRAS, TP53, ATM, MUTYH, POLE, and NTRK genes, including BRAF and NTRK fusions. BRAF V600E was the most common point mutation in the paired specimens (5/12). TERT promoter mutation C228T was detected in one case. PD-L1 expression at metastatic sites was highly positive (95%) for one patient with HTC. All specimens were stable for microsatellite instability testing, and the tumor mutation burden was low to intermediate. Therefore, the molecular profile of DTC primary and metastatic lesions can show heterogeneity, which may help explain some altered responses to therapeutic intervention.

Reciprocal signaling between mTORC1 and MNK2 controls cell growth and oncogenesis.

eIF4E plays key roles in protein synthesis and tumorigenesis. It is phosphorylated by the kinases MNK1 and MNK2. Binding of MNKs to eIF4G enhances their ability to phosphorylate eIF4E. Here, we show that mTORC1, a key regulator of mRNA translation and oncogenesis, directly phosphorylates MNK2 on Ser74. This suppresses MNK2 activity and impairs binding of MNK2 to eIF4G. These effects provide a novel mechanism by which mTORC1 signaling impairs the function of MNK2 and thereby decreases eIF4E phosphorylation. MNK2[S74A] knock-in cells show enhanced phosphorylation of eIF4E and S6K1 (i.e., increased mTORC1 signaling), enlarged cell size, and increased invasive and transformative capacities. MNK2[Ser74] phosphorylation was inversely correlated with disease progression in human prostate tumors. MNK inhibition exerted anti-proliferative effects in prostate cancer cells in vitro. These findings define a novel feedback loop whereby mTORC1 represses MNK2 activity and oncogenic signaling through eIF4E phosphorylation, allowing reciprocal regulation of these two oncogenic pathways.

Bicuculline regulated protein synthesis is dependent on Homer1 and promotes its interaction with eEF2K through mTORC1-dependent phosphorylation.

The regulation of protein synthesis is a vital and finely tuned process in cellular physiology. In neurons, this process is very precisely regulated, as which mRNAs undergo translation is highly dependent on context. One of the most prominent regulators of protein synthesis is the enzyme eukaryotic elongation factor kinase 2 (eEF2K) that regulates the elongation stage of protein synthesis. This kinase and its substrate, eukaryotic elongation factor 2 (eEF2) are important in processes such as neuronal development and synaptic plasticity. eEF2K is regulated by multiple mechanisms including Ca2+ -ions and the mTORC1 signaling pathway, both of which play key roles in neurological processes such as learning and memory. In such settings, the localized control of protein synthesis is of crucial importance. In this work, we sought to investigate how the localization of eEF2K is controlled and the impact of this on protein synthesis in neuronal cells. In this study, we used both SH-SY5Y neuroblastoma cells and mouse cortical neurons, and pharmacologically and/or genetic approaches to modify eEF2K function. We show that eEF2K activity and localization can be regulated by its binding partner Homer1b/c, a scaffolding protein known for its participation in calcium-regulated signaling pathways. Furthermore, our results indicate that this interaction is regulated by the mTORC1 pathway, through a known phosphorylation site in eEF2K (S396), and that it affects rates of localized protein synthesis at synapses depending on the presence or absence of this scaffolding protein.