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Pathology, Research and Practice Jun 2024Odontogenic tumors (OGTs), which originate from cells of odontogenic apparatus and their remnants, are rare entities. Primary intraosseous carcinoma NOS (PIOC), is one...
Odontogenic tumors (OGTs), which originate from cells of odontogenic apparatus and their remnants, are rare entities. Primary intraosseous carcinoma NOS (PIOC), is one of the OGTs, but it is even rarer and has a worse prognosis. The precise characteristics of PIOC, especially in immunohistochemical features and its pathogenesis, remain unclear. We characterized a case of PIOC arising from the left mandible, in which histopathological findings showed a transition from the odontogenic keratocyst to the carcinoma. Remarkably, the tumor lesion of this PIOC prominently exhibits malignant attributes, including invasive growth of carcinoma cell infiltration into the bone tissue, an elevated Ki-67 index, and lower signal for CK13 and higher signal for CK17 compared with the non-tumor region, histopathologically and immunohistopathologically. Further immunohistochemical analyses demonstrated increased expression of ADP-ribosylation factor (ARF)-like 4c (ARL4C) (accompanying expression of β-catenin in the nucleus) and yes-associated protein (YAP) in the tumor lesion. On the other hand, YAP was expressed and the expression of ARL4C was hardly detected in the non-tumor region. In addition, quantitative RT-PCR analysis using RNAs and dot blot analysis using genomic DNA showed the activation of Wnt/β-catenin signaling and epigenetic alterations, such as an increase of 5mC levels and a decrease of 5hmC levels, in the tumor lesion. A DNA microarray and a gene set enrichment analysis demonstrated that various types of intracellular signaling would be activated and several kinds of cellular functions would be altered in the pathogenesis of PIOC. Experiments with the GSK-3 inhibitor revealed that β-catenin pathway increased not only mRNA levels of ankyrin repeat domain1 (ANKRD1) but also protein levels of YAP and transcriptional co-activator with PDZ-binding motif (TAZ) in oral squamous cell carcinoma cell lines. These results suggested that further activation of YAP signaling by Wnt/β-catenin signaling may be associated with the pathogenesis of PIOC deriving from odontogenic keratocyst in which YAP signaling is activated.
PubMed: 38908335
DOI: 10.1016/j.prp.2024.155420 -
Neurobiology of Disease Jun 2024Leucine-rich repeat kinase 2 (LRRK2) is the most common gene responsible for familial Parkinson's disease (PD). The gene product of LRRK2 contains multiple protein...
Leucine-rich repeat kinase 2 (LRRK2) is the most common gene responsible for familial Parkinson's disease (PD). The gene product of LRRK2 contains multiple protein domains, including armadillo repeat, ankyrin repeat, leucine-rich repeat (LRR), Ras-of-complex (ROC), C-terminal of ROC (COR), kinase, and WD40 domains. In this study, we performed genetic screening of LRRK2 in our PD cohort, detecting sixteen LRRK2 rare variants. Among them, we selected seven variants that are likely to be familial and characterized them in terms of LRRK2 protein function, along with clinical information and one pathological analysis. The seven variants were S1120P and N1221K in the LRR domain; I1339M, S1403R, and V1447M in the ROC domain; and I1658F and D1873H in the COR domain. The kinase activity of the LRRK2 variants N1221K, S1403R, V1447M, and I1658F toward Rab10, a well-known phosphorylation substrate, was higher than that of wild-type LRRK2. LRRK2 D1873H showed enhanced self-association activity, whereas LRRK2 S1403R and D1873H showed reduced microtubule-binding activity. Pathological analysis of a patient with the LRRK2 V1447M variant was also performed, which revealed Lewy pathology in the brainstem. No functional alterations in terms of kinase activity, self-association activity, and microtubule-binding activity were detected in LRRK2 S1120P and I1339M variants. However, the patient with PD carrying LRRK2 S1120P variant also had a heterozygous Glucosylceramidase beta 1 (GBA1) L444P variant. In conclusion, we characterized seven LRRK2 variants potentially associated with PD. Five of the seven variants in different LRRK2 domains exhibited altered properties in kinase activity, self-association, and microtubule-binding activity, suggesting that each domain variant may contribute to disease progression in different ways.
PubMed: 38901781
DOI: 10.1016/j.nbd.2024.106571 -
Translational Cancer Research May 2024Cervical cancer, one of the prevalent malignancies among females, is closely associated with human papillomavirus (HPV) infection. Homologous to the E6-AP carboxyl...
BACKGROUND
Cervical cancer, one of the prevalent malignancies among females, is closely associated with human papillomavirus (HPV) infection. Homologous to the E6-AP carboxyl terminus (HECT) domain and ankyrin repeat containing E3 ubiquitin-protein ligase 1 (HACE1) plays pivotal roles in various cancers. This study aimed to elucidate the expression of HACE1 in cervical cancer and its correlation with clinical features.
METHODS
From The Cancer Genome Atlas Cervical Squamous Cell Carcinoma and Endocervical Adenocarcinoma (TCGA-CESC) and Gene Expression Omnibus (GEO, GSE6791) datasets, we obtained RNA-Seq profiles and associated clinical information. Differential gene analysis was conducted using the R "limma" package. Implications for HPV infection and the overall survival (OS) of cervical cancer were determined by performing differential expression analysis and the Cox proportional hazards regression model. Immunohistochemical analyses were used to validate the expression in cervical cancer and normal cervical tissue. Further, nomogram was constructed to predict OS in cervical cancer. Whether the model was credible was evaluated according to receiver operating characteristic (ROC) curves and concordance curves. To further evaluate the potential functions of HACE1, we conducted functional enrichment analysis. Finally, we assessed methylation levels in HPV+ and HPV- patients in the TCGA-CESC dataset.
RESULTS
Utilizing TCGA and GSE6791 datasets, we observed significant upregulation of HACE1 in cervical cancer patients, particularly linked to HPV infection. Immunohistochemical staining revealed enhanced HACE1 expression in tumor tissues. Further analysis demonstrated a significant positive correlation between elevated HACE1 and HPV-associated proteins (E1, E6, and E7). Moreover, high HACE1 expression was associated with adverse prognosis in cervical cancer patients. Multivariate Cox analysis indicated that HACE1 could serve as an independent prognostic factor. We developed a prognostic model integrating HPV subtypes, the International Federation of Gynecology and Obstetrics (FIGO) staging, and HACE1, exhibiting strong predictive efficacy for cervical cancer prognosis. Gene enrichment analysis indicated HACE1's potential involvement in multiple signaling pathways during cervical cancer progression, while the demethylation of cg03002526 in HPV-positive patients might contribute to HACE1 upregulation.
CONCLUSIONS
Our study reveals that HACE1 upregulation is associated with cervical cancer, particularly in HPV-positive patients. HACE1 emerges as an independent prognostic factor, linked to unfavorable outcomes.
PubMed: 38881936
DOI: 10.21037/tcr-23-2120 -
The Journal of Biological Chemistry Jun 2024Leucine rich repeat kinase 2 (LRRK2) is a large multidomain protein containing two catalytic domains, a kinase and a GTPase, as well as protein interactions domains,...
Leucine rich repeat kinase 2 (LRRK2) is a large multidomain protein containing two catalytic domains, a kinase and a GTPase, as well as protein interactions domains, including a WD40 domain. The association of increased LRRK2 kinase activity with both the familial and sporadic forms of Parkinson's disease (PD) has led to intense interest in determining its cellular function. However, small molecule probes that can bind to LRRK2 and report on or affect its cellular activity are needed. Here, we report the identification and characterization of the first high-affinity LRRK2-binding designed ankyrin-repeat protein (DARPin), named E11. Using cryo-EM, we show that DARPin E11 binds to the LRRK2 WD40 domain. LRRK2 bound to DARPin E11 showed improved behavior on cryo-EM grids, resulting in higher resolution LRRK2 structures. DARPin E11 did not affect the catalytic activity of a truncated form of LRRK2 in vitro but decreased the phosphorylation of Rab8A, a LRRK2 substrate, in cells. We also found that DARPin E11 disrupts the formation of microtubule-associated LRRK2 filaments in cells, which are known to require WD40-based dimerization. Thus, DARPin E11 is a new tool to explore the function and dysfunction of LRRK2 and guide the development of LRRK2 kinase inhibitors that target the WD40 domain instead of the kinase.
PubMed: 38876305
DOI: 10.1016/j.jbc.2024.107469 -
Open Forum Infectious Diseases Jun 2024The coronavirus disease 2019 (COVID-19) pandemic was characterized by rapid evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants, affecting...
The Designed Ankyrin Repeat Protein Antiviral Ensovibep for Nonhospitalized Patients With Coronavirus Disease 2019: Results From EMPATHY, a Randomized, Placebo-Controlled Phase 2 Study.
BACKGROUND
The coronavirus disease 2019 (COVID-19) pandemic was characterized by rapid evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants, affecting viral transmissibility, virulence, and response to vaccines/therapeutics. EMPATHY (NCT04828161), a phase 2 study, investigated the safety/efficacy of ensovibep, a multispecific designed ankyrin repeat protein (DARPin) with multivariant in vitro activity, in ambulatory patients with mild to moderate COVID-19.
METHODS
Nonhospitalized, symptomatic patients (N = 407) with COVID-19 were randomized to receive single-dose intravenous ensovibep (75, 225, or 600 mg) or placebo and followed until day 91. The primary endpoint was time-weighted change from baseline in log SARS-CoV-2 viral load through day 8. Secondary endpoints included proportion of patients with COVID-19-related hospitalizations, emergency room (ER) visits, and/or all-cause mortality to day 29; time to sustained clinical recovery to day 29; and safety to day 91.
RESULTS
Ensovibep showed superiority versus placebo in reducing log SARS-CoV-2 viral load; treatment differences versus placebo in time-weighted change from baseline were -0.42 ( = .002), -0.33 ( = .014), and -0.59 ( < .001) for 75, 225, and 600 mg, respectively. Ensovibep-treated patients had fewer COVID-19-related hospitalizations, ER visits, and all-cause mortality (relative risk reduction: 78% [95% confidence interval, 16%-95%]) and a shorter median time to sustained clinical recovery than placebo. Treatment-emergent adverse events occurred in 44.3% versus 54.0% of patients in the ensovibep and placebo arms; grade 3 events were consistent with COVID-19 morbidity. Two deaths were reported with placebo and none with ensovibep.
CONCLUSIONS
All 3 doses of ensovibep showed antiviral efficacy and clinical benefits versus placebo and an acceptable safety profile in nonhospitalized patients with COVID-19.
PubMed: 38854392
DOI: 10.1093/ofid/ofae233 -
Biomedicine & Pharmacotherapy =... Jul 2024Multiple myeloma (MM) progression is closely dependent on cells in the bone marrow (BM) microenvironment, including fibroblasts (FBs) and immune cells. In their BM...
Multiple myeloma (MM) progression is closely dependent on cells in the bone marrow (BM) microenvironment, including fibroblasts (FBs) and immune cells. In their BM niche, MM cells adhere to FBs sustaining immune evasion, drug resistance and the undetectable endurance of tumor cells known as minimal residual disease (MRD). Here, we describe the novel bi-specific designed ankyrin repeat protein (DARPin) α-FAPx4-1BB (MP0310) with FAP-dependent 4-1BB agonistic activity. The α-FAPx4-1BB DARPin simultaneously binds to FAP and 4-1BB overexpressed by activated FBs and immune cells, respectively. Although flow cytometry analysis showed that T and NK cells from MM patients were not activated and did not express 4-1BB, stimulation with daratumumab or elotuzumab, monoclonal antibodies (mAbs) currently used for the treatment of MM, significantly upregulated 4-1BB both in vitro and in MM patients following mAb-based therapy. The mAb-induced 4-1BB overexpression allowed the engagement of α-FAPx4-1BB that acted as a bridge between FAPFBs and 4-1BBNK cells. Therefore, α-FAPx4-1BB enhanced both the adhesion of daratumumab-treated NK cells on FBs as well as their activation by improving release of CD107a and perforin, hence MM cell killing via antibody-mediated cell cytotoxicity (ADCC). Interestingly, α-FAPx4-1BB significantly potentiated daratumumab-mediated ADCC in the presence of FBs, suggesting that it may overcome the BM FBs' immunosuppressive effect. Overall, we speculate that treatment with α-FAPx4-1BB may represent a valuable strategy to improve mAb-induced NK cell activity fostering MRD negativity in MM patients through the eradication of latent MRD cells.
Topics: Killer Cells, Natural; Multiple Myeloma; Humans; Antibodies, Monoclonal, Humanized; Antibodies, Monoclonal; Cell Line, Tumor; Tumor Necrosis Factor Receptor Superfamily, Member 9; Membrane Proteins; Endopeptidases
PubMed: 38850654
DOI: 10.1016/j.biopha.2024.116877 -
BMC Genomics Jun 2024Nitzschia closterium f. minutissima is a commonly available diatom that plays important roles in marine aquaculture. It was originally classified as Nitzschia...
BACKGROUND
Nitzschia closterium f. minutissima is a commonly available diatom that plays important roles in marine aquaculture. It was originally classified as Nitzschia (Bacillariaceae, Bacillariophyta) but is currently regarded as a heterotypic synonym of Phaeodactylum tricornutum. The aim of this study was to obtain the draft genome of the marine microalga N. closterium f. minutissima to understand its phylogenetic placement and evolutionary specialization. Given that the ornate hierarchical silicified cell walls (frustules) of diatoms have immense applications in nanotechnology for biomedical fields, biosensors and optoelectric devices, transcriptomic data were generated by using reference genome-based read mapping to identify significantly differentially expressed genes and elucidate the molecular processes involved in diatom biosilicification.
RESULTS
In this study, we generated 13.81 Gb of pass reads from the PromethION sequencer. The draft genome of N. closterium f. minutissima has a total length of 29.28 Mb, and contains 28 contigs with an N50 value of 1.23 Mb. The GC content was 48.55%, and approximately 18.36% of the genome assembly contained repeat sequences. Gene annotation revealed 9,132 protein-coding genes. The results of comparative genomic analysis showed that N. closterium f. minutissima was clustered as a sister lineage of Phaeodactylum tricornutum and the divergence time between them was estimated to be approximately 17.2 million years ago (Mya). CAFF analysis demonstrated that 220 gene families that significantly changed were unique to N. closterium f. minutissima and that 154 were specific to P. tricornutum, moreover, only 26 gene families overlapped between these two species. A total of 818 DEGs in response to silicon were identified in N. closterium f. minutissima through RNA sequencing, these genes are involved in various molecular processes such as transcription regulator activity. Several genes encoding proteins, including silicon transporters, heat shock factors, methyltransferases, ankyrin repeat domains, cGMP-mediated signaling pathways-related proteins, cytoskeleton-associated proteins, polyamines, glycoproteins and saturated fatty acids may contribute to the formation of frustules in N. closterium f. minutissima.
CONCLUSIONS
Here, we described a draft genome of N. closterium f. minutissima and compared it with those of eight other diatoms, which provided new insight into its evolutionary features. Transcriptome analysis to identify DEGs in response to silicon will help to elucidate the underlying molecular mechanism of diatom biosilicification in N. closterium f. minutissima.
Topics: Diatoms; Phylogeny; Gene Expression Profiling; Genome; Transcriptome; Molecular Sequence Annotation
PubMed: 38840265
DOI: 10.1186/s12864-024-10479-9 -
The Biological Bulletin Aug 2023AbstractWe describe the cloning and expression of a nonreceptor tyrosine kinase, (), a () gene, identified in a subtractive screen for maternal ascidian cDNAs in , an...
AbstractWe describe the cloning and expression of a nonreceptor tyrosine kinase, (), a () gene, identified in a subtractive screen for maternal ascidian cDNAs in , an ascidian species with a tadpole larva. The gene encodes a 4-kb mRNA expressed in gonads, eggs, and embryos in the tailed but is not detected in eggs or embryos of the closely related tailless species . There is a large insertion in in the genome, as shown by transcriptome and genome analyses, resulting in it becoming a pseudogene. The amino acid sequence encodes a nonreceptor tyrosine kinase with an N-terminal region containing two SH2 domains and five ankyrin repeats, similar to the gene found in other ascidians. Thus, the ascidian genes are members of the SHARK (Src-homology ankyrin-repeat containing tyrosine kinase) family of nonreceptor tyrosine kinases, which are found throughout invertebrates and missing from vertebrates. We show that is lacking the tyrosine kinase domain in the tailless , although the truncated mRNA is still expressed in transcriptome data. This maternal and zygotic tyrosine kinase is another described pseudogene from and appears not to be necessary for adult development.
Topics: Animals; Urochordata; Protein-Tyrosine Kinases; Amino Acid Sequence; Zygote; Pseudogenes; Phylogeny
PubMed: 38820291
DOI: 10.1086/730536 -
World Journal of Stem Cells May 2024Thrombocytopenia 2, an autosomal dominant inherited disease characterized by moderate thrombocytopenia, predisposition to myeloid malignancies and normal platelet size...
BACKGROUND
Thrombocytopenia 2, an autosomal dominant inherited disease characterized by moderate thrombocytopenia, predisposition to myeloid malignancies and normal platelet size and function, can be caused by 5'-untranslated region (UTR) point mutations in ankyrin repeat domain containing 26 (ANKRD26). Runt related transcription factor 1 (RUNX1) and friend leukemia integration 1 (FLI1) have been identified as negative regulators of . However, the positive regulators of are still unknown.
AIM
To prove the positive regulatory effect of GATA binding protein 2 (GATA2) on transcription.
METHODS
Human induced pluripotent stem cells derived from bone marrow (hiPSC-BM) and urothelium (hiPSC-U) were used to examine the expression pattern in the early stage of differentiation. Then, transcriptome sequencing of these iPSCs and three public transcription factor (TF) databases (Cistrome DB, animal TFDB and ENCODE) were used to identify potential TF candidates for . Furthermore, overexpression and dual-luciferase reporter experiments were used to verify the regulatory effect of the candidate TFs on . Moreover, using the GENT2 platform, we analyzed the relationship between expression and overall survival in cancer patients.
RESULTS
In hiPSC-BMs and hiPSC-Us, we found that the transcription levels of varied in the absence of RUNX1 and FLI1. We sequenced hiPSC-BM and hiPSC-U and identified 68 candidate TFs for . Together with three public TF databases, we found that GATA2 was the only candidate gene that could positively regulate . Using dual-luciferase reporter experiments, we showed that GATA2 directly binds to the 5'-UTR of and promotes its transcription. There are two identified binding sites of GATA2 that are located 2 kb upstream of the TSS of . In addition, we discovered that high expression is always related to a more favorable prognosis in breast and lung cancer patients.
CONCLUSION
We first discovered that the transcription factor GATA2 plays a positive role in transcription and identified its precise binding sites at the promoter region, and we revealed the importance of ANKRD26 in many tissue-derived cancers.
PubMed: 38817334
DOI: 10.4252/wjsc.v16.i5.538