Heterogeneity of cancer stem cell-related marker expression is associated with three-dimensional structures in malignant pleural effusion produced by lung adenocarcinoma.

INTRODUCTION: Cancer stem cells have been described in lung adenocarcinoma-associated malignant pleural effusion. They show clinically important features, including the ability to initiate new tumours and resistance to treatments. However, their correlation with the three-dimensional tumour structures in the effusion is not well understood. METHODS: Cell blocks produced from lung adenocarcinoma patients' pleural effusion were examined for cancer stem cell-related markers Nanog and CD133 using immunocytochemistry. The three-dimensional cancer cell structures and CD133 expression patterns were visualized with tissue-clearing technology. The expression patterns were correlated with tumour cell structures, genetic variants and clinical outcomes. RESULTS: Thirty-nine patients were analysed. Moderate-to-strong Nanog expression was detected in 27 cases (69%), while CD133 was expressed by more than 1% of cancer cells in 11 cases (28%). Nanog expression was more homogenous within individual specimens, while CD133 expression was detected in single tumour cells or cells within small clusters instead of larger structures in 8 of the 11 positive cases (73%). Although no statistically significant correlation between the markers and tumour genetic variants or patient survival was observed, we recorded seven cases with follow-up specimens after cancer treatment, and four (57%) showed a change in stem cell-related marker expression corresponding to treatment response. CONCLUSIONS: Lung adenocarcinoma cells in the pleural effusion show variable expression of cancer stem cell-related markers, some showing a correlation with the size of cell clusters. Their expression level is potentially correlated with cancer treatment effects.

Genomic Profiling With Large-Scale Next-Generation Sequencing Panels Distinguishes Separate Primary Lung Adenocarcinomas From Intrapulmonary Metastases.

The distinction between different separate primary lung cancers (SPLCs) and intrapulmonary metastases (IPMs) is a challenging but clinically significant issue. Histopathology-based classification is the current practice; however, it is subjective and affected by interobserver variability. Recently, next-generation sequencing (NGS) panels have been used in lung cancer diagnostics. This study aimed to investigate the value of large-scale NGS panels for distinguishing between SPLCs and IPMs. A total of 32 patients with 69 lung adenocarcinomas were included. Comprehensive histopathologic assessments of multiple pulmonary adenocarcinomas were performed independently by 3 pathologists. The consensus of histopathologic classification was determined by a majority vote. Genomic analysis was performed using an amplicon-based large-scale NGS panel, targeting single-nucleotide variants and short insertions and deletions in 409 genes. Tumor pairs were classified as SPLCs or IPMs according to a predefined molecular classification algorithm. Using NGS and our molecular classification algorithm, 97.6% of the tumor pairs can be unambiguously classified as SPLCs or IPMs. The molecular classification was predictive of postoperative clinical outcomes in terms of overall survival (P = .015) and recurrence-free interval (P = .0012). There was a moderate interobserver agreement regarding histopathologic classification (κ = 0.524 at the tumor pair level). The concordance between histopathologic and molecular classification was 100% in cases where pathologists reached a complete agreement but only 53.3% where they did not. This study showed that large-scale NGS panels are a powerful modality that can help distinguish SPLCs from IPMs in patients with multiple lung adenocarcinomas and objectively provide accurate risk stratification.

Application of artificial intelligence to stereotactic radiosurgery for intracranial lesions: detection, segmentation, and outcome prediction.

BACKGROUND: Rapid evolution of artificial intelligence (AI) prompted its wide application in healthcare systems. Stereotactic radiosurgery served as a good candidate for AI model development and achieved encouraging result in recent years. This article aimed at demonstrating current AI application in radiosurgery. METHODS: Literatures published in PubMed during 2010-2022, discussing AI application in stereotactic radiosurgery were reviewed. RESULTS: AI algorithms, especially machine learning/deep learning models, have been administered to different aspect of stereotactic radiosurgery. Spontaneous tumor detection and automated lesion delineation or segmentation were two of the promising application, which could be further extended to longitudinal treatment follow-up. Outcome prediction utilized machine learning algorithms with radiomic-based analysis was another well-established application. CONCLUSIONS: Stereotactic radiosurgery has taken a lead role in AI development. Current achievement, limitation, and further investigation was summarized in this article.

Stereotactic radiosurgery for pituitary and cavernous sinus metastases.

PURPOSE: Metastases extending to the pituitary gland and cavernous sinus are extremely rare; however, advances in neuroimaging have increased the reported incidence. Stereotactic radiosurgery (SRS) affords the precise delivery of focused radiation to minimize adverse radiation effects. This study assessed the efficacy and safety of SRS in the treatment of pituitary and cavernous sinus metastases. METHODS: Analysis was performed on 23 patients with pituitary and cavernous sinus metastases who underwent treatment using SRS between 1996 and 2021. The cohort was categorized into 2 groups in terms of metastasis location: pituitary involvement (Group 1, n = 11) and cavernous sinus involvement (Group 2, n = 12). Overall survival, local tumor control, and distal tumor control rates were compared between the two groups using Kaplan-Meier analysis. RESULTS: The median age of the cohort was 52.2 years and the median tumor volume was 4.5 mL. Overall survival rates were as follows: 1 year (72.9%), 2 years (51.8%), and 3 years (45.3%). Local tumor control rates were as follows: 1 year (82.3%), 2 years (82.3%), and 3 years (65.9%). Visual deficit and hypopituitarism were the most common presentations in Group 1, whereas cranial nerve deficit was the most common presentation in Group 2. CONCLUSIONS: SRS appears to be a safe and effective therapy for the treatment of pituitary and cavernous sinus metastases. GKRS is a relatively simple procedure, which places minimal stress on the patient, thereby facilitating further anti-cancer treatment. Considering the limited survival duration in cases of metastasis, it is very likely that post-GKRS complications (e.g., new onset cranial nerve deficit and hypopituitarism) would not become an issue before patient passes away.

A mosaic analysis system with Cre or Tomato expression in the mouse.

Somatic mutations are major genetic contributors to cancers and many other age-related diseases. Many disease-causing somatic mutations can initiate clonal growth prior to the appearance of any disease symptoms, yet experimental models that can be used to examine clonal abnormalities are limited. We describe a mosaic analysis system with Cre or Tomato (MASCOT) for tracking mutant cells and demonstrate its utility for modeling clonal hematopoiesis. MASCOT can be induced to constitutively express either Cre-GFP or Tomato for lineage tracing of a mutant and a reference group of cells simultaneously. We conducted mosaic analysis to assess functions of the Id3 and/or Tet2 gene in hematopoietic cell development and clonal hematopoiesis. Using Tomato-positive cells as a reference population, we demonstrated the high sensitivity of this system for detecting cell-intrinsic phenotypes during short-term or long-term tracking of hematopoietic cells. Long-term tracking of Tet2 mutant or Tet2/Id3 double-mutant cells in our MASCOT model revealed a dynamic shift from myeloid expansion to lymphoid expansion and subsequent development of lymphoma. This work demonstrates the utility of the MASCOT method in mosaic analysis of single or combined mutations, making the system suitable for modeling somatic mutations identified in humans.

A Promiscuity Locus Confers Lotus burttii Nodulation with Rhizobia from Five Different Genera.

Legumes acquire access to atmospheric nitrogen through nitrogen fixation by rhizobia in root nodules. Rhizobia are soil-dwelling bacteria and there is a tremendous diversity of rhizobial species in different habitats. From the legume perspective, host range is a compromise between the ability to colonize new habitats, in which the preferred symbiotic partner may be absent, and guarding against infection by suboptimal nitrogen fixers. Here, we investigate natural variation in rhizobial host range across Lotus species. We find that Lotus burttii is considerably more promiscuous than Lotus japonicus, represented by the Gifu accession, in its interactions with rhizobia. This promiscuity allows Lotus burttii to form nodules with Mesorhizobium, Rhizobium, Sinorhizobium, Bradyrhizobium, and Allorhizobium species that represent five distinct genera. Using recombinant inbred lines, we have mapped the Gifu/burttii promiscuity quantitative trait loci (QTL) to the same genetic locus regardless of rhizobial genus, suggesting a general genetic mechanism for symbiont-range expansion. The Gifu/burttii QTL now provides an opportunity for genetic and mechanistic understanding of promiscuous legume-rhizobia interactions. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Computer-assisted three-dimensional quantitation of programmed death-ligand 1 in non-small cell lung cancer using tissue clearing technology.

Immune checkpoint blockade therapy has revolutionized non-small cell lung cancer treatment. However, not all patients respond to this therapy. Assessing the tumor expression of immune checkpoint molecules, including programmed death-ligand 1 (PD-L1), is the current standard in predicting treatment response. However, the correlation between PD-L1 expression and anti-PD-1/PD-L1 treatment response is not perfect. This is partly caused by tumor heterogeneity and the common practice of assessing PD-L1 expression based on limited biopsy material. To overcome this problem, we developed a novel method that can make formalin-fixed, paraffin-embedded tissue translucent, allowing three-dimensional (3D) imaging. Our protocol can process tissues up to 150 µm in thickness, allowing anti-PD-L1 staining of the entire tissue and producing high resolution 3D images. Compared to a traditional 4 µm section, our 3D image provides 30 times more coverage of the specimen, assessing PD-L1 expression of approximately 10 times more cells. We further developed a computer-assisted PD-L1 quantitation method to analyze these images, and we found marked variation of PD-L1 expression in 3D. In 5 of 33 needle-biopsy-sized specimens (15.2%), the PD-L1 tumor proportion score (TPS) varied by greater than 10% at different depth levels. In 14 cases (42.4%), the TPS at different depth levels fell into different categories (< 1%, 1-49%, or ≥ 50%), which can potentially influence treatment decisions. Importantly, our technology permits recovery of the processed tissue for subsequent analysis, including histology examination, immunohistochemistry, and mutation analysis. In conclusion, our novel method has the potential to increase the accuracy of tumor PD-L1 expression assessment and enable precise deployment of cancer immunotherapy.

The whole-genome landscape of Burkitt lymphoma subtypes.

Burkitt lymphoma (BL) is an aggressive, MYC-driven lymphoma comprising 3 distinct clinical subtypes: sporadic BLs that occur worldwide, endemic BLs that occur predominantly in sub-Saharan Africa, and immunodeficiency-associated BLs that occur primarily in the setting of HIV. In this study, we comprehensively delineated the genomic basis of BL through whole-genome sequencing (WGS) of 101 tumors representing all 3 subtypes of BL to identify 72 driver genes. These data were additionally informed by CRISPR screens in BL cell lines to functionally annotate the role of oncogenic drivers. Nearly every driver gene was found to have both coding and non-coding mutations, highlighting the importance of WGS for identifying driver events. Our data implicate coding and non-coding mutations in IGLL5, BACH2, SIN3A, and DNMT1. Epstein-Barr virus (EBV) infection was associated with higher mutation load, with type 1 EBV showing a higher mutational burden than type 2 EBV. Although sporadic and immunodeficiency-associated BLs had similar genetic profiles, endemic BLs manifested more frequent mutations in BCL7A and BCL6 and fewer genetic alterations in DNMT1, SNTB2, and CTCF. Silencing mutations in ID3 were a common feature of all 3 subtypes of BL. In vitro, mass spectrometry-based proteomics demonstrated that the ID3 protein binds primarily to TCF3 and TCF4. In vivo knockout of ID3 potentiated the effects of MYC, leading to rapid tumorigenesis and tumor phenotypes consistent with those observed in the human disease.

Diffuse Intratumoral Stromal Inflammation in Ovarian Clear Cell Carcinoma is Associated With Loss of Mismatch Repair Protein and High PD-L1 Expression.

Ovarian clear cell carcinoma (OCCC) is an aggressive chemotherapy-resistant cancer with limited treatment options, and some OCCCs have mismatch repair (MMR) deficiency (MMRD). Emerging evidence has revealed that various cancers with MMRD are susceptible to anti-programmed death-1/programmed death ligand-1 (anti-PD-1/PD-L1) immunotherapy, and certain histologic features are associated with MMRD. However, few studies have addressed this in OCCC. We reviewed 76 OCCCs for tumor-associated inflammation (intratumoral stromal inflammation and peritumoral lymphocytes) and performed immunohistochemistry for 4 MMR proteins and PD-L1. MMR-deficient OCCCs were analyzed for microsatellite instability (MSI), and those with MLH1 loss were tested for MLH1 promoter methylation. No patients fulfilled the Amsterdam II criteria for the diagnosis of Lynch syndrome. Four (5.3%) tumors showed diffuse intratumoral stromal inflammation obliterating the tumor-stroma interfaces, and none had peritumoral lymphoid aggregates. MMRD was found in 2 (2.6%) tumors; one had MLH1/PMS2 loss (MSI-high and MLH1 promoter methylation was detected) and the other had MSH2/MSH6 loss (MSI-low). Twenty (26.3%) tumors showed tumoral PD-L1 expression ≥1%. Both MMR-deficient tumors showed diffuse intratumoral stromal inflammation and tumoral PD-L1 expression ≥50%. Three of the 4 (75%) tumors with diffuse intratumoral stromal inflammation also showed tumoral PD-L1 expression ≥50%. None of the tumors without diffuse intratumoral stromal inflammation showed MMRD (P=0.021) or tumoral PD-L1 expression ≥50% (P=0.0001). We identified a strong correlation among diffuse intratumoral stromal inflammation, MMRD, and high tumoral PD-L1 expression in a small but significant subset of OCCCs. Histologic evaluation can facilitate patient selection for subsequent anti-PD-1/PD-L1 immunotherapy.

Gene Expression Profile in Primary Tumor Is Associated with Brain-Tropism of Metastasis from Lung Adenocarcinoma.

Lung adenocarcinoma has a strong propensity to metastasize to the brain. The brain metastases are difficult to treat and can cause significant morbidity and mortality. Identifying patients with increased risk of developing brain metastasis can assist medical decision-making, facilitating a closer surveillance or justifying a preventive treatment. We analyzed 27 lung adenocarcinoma patients who received a primary lung tumor resection and developed metastases within 5 years after the surgery. Among these patients, 16 developed brain metastases and 11 developed non-brain metastases only. We performed targeted DNA sequencing, RNA sequencing and immunohistochemistry to characterize the difference between the primary tumors. We also compared our findings to the published data of brain-tropic and non-brain-tropic lung adenocarcinoma cell lines. The results demonstrated that the targeted tumor DNA sequencing did not reveal a significant difference between the groups, but the RNA sequencing identified 390 differentially expressed genes. A gene expression signature including CDKN2A could identify 100% of brain-metastasizing tumors with a 91% specificity. However, when compared to the differentially expressed genes between brain-tropic and non-brain-tropic lung cancer cell lines, a different set of genes was shared between the patient data and the cell line data, which include many genes implicated in the cancer-glia/neuron interaction. Our findings indicate that it is possible to identify lung adenocarcinoma patients at the highest risk for brain metastasis by analyzing the primary tumor. Further investigation is required to elucidate the mechanism behind these associations and to identify potential treatment targets.

Repeated gamma knife radiosurgery enables longer tumor control in cases of highly-recurrent intracranial ependymoma.

PURPOSE: Stereotactic radiosurgery (SRS) is a potential re-irradiation treatment for recurrent intracranial ependymoma after prior radiation therapy. The purpose of this study was to examine the efficacy and safety of repeated SRS in the treatment of recurrent intracranial ependymomas. METHODS: This is a retrospective study of consecutive patients with residual or recurrent intracranial ependymomas who were treated with SRS between 1993 and 2018. Tumor progression was defined as a ≥ 10% increase in tumor volume. Tumor regression was defined as a ≥ 10% reduction in tumor volume. A tumor that remained within 10% of its original volume was defined as stable. Tumor control comprised tumor regression and stability. Time-dependent analyses were performed using two treatment failure endpoint definitions: (1) evidence of local tumor progression or distant metastasis (single SRS analysis), and (2) lack of tumor response to SRS (repeated SRS analysis). These analyses were adjusted for the competing risk of death. RESULTS: The study comprised 37 patients (65 intracranial ependymomas) who underwent multiple SRS sessions (range: 1-7). Median age was 10.2 years (range: 0.8-53.8 years), and median tumor volume was 1.5 mL (range: 0.01-22.5 mL). The median radiation dose was 13.3 Gy (range: 7.9-22.0 Gy) at a median isodose line of 57% (range: 50-90%). Overall tumor control rates in the single SRS analysis adjusting for the competing risk of death were 53.6%, 30.5%, and 23.6% at 1, 3, and 5 years, respectively. Overall tumor control rates in the repeated SRS analysis adjusting for the competing risk of death were 70.6%, 50.4%, and 43.1% at 1, 3, and 5 years, respectively. Prior gross total resection was the only independent predictor of overall tumor control after SRS (aHR = 25.62 (1.55-422.1), p = 0.02). CONCLUSIONS: Repeated GKRS appeared to be an effective treatment strategy for recurrent or residual intracranial ependymomas, with acceptable complication rates.

Novel regulation of PKC-induced inflammation by Akt and protein phosphatase 2A in ovarian granulosa cells.

PKC-mediated inflammation is important in ovarian physiology. The roles of Akt and protein phosphatase 2A (PP2A) in PKC-mediated inflammation in ovarian granulosa cells (GCs) remain mostly unclear. PKC activator phorbol 12-myristate 13-acetate induced the Akt phosphorylation in rat primary GCs but reduced the Akt phosphorylation in KGN human GCs. In rat GCs, an inhibitory effect of PI3K inhibitor wortmannin and a stimulatory effect of Akt activator SC79 on PKC-induced cyclooxygenase-2 (COX-2)/PGE2production were noted; wortmannin and SC79 acted oppositely in human GCs. In rat GCs, PP2A inhibitor okadaic acid further enhanced the PKC-mediated promoter activation and elevation of mRNA and protein levels of the COX-2 gene, whereas PP2A activator sodium selenate attenuated the PKC-mediated COX-2 expression and promoter activation. PKC activation did not affect PP2A phosphorylation, but okadaic acid indeed augmented the PKC-induced NF-κB nuclear translocation. Thus, PP2A appears to act as a negative modulator in PKC-mediated cellular inflammation in rat GCs, at least in part due to its attenuating effect on the PKC-induced NF-κB activation.

A subcompatible rhizobium strain reveals infection duality in Lotus.

Lotus species develop infection threads to guide rhizobia into nodule cells. However, there is evidence that some species have a genetic repertoire to allow other modes of infection. By conducting confocal and electron microscopy, quantification of marker gene expression, and phenotypic analysis of transgenic roots infected with mutant rhizobia, we elucidated the infection mechanism used by Rhizobium leguminosarum Norway to colonize Lotus burttii. Rhizobium leguminosarum Norway induces a distinct host transcriptional response compared with Mesorhizobium loti. It infects L. burttii utilizing an epidermal and transcellular infection thread-independent mechanism at high frequency. The entry into plant cells occurs directly from the apoplast and is primarily mediated by 'peg'-like structures, the formation of which is dependent on the production of Nod factor by the rhizobia. These results demonstrate that Lotus species can exhibit duality in their infection mechanisms depending on the rhizobial strain that they encounter. This is especially relevant in the context of interactions in the rhizosphere where legumes do not encounter single strains, but complex rhizobial communities. Additionally, our findings support a perception mechanism at the nodule cell entry interface, reinforcing the idea that there are successive checkpoints during rhizobial infection.

Evolution of microRNA827 targeting in the plant kingdom.

Unlike most ancient microRNAs, which conservatively target homologous genes across species, microRNA827 (miR827) targets two different types of SPX (SYG1/PHO81/XPR1)-domain-containing genes, NITROGEN LIMITATION ADAPTATION (NLA) and PHOSPHATE TRANSPORTER 5 (PHT5), in Arabidopsis thaliana and Oryza sativa to regulate phosphate (Pi) transport and storage, respectively. However, how miR827 shifted its target preference and its evolutionary history are unknown. Based on target prediction analysis, we found that in most angiosperms, miR827 conservatively targets PHT5 homologs, but in Brassicaceae and Cleomaceae it preferentially targets NLA homologs, and we provide evidence for the transition of target preference during Brassicales evolution. Intriguingly, we found a lineage-specific loss of the miR827-regulatory module in legumes. Analysis of miR827-mediated cleavage efficiency and the expression of PHT5 in A. thaliana indicated that accumulation of mutations in the target site and the exclusion of the target site by alternative transcriptional initiation eliminated PHT5 targeting by miR827. Here, we identified a transition of miR827 target preference during plant evolution and revealed the uniqueness of miR827-mediated regulation among conserved plant miRNAs. Despite the change in its target preference, upregulation of miR827 by Pi starvation and its role in regulating cellular Pi homeostasis were retained.

Haloperidol Abrogates Matrix Metalloproteinase-9 Expression by Inhibition of NF-κB Activation in Stimulated Human Monocytic Cells.

Much evidence has indicated that matrix metalloproteinases (MMPs) participate in the progression of neuroinflammatory disorders. The present study was undertaken to investigate the inhibitory effect and mechanism of the antipsychotic haloperidol on MMP activation in the stimulated THP-1 monocytic cells. Haloperidol exerted a strong inhibition on tumor necrosis factor- (TNF-) α-induced MMP-9 gelatinolysis of THP-1 cells. A concentration-dependent inhibitory effect of haloperidol was observed in TNF-α-induced protein and mRNA expression of MMP-9. On the other hand, haloperidol slightly affected cell viability and tissue inhibition of metalloproteinase-1 levels. It significantly inhibited the degradation of inhibitor-κB-α (IκBα) in activated cells. Moreover, it suppressed activated nuclear factor-κB (NF-κB) detected by a mobility shift assay, NF-κB reporter gene, and chromatin immunoprecipitation analyses. Consistent with NF-κB inhibition, haloperidol exerted a strong inhibition of lipopolysaccharide- (LPS-) induced MMP-9 gelatinolysis but not of transforming growth factor-ß1-induced MMP-2. In in vivo studies, administration of haloperidol significantly attenuated LPS-induced intracerebral MMP-9 activation of the brain homogenate and the in situ in C57BL/6 mice. In conclusion, the selective anti-MMP-9 activation of haloperidol could possibly involve the inhibition of the NF-κB signal pathway. Hence, it was found that haloperidol treatment may represent a bystander of anti-MMP actions for its conventional psychotherapy.

MicroRNA396-Targeted SHORT VEGETATIVE PHASE Is Required to Repress Flowering and Is Related to the Development of Abnormal Flower Symptoms by the Phyllody Symptoms1 Effector.

Leafy flowers are the major symptoms of peanut witches' broom (PnWB) phytoplasma infection in Catharanthus roseus. The orthologs of the phyllody symptoms1 (PHYL1) effector of PnWB from other species of phytoplasma can trigger the proteasomal degradation of several MADS box transcription factors, resulting in leafy flower formation. In contrast, the flowering negative regulator gene SHORT VEGETATIVE PHASE (SVP) was up-regulated in PnWB-infected C. roseus plants, but most microRNA (miRNA) genes had repressed expression. Coincidentally, transgenic Arabidopsis (Arabidopsis thaliana) plants expressing the PHYL1 gene of PnWB (PHYL1 plants), which show leafy flower phenotypes, up-regulate SVP of Arabidopsis (AtSVP) but repress a putative regulatory miRNA of AtSVP, miR396. However, the mechanism by which PHYL1 regulates AtSVP and miR396 is unknown, and the evidence of miR396-mediated AtSVP degradation is lacking. Here, we show that miR396 triggers AtSVP messenger RNA (mRNA) decay using genetic approaches, a reporter assay, and high-throughput degradome profiles. Genetic evidence indicates that PHYL1 plants and atmir396a-1 mutants have higher AtSVP accumulation, whereas the transgenic plants overexpressing MIR396 display lower AtSVP expression. The reporter assay indicated that target-site mutation results in decreasing the miR396-mediated repression efficiency. Moreover, degradome profiles revealed that miR396 triggers AtSVP mRNA decay rather than miRNA-mediated cleavage, implying that AtSVP caused miR396-mediated translation inhibition. We hypothesize that PHYL1 directly or indirectly interferes with miR396-mediated AtSVP mRNA decay and synergizes with other effects (e.g. MADS box transcription factor degradation), resulting in abnormal flower formation. We anticipate our findings to be a starting point for studying the posttranscriptional regulation of PHYL1 effectors in symptom development.

Id3 and Id2 act as a dual safety mechanism in regulating the development and population size of innate-like γδ T cells.

The innate-like T cells expressing Vγ1.1 and Vδ6.3 represent a unique T cell lineage sharing features with both the γδ T and the invariant NKT cells. The population size of Vγ1.1(+)Vδ6.3(+) T cells is tightly controlled and usually contributes to a very small proportion of thymic output, but the underlying mechanism remains enigmatic. Deletion of Id3, an inhibitor of E protein transcription factors, can induce an expansion of the Vγ1.1(+)Vδ6.3(+) T cell population. This phenotype is much stronger on the C57BL/6 background than on the 129/sv background. Using quantitative trait linkage analysis, we identified Id2, a homolog of Id3, to be the major modifier of Id3 in limiting Vγ1.1(+)Vδ6.3(+) T cell expansion. The Vγ1.1(+)Vδ6.3(+) phenotype is attributed to an intrinsic weakness of Id2 transcription from Id2 C57BL/6 allele, leading to an overall reduced dosage of Id proteins. However, complete removal of both Id2 and Id3 genes in developing T cells suppressed the expansion of Vγ1.1(+)Vδ6.3(+) T cells because of decreased proliferation and increased cell death. We showed that conditional knockout of Id2 alone is sufficient to promote a moderate expansion of γδ T cells. These regulatory effects of Id2 and Id3 on Vγ1.1(+)Vδ6.3(+) T cells are mediated by titration of E protein activity, because removing one or more copies of E protein genes can restore Vγ1.1(+)Vδ6.3(+) T cell expansion in Id2 and Id3 double conditional knockout mice. Our data indicated that Id2 and Id3 collaboratively control survival and expansion of the γδ lineage through modulating a proper threshold of E proteins.

Inhibition of PKC-Induced COX-2 and IL-8 Expression in Human Breast Cancer Cells by Glucosamine.

Breast cancer is a common cancer leading to many deaths among females. Cyclooxygenase-2 (COX-2) and interleukin-8 (IL-8) are two highly expressed inflammatory mediators to be induced by the protein kinase C (PKC) signaling via various inflammatory stimuli and both contribute significantly to cancer metastasis/progression. Glucosamine has been shown to act as an anti-inflammation molecule. The aim of this study was to clarify the role and acting mechanism of glucosamine during the PKC-regulation of COX-2/IL-8 expression and the associated impact on breast cancer. In MCF-7 breast cancer cells, glucosamine effectively suppresses the PKC induction of COX-2 and IL-8 promoter activity, mRNA and protein levels, as well as the production of prostaglandin E(2) (PGE(2)) and IL-8. Glucosamine is able to promote COX-2 protein degradation in a calpain-dependent manner and IL-8 protein degradation in calpain-dependent and proteasome-dependent manners. The MAPK and NF-κB pathways are involved in PKC-induced COX-2 expression, but only the NF-κB pathway is involved in PKC-induced IL-8 expression. Glucosamine attenuates PKC-mediated IκBα phosphorylation, nuclear NF-κB translocation, and NF-κB reporter activation. Both PGE(2) and IL-8 promote cell proliferation and IL-8 induces cell migration; thus, glucosamine appears to suppress PKC-induced cell proliferation and migration. Furthermore, glucosamine significantly inhibits the growth of breast cancer xenografts and this is accompanied by a reduction in COX-2 and IL-8 expression. In conclusion, glucosamine seems to attenuate the inflammatory response in vitro and in vivo and this occurs, at least in part by targeting to the NF-κB signaling pathway, resulting in an inhibition of breast cancer cell growth.

High-throughput transcriptome analysis of the leafy flower transition of Catharanthus roseus induced by peanut witches'-broom phytoplasma infection.

Peanut witches'-broom (PnWB) phytoplasma are obligate bacteria that cause leafy flower symptoms in Catharanthus roseus. The PnWB-mediated leafy flower transitions were studied to understand the mechanisms underlying the pathogen-host interaction; however, our understanding is limited because of the lack of information on the C. roseus genome. In this study, the whole-transcriptome profiles from healthy flowers (HFs) and stage 4 (S4) PnWB-infected leafy flowers of C. roseus were investigated using next-generation sequencing (NGS). More than 60,000 contigs were generated using a de novo assembly approach, and 34.2% of the contigs (20,711 genes) were annotated as putative genes through name-calling, open reading frame determination and gene ontology analyses. Furthermore, a customized microarray based on this sequence information was designed and used to analyze samples further at various stages of PnWB infection. In the NGS profile, 87.8% of the genes showed expression levels that were consistent with those in the microarray profiles, suggesting that accurate gene expression levels can be detected using NGS. The data revealed that defense-related and flowering gene expression levels were altered in S4 PnWB-infected leafy flowers, indicating that the immunity and reproductive stages of C. roseus were compromised. The network analysis suggested that the expression levels of >1,000 candidate genes were highly associated with CrSVP1/2 and CrFT expression, which might be crucial in the leafy flower transition. In conclusion, this study provides a new perspective for understanding plant pathology and the mechanisms underlying the leafy flowering transition caused by host-pathogen interactions through analyzing bioinformatics data obtained using a powerful, rapid high-throughput technique.

Transcriptional regulator Id2 is required for the CD4 T cell immune response in the development of experimental autoimmune encephalomyelitis.

An effective immune response to Ag challenge is critically dependent on the size of the effector cell population generated from clonal activation of Ag-specific T cells. The transcription network involved in regulating the size of the effector population, particularly for CD4 Th cells, is poorly understood. In this study, we investigate the role of Id2, an inhibitor of E protein transcription factors, in the generation of CD4 effectors. Using a T cell-specific conditional Id2 knockout mouse model, we show that inhibitor of DNA binding (Id)2 is essential for the development of experimental autoimmune encephalomyelitis. Although Ag-specific and IL-17-producing CD4 T cells are produced in these mice, the activated CD4 T cells form a smaller pool of effector cells in the peripheral lymphoid organs, exhibit reduced proliferation and increased cell death, and are largely absent in the CNS. In the absence of Id2, E protein targets, including the proapoptotic protein Bim and SOCS3, are expressed at higher levels among activated CD4 T cells. This study reveals a critical role of Id2 in the control of effector CD4 T cell population size and the development of a Th17-mediated autoimmune disease.