Orphan GPCR GPRC5C Facilitates Angiotensin II-Induced Smooth Muscle Contraction.

BACKGROUND: GPCRs (G-protein-coupled receptors) play a central role in the regulation of smooth muscle cell (SMC) contractility, but the function of SMC-expressed orphan GPCR class C group 5 member C (GPRC5C) is unclear. The aim of this project is to define the role of GPRC5C in SMC in vitro and in vivo. METHODS: We studied the role of GPRC5C in the regulation of SMC contractility and differentiation in human and murine SMC in vitro, as well as in tamoxifen-inducible, SMC-specific GPRC5C knockout mice under basal conditions and in vascular disease in vivo. RESULTS: Mesenteric arteries from tamoxifen-inducible, SMC-specific GPRC5C knockout mice showed ex vivo significantly reduced angiotensin II (Ang II)-dependent calcium mobilization and contraction, whereas responses to other relaxant or contractile factors were normal. In vitro, the knockdown of GPRC5C in human aortic SMC resulted in diminished Ang II-dependent inositol phosphate production and lower myosin light chain phosphorylation. In line with this, tamoxifen-inducible, SMC-specific GPRC5C knockout mice showed reduced Ang II-induced arterial hypertension, and acute inactivation of GPRC5C was able to ameliorate established arterial hypertension. Mechanistically, we show that GPRC5C and the Ang II receptor AT1 dimerize, and knockdown of GPRC5C resulted in reduced binding of Ang II to AT1 receptors in HEK293 cells, human and murine SMC, and arteries from tamoxifen-inducible, SMC-specific GPRC5C knockout mice. CONCLUSIONS: Our data show that GPRC5C regulates Ang II-dependent vascular contraction by facilitating AT1 receptor-ligand binding and signaling.

Interploidy Introgression Shaped Adaptation during the Origin and Domestication History of Brassica napus.

Polyploidy is recurrent across the tree of life and known as an evolutionary driving force in plant diversification and crop domestication. How polyploid plants adapt to various habitats has been a fundamental question that remained largely unanswered. Brassica napus is a major crop cultivated worldwide, resulting from allopolyploidy between unknown accessions of diploid B. rapa and B. oleracea. Here, we used whole-genome resequencing data of accessions representing the majority of morphotypes and ecotypes from the species B. rapa, B. oleracea, and B. napus to investigate the role of polyploidy during domestication. To do so, we first reconstructed the phylogenetic history of B. napus, which supported the hypothesis that the emergence of B. napus derived from the hybridization of European turnip of B. rapa and wild B. oleracea. These analyses also showed that morphotypes of swede and Siberian kale (used as vegetable and fodder) were domesticated before rapeseed (oil crop). We next observed that frequent interploidy introgressions from sympatric diploids were prominent throughout the domestication history of B. napus. Introgressed genomic regions were shown to increase the overall genetic diversity and tend to be localized in regions of high recombination. We detected numerous candidate adaptive introgressed regions and found evidence that some of the genes in these regions contributed to phenotypic diversification and adaptation of different morphotypes. Overall, our results shed light on the origin and domestication of B. napus and demonstrate interploidy introgression as an important mechanism that fuels rapid diversification in polyploid species.

Gene coexpression network during ontogeny in the yellow fever mosquito, Aedes aegypti.

BACKGROUND: The behaviors and ontogeny of Aedes aegypti are closely related to the spread of diseases caused by dengue (DENV), chikungunya (CHIKV), Zika (ZIKV), and yellow fever (YFV) viruses. During the life cycle, Ae. aegypti undergoes drastic morphological, metabolic, and functional changes triggered by gene regulation and other molecular mechanisms. Some essential regulatory factors that regulate insect ontogeny have been revealed in other species, but their roles are still poorly investigated in the mosquito. RESULTS: Our study identified 6 gene modules and their intramodular hub genes that were highly associated with the ontogeny of Ae. aegypti in the constructed network. Those modules were found to be enriched in functional roles related to cuticle development, ATP generation, digestion, immunity, pupation control, lectins, and spermatogenesis. Additionally, digestion-related pathways were activated in the larvae and adult females but suppressed in the pupae. The integrated protein‒protein network also identified cilium-related genes. In addition, we verified that the 6 intramodular hub genes encoding proteins such as EcKinase regulating larval molt were only expressed in the larval stage. Quantitative RT‒PCR of the intramodular hub genes gave similar results as the RNA-Seq expression profile, and most hub genes were ontogeny-specifically expressed. CONCLUSIONS: The constructed gene coexpression network provides a useful resource for network-based data mining to identify candidate genes for functional studies. Ultimately, these findings will be key in identifying potential molecular targets for disease control.

PlaASDB: a comprehensive database of plant alternative splicing events in response to stress.

BACKGROUND: Alternative splicing (AS) is a co-transcriptional regulatory mechanism of plants in response to environmental stress. However, the role of AS in biotic and abiotic stress responses remains largely unknown. To speed up our understanding of plant AS patterns under different stress responses, development of informative and comprehensive plant AS databases is highly demanded. DESCRIPTION: In this study, we first collected 3,255 RNA-seq data under biotic and abiotic stresses from two important model plants (Arabidopsis and rice). Then, we conducted AS event detection and gene expression analysis, and established a user-friendly plant AS database termed PlaASDB. By using representative samples from this highly integrated database resource, we compared AS patterns between Arabidopsis and rice under abiotic and biotic stresses, and further investigated the corresponding difference between AS and gene expression. Specifically, we found that differentially spliced genes (DSGs) and differentially expressed genes (DEG) share very limited overlapping under all kinds of stresses, suggesting that gene expression regulation and AS seemed to play independent roles in response to stresses. Compared with gene expression, Arabidopsis and rice were more inclined to have conserved AS patterns under stress conditions. CONCLUSION: PlaASDB is a comprehensive plant-specific AS database that mainly integrates the AS and gene expression data of Arabidopsis and rice in stress response. Through large-scale comparative analyses, the global landscape of AS events in Arabidopsis and rice was observed. We believe that PlaASDB could help researchers understand the regulatory mechanisms of AS in plants under stresses more conveniently. PlaASDB is freely accessible at http://zzdlab.com/PlaASDB/ASDB/index.html .

Cytotoxic Terpenes from the Flowers of Inula japonica against Human Hepatocarcinoma Cells.

Two new 1,10-seco-eudesmanolides (1 and 2) were isolated from the flowers of Inula japonica together with two eudesmanolide analogs (3 and 4) and two monoterpene derivatives (5 and 6). Their structures were established on the basis of detailed spectroscopic analyses and electronic circular dichroism data. All isolates were evaluated for their antiproliferative activities against human hepatocarcinoma HepG2 and SMMC-7721 cells. Japonipene B (3) exhibited the most potent effect with the IC50 values of 14.60±1.62 and 22.06±1.34 µM against HepG2 and SMMC-7721 cells, respectively. Furthermore, japonipene B (3) showed significant efficacies of arresting the cell cycle at the S/G2-M stages, inducing mitochondria-mediated apoptosis, and inhibiting cell migration in HepG2 cells.

Delphinieae flowers originated from the rewiring of interactions between duplicated and diversified floral organ identity and symmetry genes.

Species of the tribe Delphinieae (Ranunculaceae) have long been the focus of morphological, ecological, and evolutionary studies due to their highly specialized, nearly zygomorphic (bilaterally symmetrical) spiral flowers with nested petal and sepal spurs and reduced petals. The mechanisms underlying the development and evolution of Delphinieae flowers, however, remain unclear. Here, by conducting extensive phylogenetic, comparative transcriptomic, expression, and functional studies, we clarified the evolutionary histories, expression patterns, and functions of floral organ identity and symmetry genes in Delphinieae. We found that duplication and/or diversification of APETALA3-3 (AP3-3), AGAMOUS-LIKE6 (AGL6), CYCLOIDEA (CYC), and DIVARICATA (DIV) lineage genes was tightly associated with the origination of Delphinieae flowers. Specifically, an AGL6-lineage member (such as the Delphinium ajacis AGL6-1a) represses sepal spur formation and petal development in the lateral and ventral parts of the flower while determining petal identity redundantly with AGL6-1b. By contrast, two CYC2-like genes, CYC2b and CYC2a, define the dorsal and lateral-ventral identities of the flower, respectively, and form complex regulatory links with AP3-3, AGL6-1a, and DIV1. Therefore, duplication and diversification of floral symmetry genes, as well as co-option of the duplicated copies into the preexisting floral regulatory network, have been key for the origin of Delphinieae flowers.

Investigation of Brassica and its relative genomes in the post-genomics era.

The Brassicaceae family includes many economically important crop species, as well as cosmopolitan agricultural weed species. In addition, Arabidopsis thaliana, a member of this family, is used as a molecular model plant species. The genus Brassica is mesopolyploid, and the genus comprises comparatively recently originated tetrapolyploid species. With these characteristics, Brassicas have achieved the commonly accepted status of model organisms for genomic studies. This paper reviews the rapid research progress in the Brassicaceae family from diverse omics studies, including genomics, transcriptomics, epigenomics, and three-dimensional (3D) genomics, with a focus on cultivated crops. The morphological plasticity of Brassicaceae crops is largely due to their highly variable genomes. The origin of several important Brassicaceae crops has been established. Genes or loci domesticated or contributing to important traits are summarized. Epigenetic alterations and 3D structures have been found to play roles in subgenome dominance, either in tetraploid Brassica species or their diploid ancestors. Based on this progress, we propose future directions and prospects for the genomic investigation of Brassicaceae crops.

Sympatric Recombination in Zoonotic Cryptosporidium Leads to Emergence of Populations with Modified Host Preference.

Genetic recombination plays a critical role in the emergence of pathogens with phenotypes such as drug resistance, virulence, and host adaptation. Here, we tested the hypothesis that recombination between sympatric ancestral populations leads to the emergence of divergent variants of the zoonotic parasite Cryptosporidium parvum with modified host ranges. Comparative genomic analyses of 101 isolates have identified seven subpopulations isolated by distance. They appear to be descendants of two ancestral populations, IIa in northwestern Europe and IId from southwestern Asia. Sympatric recombination in areas with both ancestral subtypes and subsequent selective sweeps have led to the emergence of new subpopulations with mosaic genomes and modified host preference. Subtelomeric genes could be involved in the adaptive selection of subpopulations, while copy number variations of genes encoding invasion-associated proteins are potentially associated with modified host ranges. These observations reveal ancestral origins of zoonotic C. parvum and suggest that pathogen import through modern animal farming might promote the emergence of divergent subpopulations of C. parvum with modified host preference.

Automated Computer Software Assessment of 5-Hydroxytryptamine 2A Receptor-Mediated Head Twitch Responses from Video Recordings of Mice.

Psychedelics are a class of drugs that produce unique subjective effects via agonist actions at the 5-hydroxytryptamine 2A receptor (5-HT2A). The 5-HT2A-mediated head twitch response (HTR) in rodents is used as a reliable proxy for psychedelic drug activity in humans, but existing methods for measuring HTRs require surgery or time-consuming visual scoring. In the present work, we validated a simple noninvasive method for quantitating HTRs using computer-based analysis of experimental video recordings. Male C57BL/6J mice received injections of the 5-HT2 receptor agonist (±)2,5-dimethoxy-4-iodoamphetamine (DOI; 0.03-3 mg/kg, s.c.) and were placed into cylindrical arenas. High frame rate videos were recorded via cameras mounted above the arenas. Antagonist experiments, which entailed pretreatment with the 5-HT2A antagonist M100907 (0.01 or 0.1 mg/kg s.c.) prior to DOI (1 mg/kg s.c.), were also recorded. The experimental videos were analyzed for HTRs using a newly developed feature of a commercial software package and compared to visual scoring carried out by trained observers. As expected, DOI produced dose-related increases in HTRs, which were blocked by M100907. Computer scoring was positively correlated with visual scoring, and no statistical difference between the two methods was found. The software captured nearly all visually observed HTRs, false positives induced by other behaviors (e.g., grooming) were rare and easily identified, and results were improved by optimizing lighting conditions. Our findings demonstrate the utility of combining high frame rate video recordings with commercial software analyses to measure HTRs, validating an additional reliable method to study psychedelic-like drug activity in mice.

Proteome-wide prediction and analysis of the Cryptosporidium parvum protein-protein interaction network through integrative methods.

As one of the most studied Apicomplexan parasite Cryptosporidium, Cryptosporidium parvum (C. parvum) causes worldwide serious diarrhea disease cryptosporidiosis, which can be deadly to immunodeficiency individuals, newly born children, and animals. Proteome-wide identification of protein-protein interactions (PPIs) has proven valuable in the systematic understanding of the genome-phenome relationship. However, the PPIs of C. parvum are largely unknown because of the limited experimental studies carried out. Therefore, we took full advantage of three bioinformatics methods, i.e., interolog mapping (IM), domain-domain interaction (DDI)-based inference, and machine learning (ML) method, to jointly predict PPIs of C. parvum. Due to the lack of experimental PPIs of C. parvum, we used the PPI data of Plasmodium falciparum (P. falciparum), which owned the largest number of PPIs in Apicomplexa, to train an ML model to infer C. parvum PPIs. We utilized consistent results of these three methods as the predicted high-confidence PPI network, which contains 4,578 PPIs covering 554 proteins. To further explore the biological significance of the constructed PPI network, we also conducted essential network and protein functional analysis, mainly focusing on hub proteins and functional modules. We anticipate the constructed PPI network can become an important data resource to accelerate the functional genomics studies of C. parvum as well as offer new hints to the target discovery in developing drugs/vaccines.

ASTool: An Easy-to-Use Tool to Accurately Identify Alternative Splicing Events from Plant RNA-Seq Data.

Alternative splicing (AS) is an essential co-transcriptional regulatory mechanism in eukaryotes. The accumulation of plant RNA-Seq data provides an unprecedented opportunity to investigate the global landscape of plant AS events. However, most existing AS identification tools were originally designed for animals, and their performance in plants was not rigorously benchmarked. In this work, we developed a simple and easy-to-use bioinformatics tool named ASTool for detecting AS events from plant RNA-Seq data. As an exon-based method, ASTool can detect 4 major AS types, including intron retention (IR), exon skipping (ES), alternative 5' splice sites (A5SS), and alternative 3' splice sites (A3SS). Compared with existing tools, ASTool revealed a favorable performance when tested in simulated RNA-Seq data, with both recall and precision values exceeding 95% in most cases. Moreover, ASTool also showed a competitive computational speed and consistent detection results with existing tools when tested in simulated or real plant RNA-Seq data. Considering that IR is the most predominant AS type in plants, ASTool allowed the detection and visualization of novel IR events based on known splice sites. To fully present the functionality of ASTool, we also provided an application example of ASTool in processing real RNA-Seq data of Arabidopsis in response to heat stress.

Expanding the genetic variation of Brassica juncea by introgression of the Brassica rapa Genome.

Brassica juncea is an important vegetable and oil crop cultivated worldwide. To increase its genetic variation, we introgressed the A-genome of Brassica rapa into B. juncea. We used three each of heading and semi-heading B. juncea accessions as recipient parents and a B. rapa line, B9008, as the donor parent. We obtained 101 BC1S1 lines in total with expanded phenotypic variations such as leafy head shapes. We developed 132 SNP markers, which could distinguish the A genome of B. juncea from B. rapa genome, and tracked the introgression of B. rapa segments in the new B. juncea germplasm. On average, 59.2% of the B. juncea A genome in the B. juncea introgression lines was covered by the donor segments. We also identified three markers whose donor genotype frequencies were significantly lower than the theoretical value, suggesting strong selection of the recipient genotype during the introgression process. We provide an effective strategy to evaluate the diversity of the new germplasm based on the combination of parental re-sequencing data and marker genotyping results. Further genetic analysis of 1642 SNPs showed that the genetic diversity of the new B. juncea germplasm with the introgressed B. rapa genome was significantly increased. This study illustrated the potential for expanding the genetic diversity of B. juncea through the introgression of the B. rapa genome.

BRAD V3.0: an upgraded Brassicaceae database.

The Brassicaceae Database (BRAD version 3.0, BRAD V3.0; http://brassicadb.cn) has evolved from the former Brassica Database (BRAD V2.0), and represents an important community portal hosting genome information for multiple Brassica and related Brassicaceae plant species. Since the last update in 2015, the complex genomes of numerous Brassicaceae species have been decoded, accompanied by many omics datasets. To provide an up-to-date service, we report here a major upgrade of the portal. The Model-View-ViewModel (MVVM) framework of BRAD has been re-engineered to enable easy and sustainable maintenance of the database. The collection of genomes has been increased to 26 species, along with optimization of the user interface. Features of the previous version have been retained, with additional new tools for exploring syntenic genes, gene expression and variation data. In the 'Syntenic Gene @ Subgenome' module, we added features to view the sequence alignment and phylogenetic relationships of syntenic genes. New modules include 'MicroSynteny' for viewing synteny of selected fragment pairs, and 'Polymorph' for retrieval of variation data. The updated BRAD provides a substantial expansion of genomic data and a comprehensive improvement of the service available to the Brassicaceae research community.

Long Non-coding RNA SNHG12, a New Therapeutic Target, Regulates miR-199a-5p/Klotho to Promote the Growth and Metastasis of Intrahepatic Cholangiocarcinoma Cells.

Background: Small nucleolar RNA host gene 12 (SNHG12) is a newly identified long non-coding RNA (lncRNA) whose involvements have been explored in several cancers. Our study aimed to explore the functions of SNHG12 on intrahepatic cholangiocarcinoma (ICC) progression and its interaction with miR-199a-5p and Klotho. Methods: RT-PCR was performed to examine the expressions of SNHG12, miR-199a-5p and Klotho in ICC cells. Cell counting kit-8 (CCK-8), colony formation assays and transwell assays were applied to analyze the proliferation, migration and invasion of ICC cells. Luciferase assays, RIP assays and RNA pull-down assays were carried out to demonstrate the direct binding relationships among SNHG12, miR-199a-5p and Klotho. The xenograft nude models were applied to test the effects of SNHG12 on ICC tumor growth. Results: The expression of SNHG12 and Klotho was distinctly increased in ICC cells, while miR-199a-5p expressions were decreased. Functionally, the silence of SNHG12 inhibited the proliferation and metastasis of ICC cells, while miR-199a-5p overexpression exhibited an opposite result. Mechanistically, Knockdown of SNHG12 significantly suppressed the expressions of miR-199a-5p by sponging it, and then increased Klotho expression. The final in vivo experiments suggested that the silence of SNHG12 distinctly inhibited tumor growth. Conclusion: Our findings indicated that SNHG12 inhibited cell proliferation and metastasis process of ICC cells through modulating the miR-199a-5p/Klotho axis and it is expected to become a potential therapeutic target for ICC.

Investigation on Crystallization and Magnetic Properties of (Nd, Pr, Ce)2Fe14B/α-Fe Nanocomposite Magnets by Microwave Annealing Treatment.

In the present work, the structures and magnetic properties of (Nd, Pr, Ce) 2Fe14B/α-Fe nanocomposite magnets were thoroughly investigated. The microwave annealing was applied to achieve a uniform heating effect and uniform grains. Microwave annealing is more favorable to obtain α-Fe phase than conventional annealing, which leads to the enhanced coercivity of hysteresis loops. The coercivity of nanocomposite magnets was 245 kA/m after annealing at 2000 W for 10 min.

Sesquiterpenoids isolated from the flower of Inula japonica as potential antitumor leads for intervention of paclitaxel-resistant non-small-cell lung cancer.

Three new sesquiterpene lactone dimers (1-3) were isolated from the flowers of Inula japonica together with twenty-two known sesquiterpene derivatives (4-25). Their structures were established on the basis of detailed spectroscopic analyses. All isolates were evaluated for their antiproliferative activities against paclitaxel-resistant human non-small-cell lung cancer cell line A549/PTX. The preliminary structure-activity relationship was discussed. Compound 24 exhibited the most potent effect with the IC50 value of 0.34 ± 0.10 µM, even more active than the clinically used drug paclitaxel (PTX, IC50 = 1.40 ± 0.52 µM). Compound 24 showed significant efficacy of arresting the cell cycle at the G2-M stage, inducing apoptosis through mitochondria-mediated pathway, and inhibiting cell migration and invasion. Furthermore, compound 24 could reverse multidrug resistance through suppressing the expression of ABC family proteins.

PlaPPISite: a comprehensive resource for plant protein-protein interaction sites.

BACKGROUND: Protein-protein interactions (PPIs) play very important roles in diverse biological processes. Experimentally validated or predicted PPI data have become increasingly available in diverse plant species. To further explore the biological functions of PPIs, understanding the interaction details of plant PPIs (e.g., the 3D structural contexts of interaction sites) is necessary. By integrating bioinformatics algorithms, interaction details can be annotated at different levels and then compiled into user-friendly databases. In our previous study, we developed AraPPISite, which aimed to provide interaction site information for PPIs in the model plant Arabidopsis thaliana. Considering that the application of AraPPISite is limited to one species, it is very natural that AraPPISite should be evolved into a new database that can provide interaction details of PPIs in multiple plants. DESCRIPTION: PlaPPISite (http://zzdlab.com/plappisite/index.php) is a comprehensive, high-coverage and interaction details-oriented database for 13 plant interactomes. In addition to collecting 121 experimentally verified structures of protein complexes, the complex structures of experimental/predicted PPIs in the 13 plants were also constructed, and the corresponding interaction sites were annotated. For the PPIs whose 3D structures could not be modelled, the associated domain-domain interactions (DDIs) and domain-motif interactions (DMIs) were inferred. To facilitate the reliability assessment of predicted PPIs, the source species of interolog templates, GO annotations, subcellular localizations and gene expression similarities are also provided. JavaScript packages were employed to visualize structures of protein complexes, protein interaction sites and protein interaction networks. We also developed an online tool for homology modelling and protein interaction site annotation of protein complexes. All data contained in PlaPPISite are also freely available on the Download page. CONCLUSION: PlaPPISite provides the plant research community with an easy-to-use and comprehensive data resource for the search and analysis of protein interaction details from the 13 important plant species.

Identification of Parthenolide Dimers as Activators of Pyruvate Kinase M2 in Xenografts of Glioblastoma Multiforme in Vivo.

Herein we detail the discovery of a series of parthenolide dimers as activators of PKM2 and evaluation of their anti-GBM activities. The most promising compound 5 showed high potency to activate PKM2 with an AC50 value of 15 nM, inhibited proliferation and metastasis, and induced apoptosis of GBM cells. Compound 5 could promote tetramer formation of PKM2 and reduce nucleus translocation of PKM2 in GBM cells without influence on the expression of total PKM2, thereby inhibiting the STAT3 signal pathway in vitro and in vivo. PKM2 knockdown assay demonstrated that the anti-GBM effect of 5 mainly depended on the expression of PKM2 in vitro and in vivo. Compound 16, a prodrug of 5, markedly suppressed U118 tumor xenograft growth and reduced the weight of tumor. On the basis of these investigations, we propose that 16 might be considered as a promising lead compound for discovery of anti-GBM drugs.

EX527, a Sirt-1 inhibitor, induces apoptosis in glioma via activating the p53 signaling pathway.

Sirtuin-1 (Sirt-1), an NAD-dependent deacetylase, promotes tumorigenesis in glioma; however, whether the Sirt-1 specific inhibitor, EX527 exerts antitumor effects and the underlying mechanism in glioma requires further investigation. In the present study, the proliferative and colony formation abilities of two glioma cell lines (U87MG and LN-299) were inhibited by EX527. Treatment with EX527 increased the number of apoptotic cells (Annexin V-fluorescein isothiocyanate/propidium iodide); pretreatment with the caspase inhibitor Z-VAD-FMK suppressed EX527-induced apoptosis, suggesting that EX527 induced caspase-dependent apoptosis. In addition, western blotting revealed that EX527 treatment increased the expression of cleaved-caspase-3, poly (ADP-ribose) polymerase-1, B-cell lymphoma 2 (Bcl-2)-associated-X-protein and Bcl-2-like 11 but decreased that of Bcl-2. p53 is deacetylated by Sirt-1, attenuating its function. Furthermore, EX527 upregulated the expression of p53, acetylated p53 and the p53 target gene p21. This result suggests that EX527 induced cell apoptosis by activating p53 in glioma. Of note, EX527 exhibited antitumor effects on patient-derived glioma cells under three-dimensional culture conditions. Collectively, the results of the present study indicated that EX527 may be used as an effective compound in the treatment of glioma.

[Primary appendiceal signet ring cell carcinoma:a case report].

A 52-year-old woman with epigastralgia and abdominal discomfort was admitted to our hospital. The abdominal CT scan showed that she had intestinal obstruction and peritoneal dissemination. Colonoscopy also revealed a submucosal tumor around the orifice of the appendix. Moreover, histological examination results indicated signet ring cell carcinoma. She was then treated with modified FOLFOX chemotherapy;however, the disease condition progressed after an 8-course treatment, and she died 12 months after the chemotherapy was initiated.