Impacts of spatial symmetries on stimulated Brillouin scatterings in nanoscale silicon waveguides: a theoretical and numerical study considering material anisotropies.

Fully considering the mechanical and photoelastic anisotropies of monocrystalline silicon, the impacts of spatial symmetries on the stimulated Brillouin scatterings (SBSs) in nanoscale suspended silicon waveguides are studied theoretically and numerically based on group theory. First, starting from an assumption that the principal material coordinate system can be arbitrarily orientated in a waveguide with fixed geometry, the silicon waveguides are systematically classified into a number of point groups according to their spatial symmetry features. Thereafter, the symmetry characteristics of physical fields and SBS opto-mechanical coupling characteristics in the silicon waveguides belonging to different point groups are further examined, and the major new findings can be summarized as follows: The SBS opto-mechanical couplings in several kinds of silicon waveguides with certain nontrivial symmetry features exhibit relatively predictable behaviors in that the opto-mechanical coupling coefficients can be deterministically vanishing or nonvanishing under very few constraints, which can thus serve as general symmetry selection rules for SBSs in suspended silicon waveguides. The results obtained in the present study could be a useful theoretical reference for the design of novel SBS-active silicon photonic devices.

Oncogenic pathways refine a new perspective on the classification of hepatocellular carcinoma.

BACKGROUND: Genetic alterations in oncogenic pathways are critical for cancer initiation, development, and treatment resistance. However, studies are limited regarding pathways correlated with prognosis, sorafenib, and transcatheter arterial chemoembolization (TACE) in hepatocellular carcinoma (HCC). METHODS: In this study, 1928 patients from 11 independent datasets and a clinical in-house cohort were screened to explore the relationships among canonical pathway alterations in HCC patients. The molecular mechanisms, biological functions, immune landscape, and clinical outcomes among three heterogeneous phenotypes were further explored. RESULTS: We charted the detailed landscape of pathway alterations in the TCGA-LIHC cohort, screened three pivotal pathways (p53, PI3K, and WNT), identified co-occurrence patterns and mutual exclusively, and stratified patients into three altered-pathway dominant phenotypes (ADPs). P53|PI3K ADP characterized by genomic instability (e.g., highest TMB, FGA, FGG, and FGL) indicated an unfavorable prognosis. While, patients in WNT ADP suggested a median prognosis, enhanced immune activation, and sensitivity to PD-L1 therapy. Remarkably, sorafenib and TACE exhibited efficacy for patients in WNT ADP and low frequent alteration phenotype (LFP). Additionally, ADP could work independently of common clinical traits (e.g., AJCC stage) and previous molecular classifications (e.g., iCluster, serum biomarkers). CONCLUSIONS: ADP provides a new perspective for identifying patients at high risk of recurrence and could optimize precision treatment to improve the clinical outcomes in HCC.

Durable response to afatinib in advanced lung adenocarcinoma harboring a novel NPTN-NRG1 fusion: a case report.

BACKGROUND: NRG1 fusions are rare oncogenic drivers in solid tumors, and the incidence of NRG1 fusions in non-small cell lung cancer (NSCLC) was 0.26%. It is essential to explore potential therapeutic strategies and efficacy predictors for NRG1 fusion-positive cancers. CASE PRESENTATION: We report an advanced lung adenocarcinoma patient harboring a novel NPTN-NRG1 fusion identified by RNA-based next-generation sequencing (NGS), which was not detected by DNA-based NGS at initial diagnosis. Transcriptomics data of the tissue biopsy showed NRG1α isoform accounted for 30% of total NRG1 reads, and NRG1ß isoform was undetectable. The patient received afatinib as fourth-line treatment and received a progression-free survival (PFS) of 14 months. CONCLUSIONS: This report supports afatinib can provide potential benefit for NRG1 fusion patients, and RNA-based NGS is an accurate and cost-effective strategy for fusion detection and isoform identification.

Distinct genomic features between osteosarcomas firstly metastasing to bone and to lung.

Background: Osteosarcoma initially metastasing to bone only shows distinct biological features compared to osteosarcoma that firstly metastasizes to the lung, which suggests us underlying different genomic pathogenetic mechanism. Methods: We analyzed whole-exome sequencing (WES) data for 38 osteosarcoma with paired samples in different relapse patterns. We also sought to redefine disease subclassifications for osteosarcoma based on genetic alterations and correlate these genetic profiles with clinical treatment courses to elucidate potential evolving cladograms. Results: We investigated WES of 12/38 patients with high-grade osteosarcoma (31.6%) with initial bone metastasis (group A) and 26/38 (68.4%) with initial pulmonary metastasis (group B), of whom 15/38 (39.5%) had paired samples of primary lesions and metastatic lesions. We found that osteosarcoma in group A mainly carries single-nucleotide variations displaying higher tumor mutation burden and neoantigen load and more tertiary lymphoid structures, while those in group B mainly exhibits structural variants. High conservation of reported genetic sequencing over time in their evolving cladograms. Conclusions: Osteosarcoma with mainly single-nucleotide variations other than structural variants might exhibit biological behavior predisposing toward bone metastases as well as better immunogenicity in tumor microenvironment.

Feasibility and tolerability of sintilimab plus anlotinib as the second-line therapy for patients with advanced biliary tract cancers: An open-label, single-arm, phase II clinical trial.

Patients with biliary tract cancer (BTC) were associated with poor prognosis and limited therapeutic options after first-line therapy currently. In this study, we sought to evaluate the feasibility and tolerability of sintilimab plus anlotinib as the second-line treatment for patients with advanced BTC. Eligible patients had histologically confirmed locally advanced unresectable or metastatic BTC and failed after the first-line treatment were recruited. The primary endpoint was overall survival (OS). Simultaneously, association between clinical outcomes and genomic profiling and gut microbiome were explored to identify the potential biomarkers for this regimen. Twenty patients were consecutively enrolled and received study therapy. The trail met its primary endpoint with a median OS of 12.3 months (95% CI: 10.1-14.5). Only four (20%) patients were observed of the grade 3 treatment-related adverse events (TRAEs) and no grade 4 or 5 TRAEs were detected. Mutation of AGO2 was correlated with a significantly longer OS. Abundance of Proteobacteria was associated with inferior clinical response. Therefore, sintilimab plus anlotinib demonstrated encouraging anti-tumor activity with a tolerable safety profile and deserved to be investigated in larger randomized trials for patients with advanced BTC subsequently.

Identification of a Rare BAIAP2-ROS1 Fusion and Clinical Benefit of Crizotinib in the Treatment of Advanced Lung Adenocarcinoma: A Case Report.

Objective: Previous studies have shown that fusion partners have a potential role in influencing different tumorigenic abilities of ROS1 fusion variants, as well as potential differential responses to crizotinib. Therefore, it is important to accurately identify the type of ROS1 rearrangement in NSCLC for clinical treatment selection. Materials and Methods: Deep-coverage targeting solid tumor 31 cancer-related genes panel was used to capture DNA-based NGS information to detect gene fusion. RNA fusion panel based on hybrid capture sequencing was performed to verify gene fusions from total RNA which isolated from formalin fixed paraffin-embedded (FFPE) tissue blocks. Results: Using DNA-targeted NGS method, we identified a novel BAIAP2-ROS1 fusion in a 71-year-old non-smoking female patient with stage IVB lung adenocarcinoma. Rearrangement consisted of BAIAP2 in exon1-exon13 of chr17: q23 and ROS1 in exon35-exon43 of chr6: q22, which were further confirmed by RNA-based NGS methodology. A complete kinase domain in ROS1 fusion was preserved. The patient subsequently received crizotinib and showed significant tumor reduction until 17 months, who got benefit from targeted therapy. Conclusion: This study discovered a novel BAIAP2-ROS1 rearrangement; it provides more knowledge of ROS1 fusion in clinical personalized treatment. The good response to crizotinib therapy emphasizes the importance of DNA-based and RNA-based NGS in rare fusion identification in clinical practice.

Tracking the evolution of untreated high-intermediate/high-risk diffuse large B-cell lymphoma by circulating tumour DNA.

Diffuse large B-cell lymphoma (DLBCL) is a highly heterogenous malignancy, early identification of patients for relapse remains challenging. The potential to non-invasively monitor tumour evolutionary dynamics of DLBCL needs to be further established. In the present study, 17 tumour biopsy and 38 plasma samples from 38 patients with high-intermediate/high-risk DLBCL were evaluated at baseline. Longitudinal blood samples were also collected during therapy. Circulating tumour DNA (ctDNA) was analysed using targeted sequencing based on a gene panel via a recently developed methodology, circulating single-molecule amplification and re-sequencing technology (cSMART). We found that the most frequently mutated genes were tumour protein p53 (TP53; 42·1%), histone-lysine N-methyltransferase 2D (KMT2D; 28·9%), caspase recruitment domain family member 11 (CARD11; 21·1%), cAMP response element-binding protein binding protein (CREBBP; 15·8%), ß2 -microglobulin (B2M; 15·8%), and tumour necrosis factor alpha-induced protein 3 (TNFAIP3; 15·8%). The mutation profiles between ctDNA and matched tumour tissue showed good concordance; however, more mutation sites were detected in ctDNA samples. Either TP53 or B2M mutations before treatment predicted poor prognosis. Analysis of dynamic blood samples confirmed the utility of ctDNA for the real-time assessment of treatment response and revealed that the increases in ctDNA levels and changes in KMT2D mutation status could be useful predictors of disease progression. Our present results suggest that ctDNA is a promising method for the detection of mutation spectrum and serves as a biomarker for disease monitoring and predicting clinical recurrence.

Identification of a Novel SLC8A1-ALK Fusion and Non-Canonical Expression Significantly Responding to ALK-TKIs in Lung Adenocarcinoma: A Case Report.

BACKGROUND: Approximately 2-7% of patients with non-small cell lung cancer harbor anaplastic lymphoma kinase (ALK) rearrangement events. Of note, typical ALK actionable rearrangements are sensitive to treatment with tyrosine kinase inhibitors (TKIs). However, different types of ALK fusion influence the clinical outcomes of this therapeutic approach. Approximately 10-40% of patients with ALK-fusion positive non-small cell lung cancer do not response to ALK-TKI therapy. Therefore, it is important to accurately identify the types of ALK rearrangement for appropriate selection of clinical treatment. CASE REPORT: Using a DNA-targeted next-generation sequencing technique, we found a novel solute carrier family 8 member A1 (SLC8A1)-ALK fusion type in a patient with lung adenocarcinoma. Further reverse transcriptase-polymerase chain reaction and Sanger sequencing demonstrated the rearrangement as a B-cell CLL/lymphoma 11A (BCL11A)-ALK fusion at the transcriptional level. The patient showed a rapid and strong response to treatment with crizotinib, which lasted for 9 months. The patient also responded well to treatment with alectinib after developed resistance to crizotinib. CONCLUSION: A strategy combining DNA-targeted next-generation sequencing with RNA reverse transcriptase-polymerase chain reaction and sequencing, besides fluorescence in situ hybridization and immunohistochemistry, may provide an effective and practical solution for correct identification of partner genes and fusion structures in the diagnosis of ALK rearrangements, particularly for non-canonical expression patterns of ALK fusion events. The combined approach may lead to more benefits for patients.

Pulmonary Adenofibroma: Clinicopathological and Genetic Analysis of 7 Cases With Literature Review.

INTRODUCTION: Pulmonary adenofibroma (PAF), characterized by biphasic differentiation composed of gland-like space lined by respiratory epithelium and stromal spindle cells, is a rare benign tumor of the lung. PAF was reported infrequently and inconsistently with diagnostic criteria and withstood higher risk of misdiagnosis as solitary fibrous tumors (SFTs) due to their morphological resemblance. In this study, we report seven cases of PAF with gene sequencing results and summarize the data of previous literature. MATERIALS AND METHODS: Seven cases of PAF with surgically resection samples were collected from Pathology department of West China Hospital, Sichuan University between 2009 to 2020. Immunohistochemical studies were performed in all cases and 3 cases underwent a 425-gene panel next-generation sequencing (NGS). RESULTS: Five female and two male patients were included in this study, with an average age of 51 years. All the patients were asymptomatic, and the lesion was identified on routine chest radiography. The tumor size measured by computed tomography (CT) ranged from 0.5 to 2.7 cm. Gland-like structures were mostly positive for glandular epithelium markers. The spindle cells in stroma expressed Desmin, SMA, ER and PR in 3 of 7 cases. No well-recognized molecular abnormalities can be identified by NGS in the 3 cases. To date, all the patients are alive, with no evidence of recurrence and metastasis. CONCLUSION: PAF is a unique benign pulmonary tumor with low incidence. Biphasic morphology, IHC stains along with molecular detection is of great significance to make a clear diagnosis.

Molecular characteristics and clinical outcomes of complex ALK rearrangements identified by next-generation sequencing in non-small cell lung cancers.

BACKGROUND: Complex kinase rearrangement, a mutational process involving one or two chromosomes with clustered rearrangement breakpoints, interferes with the accurate detection of kinase fusions by DNA-based next-generation sequencing (NGS). We investigated the characteristics of complex ALK rearrangements in non-small cell lung cancers using multiple molecular tests. METHODS: Samples of non-small cell lung cancer patients were analyzed by targeted-capture DNA-based NGS with probes tilling the selected intronic regions of fusion partner genes, RNA-based NGS, RT-PCR, immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). RESULTS: In a large cohort of 6576 non-small cell lung cancer patients, 343 (5.2%) cases harboring ALK rearrangements were identified. Fourteen cases with complex ALK rearrangements were identified by DNA-based NGS and classified into three types by integrating various genomic features, including intergenic (n = 3), intragenic (n = 5) and "bridge joint" rearrangements (n = 6). All thirteen cases with sufficient samples actually expressed canonical EML4-ALK fusion transcripts confirmed by RNA-based NGS. Besides, positive ALK IHC was detected in 13 of 13 cases, and 9 of 11 cases were positive in FISH testing. Patients with complex ALK rearrangements who received ALK inhibitors treatment (n = 6), showed no difference in progression-free survival (PFS) compared with patients with canonical ALK fusions n = 36, P = 0.9291). CONCLUSIONS: This study firstly reveals the molecular characteristics and clinical outcomes of complex ALK rearrangements in NSCLC, sensitive to ALK inhibitors treatment, and highlights the importance of utilizing probes tilling the selected intronic regions of fusion partner genes in DNA-based NGS for accurate fusion detection. RNA and protein level assay may be critical in validating the function of complex ALK rearrangements in clinical practice for optimal treatment decision.

Plasma EGFR mutation abundance affects clinical response to first-line EGFR-TKIs in patients with advanced non-small cell lung cancer.

BACKGROUND: Activated epidermal growth factor receptor (EGFR) mutation is the main pathogenic cause of non-small cell lung cancer (NSCLC) in Asia. However, the impact of plasma EGFR mutation abundance, especially of the ultra-low abundance of EGFR mutation detected by highly sensitive techniques on clinical outcomes of first-line EGFR tyrosine kinase inhibitors (TKIs) for advanced NSCLC patients remains unclear. METHODS: We qualitatively detected baseline EGFR status of NSCLC tissues using amplification-refractory mutation system and quantified the plasma abundance of EGFR mutations through next-generation sequencing (NGS). Every 8-12 weeks, we performed dynamic detection of plasma mutation abundance and imaging evaluation. We analyzed the association between plasma abundance of EGFR sensitizing mutations, tumor size, tumor shrinkage percentage, concomitant TP53 mutations, and clinical response to TKIs. RESULTS: This prospective study enrolled 135 patients with advanced NSCLC. The objective response rate (ORR) and disease control rate (DCR) for EGFR mutation-positive patients were 50.0% and 87.0%, respectively. When the cutoff value of plasma EGFR mutation abundance was 0.1%, the ORRs of TKI-treated patients were significantly different (60.0% for the >0.1% group vs. 21.4% for the ≤0.1% group, P=0.028). Median progression-free survival (PFS) was significantly longer for participants with a mutation abundance above 0.1% compared to those with a 0.01-0.1% abundance (log rank, P=0.0115). There was no significant association between plasma abundance of EGFR sensitizing mutations and tumor size, tumor shrinkage percentage, or concomitant TP53 mutations. Cox multivariate analysis demonstrated that plasma mutation abundance was an independent predictive factor for PFS [hazard ratio (HR) 2.41, 95% confidence interval (CI): 1.12-5.20; P=0.025]. We identified 11 participants with the acquired T790M resistance mutation according to serial dynamic plasma samples. CONCLUSIONS: Liquid biopsy screening based on highly sensitive NGS is reliable for detecting drug resistance and actionable somatic mutations. The plasma abundance of the EGFR driver mutation affected clinical response to EGFR-TKIs in advanced NSCLC patients; prolongation of PFS was also observed in patients with an ultra-low abundance of EGFR sensitizing mutations.

Next Generation Sequencing Reveals a Synchronous Trilateral Lung Adenocarcinoma Case with Distinct Driver Alterations of EGFR 19 Deletion or EGFR 20 Insertion or EZR-ROS1 Fusion.

OBJECTIVE: Synchronous multiple primary lung cancer (SMPLC) has a reported occurrence from 0.5% to 2% in lung cancer, and the surgical treatment and prognosis were quite diverse. With the discovery of driver mutations in lung adenocarcinoma (ADC), next-generation sequencing (NGS) would provide an explicit answer to the key question, whether individual tumors represent intrapulmonary metastases or independent tumors. Here, we reported a 64-year-old female diagnosed with a synchronous trilateral early-stage ADC with distinct driver alterations. MATERIALS AND METHODS: NGS test targeting 31 cancer-relevant genes and amplification RNA sequencing (if gene fusion was found on DNA level) were performed on the surgical tumor tissue. RESULTS: A 64-year-old Chinese female never smoker was found with one nodule in the right upper lobe and two nodules in the right middle lobe through chest computed tomography. The lesions were resected through video-assisted thoracic surgery and diagnosed with stage IA ADC, T1N0M0, in the postoperative pathology. NGS detected three independent driver mutations in three primary sites, respectively, EGFR 19del, EGFR 20ins and ROS1 fusion. CONCLUSION: This is the first report of a synchronous trilateral early-stage ADC with distinct driver alterations. All individual tumors were independent identified by NGS methodology, which had provided a clear answer to the key question of SMPLC in this case and should be used as a routine genetic test to explore fully pathological diagnosis and more comprehensive oncogenesis information in the early-stage ADC clinical prevention.

Identification of a Novel MPRIP-ROS1 Fusion and Clinical Efficacy of Crizotinib in an Advanced Lung Adenocarcinoma Patient: A Case Report.

OBJECTIVE: ROS1 fusions have been identified in 1-2% of non-small-cell lung cancer (NSCLC) patients; they are validated as a driver of carcinogenesis and could be subjected to inhibition by crizotinib. However, previous studies suggested a variable progression-free survival (PFS) ranging from 9.1 to 20.0 months for crizotinib treatment in ROS1-rearranged NSCLC. Here, we reported a 45-year-old female diagnosed with stage IVB lung adenocarcinoma with multiple lymph nodes and bone metastasis carrying a novel MPRIP-ROS1 fusion, which was identified by RNA-based NGS (next-generation sequencing) and was sensitive to crizotinib treatment. MATERIALS AND METHODS: A targeted NGS panel was used to analyze genomic DNA and total RNA isolated from formalin-fixed paraffin-embedded (FFPE) tissue block of the patient. An RNA fusion panel based on amplicon sequencing was designed for detection fusion variation. Fusion results were validated using reverse transcriptase polymerase chain reaction and Sanger sequencing. RESULTS: We reported a novel MPRIP-ROS1 fusion identified in this advanced lung adenocarcinoma case. The rearrangement was generated by exons 1-21 of MPRIP at chr17: p11.2 joined to exons 35-43 of ROS1 at chr6: q22.1, which retained an intact kinase domain of ROS1. The primary tumor and metastatic lymph nodes were eliminated on computed tomographic (CT) scan imaging after 2 months' crizotinib treatment, and the multiple bone metastatic lesions were significantly relieved according to bone scintigraphy images. To date, the treatment has lasted 16 months, and the patient is still in follow-up showing sustained response to crizotinib. CONCLUSION: The study identified a novel MPRIP-ROS1 fusion that was sensitive to crizotinib, which provided a new driver of lung adenocarcinoma and potential therapeutic target for crizotinib. It also expanded NSCLC treatment of ROS1 rearrangement and highlighted the importance of genetic testing for precise treatment decision-making.

Publisher Correction: Safety and feasibility of CRISPR-edited T cells in patients with refractory non-small-cell lung cancer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Safety and feasibility of CRISPR-edited T cells in patients with refractory non-small-cell lung cancer.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 editing of immune checkpoint genes could improve the efficacy of T cell therapy, but the first necessary undertaking is to understand the safety and feasibility. Here, we report results from a first-in-human phase I clinical trial of CRISPR-Cas9 PD-1-edited T cells in patients with advanced non-small-cell lung cancer (ClinicalTrials.gov NCT02793856). Primary endpoints were safety and feasibility, and the secondary endpoint was efficacy. The exploratory objectives included tracking of edited T cells. All prespecified endpoints were met. PD-1-edited T cells were manufactured ex vivo by cotransfection using electroporation of Cas9 and single guide RNA plasmids. A total of 22 patients were enrolled; 17 had sufficient edited T cells for infusion, and 12 were able to receive treatment. All treatment-related adverse events were grade 1/2. Edited T cells were detectable in peripheral blood after infusion. The median progression-free survival was 7.7 weeks (95% confidence interval, 6.9 to 8.5 weeks) and median overall survival was 42.6 weeks (95% confidence interval, 10.3-74.9 weeks). The median mutation frequency of off-target events was 0.05% (range, 0-0.25%) at 18 candidate sites by next generation sequencing. We conclude that clinical application of CRISPR-Cas9 gene-edited T cells is generally safe and feasible. Future trials should use superior gene editing approaches to improve therapeutic efficacy.

Genome-wide identification and expression analysis of the BTB domain-containing protein gene family in tomato.

BTB (broad-complex, tramtrack, and bric-a-brac) family proteins are characterized by the presence of a protein-protein interaction BTB domain. BTB proteins have diverse functions, including transcriptional regulation, protein degradation, chromatin remodeling, and cytoskeletal regulation. However, little is known about this gene family in tomato (Solanum lycopersicum), the most important model plant for crop species. In this study, 38 BTB genes were identified based on tomato whole-genome sequence. Phylogenetic analysis of BTB proteins in tomato revealed that SlBTB proteins could be divided into at least 4 subfamilies. The SlBTB proteins contains 1-3 BTB domains, and several other types of functional domains, including KCTD (Potassium channel tetramerization domain-containing), the MATH (meprin and TRAF homology), ANK (Ankyrin repeats), NPR1 (nonexpressor of pathogenesis-related proteins1), NPH3 (Nonphototropic Hypocotyl 3), TAZ zinc finger, C-terminal Kelch, Skp1 and Arm (Armadillo/beta-catenin-like repeat) domains are also found in some tomato BTB proteins. Moreover, their expression patterns in tissues/stages, in response to different abiotic stress treatments and hormones were also investigated. This study provides the first comprehensive analysis of BTB gene family in the tomato genome. The data will undoubtedly be useful for better understanding the potential functions of BTB genes, and their possible roles in mediating hormone cross-talk and abiotic stress in tomato as well as in some other relative species.

A Global View of Transcriptome Dynamics During Male Floral Bud Development in Populus tomentosa.

To obtain a comprehensive overview of the dynamic transcriptome during male floral bud development in Populus tomentosa, high-throughput RNA-seq was conducted during eight flowering-related stages. Among the 109,212 de novo assembled unigenes, 6,959 were differentially expressed during the eight stages. The overrepresented classed of genes identified by Gene Ontology (GO) enrichment included 'response to environmental stimuli' and 'plant-type spore development'. One-third of the differentially expressed genes were transcription factors (TFs). Several genes and gene families were analyzed in depth, including MADS-box TFs, Squamosa promoter binding protein-like family, receptor-like kinases, FLOWERING LOCUS T/TERMINAL-FLOWER-LIKE 1 family, key genes involved in anther and tapetum development, as well as LEAFY, WUSCHEL and CONSTANS. The results provided new insights into the roles of these and other well known gene families during the annual flowering cycle. To explore the mechanisms regulating poplar flowering, a weighted gene co-expression network was constructed using 98 floral-related genes involved in flower meristem identity and flower development. Many modules of co-expressed genes and hub genes were identified, such as APETALA1 and HUA1. This work provides many new insights on the annual flowering cycle in a perennial plant, and a major new resource for plant biology and biotechnology.

De novo Sequencing and Transcriptome Analysis Reveal Key Genes Regulating Steroid Metabolism in Leaves, Roots, Adventitious Roots and Calli of Periploca sepium Bunge.

Periploca sepium Bunge is a traditional medicinal plant, whose root bark is important for Chinese herbal medicine. Its major bioactive compounds are C21 steroids and periplocin, a kind of cardiac glycoside, which are derived from the steroid synthesis pathway. However, research on P. sepium genome or transcriptomes and their related genes has been lacking for a long time. In this study we estimated this species nuclear genome size at 170 Mb (using flow cytometry). Then, RNA sequencing of four different tissue samples of P. sepium (leaves, roots, adventitious roots, and calli) was done using the sequencing platform Illumina/Solexa Hiseq 2,500. After de novo assembly and quantitative assessment, 90,375 all-transcripts and 71,629 all-unigenes were finally generated. Annotation efforts that used a number of public databases resulted in detailed annotation information for the transcripts. In addition, differentially expressed genes (DEGs) were identified by using digital gene profiling based on the reads per kilobase of transcript per million reads mapped (RPKM) values. Compared with the leaf samples (L), up-regulated genes and down-regulated genes were eventually obtained. To deepen our understanding of these DEGs, we performed two enrichment analyses: gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Here, the analysis focused upon the expression characteristics of those genes involved in the terpene metabolic pathway and the steroid biosynthesis pathway, to better elucidate the molecular mechanism of bioactive steroid synthesis in P. sepium. The bioinformatics analysis enabled us to find many genes that are involved in bioactive steroid biosynthesis. These genes encoded acetyl-CoA acetyltransferase (ACAT), HMG-CoA synthase (HMGS), HMG-CoA reductase (HMGR), mevalonate kinase (MK), phosphomevalonate kinase (PMK), mevalonate diphosphate decarboxylase (MDD), isopentenylpyrophosphate isomerase (IPPI), farnesyl pyrophosphate synthase (FPS), squalene synthase (SS), squalene epoxidase (SE), cycloartenol synthase (CAS), sterol C-24 methyltransferase (SMT1), sterol-4alpha-methyl oxidase 1 (SMO1), sterol 14alpha-demethylase (CYP51/14-SDM), delta(14)-sterol reductase (FK/14SR), C-8,7 sterol isomerase (HYD1), sterol-4alpha-methyl oxidase 2 (SMO2), delta(7)-sterol-C5(6)-desaturase (STE1/SC5DL), 7-dehydrocholesterol reductase (DWF5/DHCR7), delta (24)-sterol reductase (DWF1/DHCR24), sterol 22-desaturase (CYP710A), progesterone 5beta-reductase (5ß-POR), 3-beta-hydroxysteroid dehydrogenase (3ß-HSD). This research will be helpful to further understand the mechanism of bioactive steroid biosynthesis in P. sepium, namely C21 steroid and periplocin biosynthesis.

Genome-Wide Identification of the Invertase Gene Family in Populus.

Invertase plays a crucial role in carbohydrate partitioning and plant development as it catalyses the irreversible hydrolysis of sucrose into glucose and fructose. The invertase family in plants is composed of two sub-families: acid invertases, which are targeted to the cell wall and vacuole; and neutral/alkaline invertases, which function in the cytosol. In this study, 5 cell wall invertase genes (PtCWINV1-5), 3 vacuolar invertase genes (PtVINV1-3) and 16 neutral/alkaline invertase genes (PtNINV1-16) were identified in the Populus genome and found to be distributed on 14 chromosomes. A comprehensive analysis of poplar invertase genes was performed, including structures, chromosome location, phylogeny, evolutionary pattern and expression profiles. Phylogenetic analysis indicated that the two sub-families were both divided into two clades. Segmental duplication is contributed to neutral/alkaline sub-family expansion. Furthermore, the Populus invertase genes displayed differential expression in roots, stems, leaves, leaf buds and in response to salt/cold stress and pathogen infection. In addition, the analysis of enzyme activity and sugar content revealed that invertase genes play key roles in the sucrose metabolism of various tissues and organs in poplar. This work lays the foundation for future functional analysis of the invertase genes in Populus and other woody perennials.

Overexpression of AtAP1M3 regulates flowering time and floral development in Arabidopsis and effects key flowering-related genes in poplar.

APETALA1 plays a crucial role in the transition from vegetative to reproductive phase and in floral development. In this study, to determine the effect of AP1 expression on flowering time and floral organ development, transgenic Arabidopsis and poplar overexpressing of AtAP1M3 (Arabidopsis AP1 mutant by dominant negative mutation) were generated. Transgenic Arabidopsis with e35Spro::AtAP1M3 displayed phenotypes with delayed-flowering compared to wild-type and flowers with abnormal sepals, petals and stamens. In addition, transgenic Arabidopsis plants exhibited reduced growth vigor compared to the wild-type plants. Ectopic expression of AtAP1M3 in poplar resulted in up- or down-regulation of some endogenous key flowering-related genes, including floral meristems identity gene LFY, B-class floral organ identity genes AP3 and PI, flowering pathway integrator FT1 and flower repressors TFL1 and SVP. These results suggest that AtAP1M3 regulates flowering time and floral development in plants.