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Arabidopsis thaliana purple acid phosphatase 2 (AtPAP2), which is anchored to the outer membranes of chloroplasts and mitochondria, affects carbon metabolism by modulating the import of some preproteins into chloroplasts and mitochondria. AtPAP9 bears a 72% amino acid sequence identity with AtPAP2, and both proteins carry a hydrophobic motif at their C-termini. Here, we show that AtPAP9 is a tail-anchored protein targeted to the outer membrane of chloroplasts. Yeast two-hybrid and bimolecular fluorescence complementation experiments demonstrated that both AtPAP9 and AtPAP2 bind to a small subunit of rubisco 1B (AtSSU1B) and a number of chloroplast proteins. Chloroplast import assays using [35S]-labeled AtSSU1B showed that like AtPAP2, AtPAP9 also plays a role in AtSSU1B import into chloroplasts. Based on these data, we propose that AtPAP9 and AtPAP2 perform overlapping roles in modulating the import of specific proteins into chloroplasts. Most plant genomes contain only one PAP-like sequence encoding a protein with a hydrophobic motif at the C-terminus. The presence of both AtPAP2 and AtPAP9 in the Arabidopsis genome may have arisen from genome duplication in Brassicaceae. Unlike AtPAP2 overexpression lines, the AtPAP9 overexpression lines did not exhibit early-bolting or high-seed-yield phenotypes. Their differential growth phenotypes could be due to the inability of AtPAP9 to be targeted to mitochondria, as the overexpression of AtPAP2 on mitochondria enhances the capacity of mitochondria to consume reducing equivalents.

Efficient photosynthesis requires a balance of ATP and NADPH production/consumption in chloroplasts, and the exportation of reducing equivalents from chloroplasts is important for balancing stromal ATP/NADPH ratio. Here, we showed that the overexpression of purple acid phosphatase 2 on the outer membranes of chloroplasts and mitochondria can streamline the production and consumption of reducing equivalents in these two organelles, respectively. A higher capacity of consumption of reducing equivalents in mitochondria can indirectly help chloroplasts to balance the ATP/NADPH ratio in stroma and recycle NADP+, the electron acceptors of the linear electron flow (LEF). A higher rate of ATP and NADPH production from the LEF, a higher capacity of carbon fixation by the Calvin–Benson–Bassham (CBB) cycle and a greater consumption of NADH in mitochondria enhance photosynthesis in the chloroplasts, ATP production in the mitochondria and sucrose synthesis in the cytosol and eventually boost plant growth and seed yields in the overexpression lines.

The challenge of monitoring in planta dynamic changes of NADP(H) and NAD(H) redox states at the subcellular level is considered a major obstacle in plant bioenergetics studies. Here, we introduced two circularly permuted yellow fluorescent protein sensors, iNAP and SoNar, into Arabidopsis thaliana to monitor the dynamic changes in NADPH and the NADH/NAD+ ratio. In the light, photosynthesis and photorespiration are linked to the redox states of NAD(P)H and NAD(P) pools in several subcellular compartments connected by the malate-OAA shuttles. We show that the photosynthetic increases in stromal NADPH and NADH/NAD+ ratio, but not ATP, disappear when glycine decarboxylation is inhibited. These observations highlight the complex interplay between chloroplasts and mitochondria during photosynthesis and support the suggestions that, under normal conditions, photorespiration supplies a large amount of NADH to mitochondria, exceeding its NADH-dissipating capacity, and the surplus NADH is exported from the mitochondria to the cytosol through the malate-OAA shuttle., NADP(H) and NAD(H) are crucial energy molecules in plant metabolism. Here, via the use of circularly permutated fluorescent protein sensors, the authors demonstrate dynamic changes in NADPH and the NADH/NAD+ ratio during photosynthesis and photorespiration at the subcellular level in planta.

Natural antineoplastic drug development is crucial to treatment of clinical oncology. Dihydromyricetin, a bioactive flavonoid compound was extracted from the stems and leaves of Ampelopsis grossedentata. It exhibited anticancer activity and induced apoptosis in human hepatocellular carcinoma cells according to our previous studies. In this study, we demonstrated that DHM could significantly inhibit proliferation and induce apoptosis in HepG2 cells with MTT and Flow Cytometry methods. It is very interesting that we found DHM could regulate TGF-β signal pathway and which has a crosstalk with P53, Smad3 and P-Smad2/3 proteins. Meanwhile, we confirmed that DHM showed antitumor activity by regulating the activation of the p53-dependent pathways (MDM2, P-MDM2, BAX and Bcl-2). These findings defined and supported a novel mechanism that DHM could induce cell apoptosis by reducing TGF-β via p53 signal pathway in HepG2 cells.

e15092 Background: Receptor tyrosine kinases (RTKs) are a class of tyrosine kinases that regulate cell-to-cell communication and control a variety of complex biological functions. Dysregulation of RTK signaling partly due to chromosomal rearrangements leads to novel tyrosine kinase fusion oncoproteins which are possibly driver alterations to cancers. Targeting some RTK fusions with specific tyrosine kinases inhibitors (TKIs) is an effective therapeutic strategy across a spectrum of RTK fusion-related cancers. However, there is still a paucity of extensive RTK fusion investigations in breast cancer. We aimed to characterize RTK fusions in Chinese breast cancer patients. Methods: An in-house sequencing database of 1440 Chinese breast cancer patients using a 520-gene NGS sequencing panel was thoroughly reviewed. RTK fusion was defined as an in-frame fusion with the tyrosine kinase domain of the RTK completely retained with the only exception of ERBB2 fusion which was not counted due to its unclear significance. Concomitant mutations and TMB were also analyzed and calculated. Patients’ clinical characteristics were retrieved from case records. Results: 27 RTK fusion-positive breast cancers (12 tissues + 15 plasmas) were identified, patients had a median age of 52 years. Triple-negative breast cancer subtype comprised 37% with luminal and HER2 positive subtypes being 40.8% and 22.2%, respectively. 77.8% of patients were at stage IV and 22.2% at stage I-III. Ten were treatment naïve. RTK fusions occurred in 2% of breast cancers in our database, compared with the prevalence of 0.6% and 1.3% in MSKCC and TCGA, respectively. In the subset of stage IV patients, our database showed a significantly higher RTK fusion frequency than that in MSKCC (3.2% vs. 0.6%, p = 0.013). FGFR2 fusions were seen most commonly (n = 7), followed by RET (n = 4), ROS1 (n = 3), NTRK3 (n = 3), BRAF (n = 2), and NTRK1 (n = 2). Other RTK fusions including ALK, EGFR, FGFR1, FGFR3, MET, and NTRK2 were identified in one patient each. Of note, the normalized abundance of RTK fusion (fusion AF/max AF) correlated negatively with TMB (r = -0.48, p = 0.017). Patients with TMB < 4 (Muts/Mb) displayed a higher fusion abundance than those with TMB ≥ 4 (Muts/Mb) (p = 0.018), suggesting a higher likelihood of subclonal nature for RTK fusions in TMB-high patients. Moreover, CREBBP mutation only co-occurred with FGFR2 fusion (p = 0.012), while NTRK3 fusion and TP53 mutation were mutually exclusive (p = 0.019). Conclusions: This is the first study comprehensively delineating the prevalence and spectrum of RTK fusions in Chinese breast cancers. Further study is ongoing to identify the enriched subpopulation which may benefit from RTK fusion inhibitors.

e15535 Background: To improve the prognosis of resected lung cancer patients, growing efforts are being invested in finding the most optimal approach to detect MRD and predict relapse. In our study, we aimed to investigate the utility of ctDNA methylation profiling in detecting MRD from patients with resected early-stage NSCLC. Methods: Surgically-resected tumor tissues were obtained from 65 patients diagnosed with resectable stage IA-III NSCLC with various histological subtypes. Matched blood samples were also collected before surgery (baseline) and during regular follow-up after 2-8 weeks of surgery. Comprehensive somatic mutation and methylation level profile from circulating tumor DNA (ctDNA) were performed using unique molecular identifier-based targeted sequencing and targeted bisulfite sequencing, respectively. A tumor-informed MRD prediction model was constructed based on methylation levels obtained from the patient’s resected tumor tissue to calculate the corresponding methylation signal intensity from the matched plasma sample of the patient at baseline or other time points during follow-up, which is reflected as MRD score. Results: Of the 28 patients with baseline ctDNA methylation data, 28.6% (8/28) of the patients had elevated ctDNA methylation levels at first post-operative follow-up (F1) as compared to baseline, indicating the possibility of MRD. Meanwhile, of the 20 patients (71.4%, 20/28) who had reduced ctDNA methylation levels at F1, elevation of ctDNA methylation level was detected from 3 and 1 patients at second (F2) and third (F3) follow-up, respectively. Based on the MRD prediction model, 17.9% (5/28) of the patients had higher MRD scores at F1. Of the 23 patients with lower MRD scores at F1, 5, 1 and 1 patients had an elevation in MRD scores at F2, F3, and F4, respectively, which indicates a possible MRD. Disease relapse was radiologically confirmed after 10-16 months post-surgery in three patients with concomitant elevation of ctDNA methylation level and MRD score during follow-up between 2-9 months prior to radiologic relapse. Conclusions: Our results demonstrate that post-operative ctDNA methylation levels could be used to detect MRD in patients with resected NSCLC. Moreover, ctDNA methylation-based prediction model of MRD could serve as a potential model to predict relapse in early-stage as well as disease progression in advanced-stage NSCLC.

e18085 Background: BRCA1/2 mutated cancers are homologous recombination repair (HRR) deficient and often sensitive to drugs targeting DNA repair pathway. Emerging evidences suggest tumors with mutations in other HRR genes might also respond favorably to DNA repair pathway targeted drugs; however, the results were inconclusive. Genomic instability is characterized by telomeric allelic imbalance (TAI), large-scale transition (LST) and loss of heterozygosity (LOH). Here, we developed an HRD score based on assessing the status of these patterns in tumors. Methods: 73 cancer patients, including 36 breast cancer (BC), 35 ovarian cancer (OC) and 2 prostate cancer (PC) patients were recruited. 28 were BRCA-deficient tumors (10 BCs, 17OCs and 1 PC) harboring pathogenic/likely pathogenic (P/LP) germline mutation in BRCA1/2 with LOH. 13 BRCA-mutated tumors (10 BCs, 2 OCs and 1 PC) carried P/LP BRCA1/2 mutation without LOH. The rest 33 were HRR wild type controls (16 BCs and 16 OCs). All samples were profiled using a panel consisting of 24 classic HRR genes and their adjacent SNPs. The scores for TAI, LST and LOH were developed separately and an incorporated HRD score based on the 3 patterns was established. Clinical data of 42 OC pts were collected to validate the algorithm. Results: All pairwise correlations of TAI, LST and LOH scores showed high correlations (TAI vs. LOH: r = 0.89; TAI vs. LST: r = 0.92; LST vs. LOH: r = 0.83). The univariate logistic regression was used to assess the correlation of the score with BRCA deficiency. In the cohort of BRCA-deficient vs. HRR-negative, the scores of TAI (P = 9.72 x 10−4), LST (P = 8.75 x 10−5), LOH (P = 0.01) and HRD (P = 3.35 x 10−4) all showed significant associations with BRCA-deficiency. In the cohort of BRCA-deficient vs. BRCA-mutated +HRR-negative, all the scores also highly correlated with BRCA-deficiency (TAI: P = 2.35 x 10−4, LST: P = 1.48 x 10−5; LOH: P = 2.36 x 10−4, HRD: P = 6.29 x 10−5). Using 42 as a cutoff, it achieved sensitivities of 94.1%, 60% and 82% for identifying BRCA-deficient tumors in the 35 OCs, 36 BCs and in all 73 tumors, respectively. We further validated the predictive value of HRD score in an additional 42 OC pts and found significantly higher HRD sore in pts sensitive to platinum therapy (P = 0.036). Patients having an HRD score > 42 had a significantly longer PFS than pts with a score < 42 (p = 0.04). Conclusions: Our study developed a HRD score base on assessing 3 patterns of genomic instability (TAI, LST and LOH). The HRD score revealed a promising value in detecting HR-deficient tumors (especially BRCA-deficient).

Rationale: Medullary thyroid carcinoma (MTC) is a rare type thyroid carcinoma originating from the thyroid parafollicular cells (C cells). Chemotherapy has a limited efficacy for treating persistent or recurrent MTC. Patient concerns: A 46-year-old woman who underwent thyroidectomy for MTC in December 2007. She began experience recurring diarrhea in January 2015 and started to cough and feel shortness of breath in March 2016. Diagnoses: A chestcomputed tomography (CT) scan showed metastases in the bilateral lungs, pulmonary hilum, and mediastinal lymph nodes. Percutaneous biopsy of the pulmonary occupying lesions performed on March 21, 2016 indicated medullary carcinoma metastases at the right pulmonary hilum. Interventions: This patient was treated with oral apatinib (500 mg daily). Outcomes: The patient's symptoms of diarrhea, coughing, and shortness of breath disappeared. CT reexaminations for efficacy assessment at 1, 2, and 3 months after the treatment indicated partial remission. Systemic migrating bone and joint pains occurred during the treatment, which were considered to be adverse events of apatinib. Lessons: Treatment of MTC with apatinib has been shown to be effective in our case. Tyrosine kinase inhibitors (TKIs) that suppress rearranged during transfection (RET) and vascular endothelial growth factor receptor (VEGFR) should be considered as a effective therapeutic approaches.

Significance By studying in vivo changes of ATP levels in the plastids and cytosol of Arabidopsis thaliana using a FRET-based ATP sensor, we show that the plastidic ATP concentrations in cotyledon, hypocotyl, and root of 10-day-old seedlings are significantly lower than the cytosolic concentrations. We show that chloroplasts consume ATP rapidly and the import of ATP into mature chloroplasts is impeded by the low density of NTT transporter. Hence, unlike in diatoms, where ATP is imported into chloroplasts to support the Calvin–Benson–Bassham (CBB) cycle, mature chloroplasts of Arabidopsis do not balance the ATP:NADPH ratio by importing ATP from the cytosol. Rather, chloroplasts can export surplus reducing equivalents, which can be used by the mitochondria to supply ATP to the cytosol., Matching ATP:NADPH provision and consumption in the chloroplast is a prerequisite for efficient photosynthesis. In terms of ATP:NADPH ratio, the amount of ATP generated from the linear electron flow does not meet the demand of the Calvin–Benson–Bassham (CBB) cycle. Several different mechanisms to increase ATP availability have evolved, including cyclic electron flow in higher plants and the direct import of mitochondrial-derived ATP in diatoms. By imaging a fluorescent ATP sensor protein expressed in living Arabidopsis thaliana seedlings, we found that MgATP2− concentrations were lower in the stroma of mature chloroplasts than in the cytosol, and exogenous ATP was able to enter chloroplasts isolated from 4- and 5-day-old seedlings, but not chloroplasts isolated from 10- or 20-day-old photosynthetic tissues. This observation is in line with the previous finding that the expression of chloroplast nucleotide transporters (NTTs) in Arabidopsis mesophyll is limited to very young seedlings. Employing a combination of photosynthetic and respiratory inhibitors with compartment-specific imaging of ATP, we corroborate the dependency of stromal ATP production on mitochondrial dissipation of photosynthetic reductant. Our data suggest that, during illumination, the provision and consumption of ATP:NADPH in chloroplasts can be balanced by exporting excess reductants rather than importing ATP from the cytosol.

The nucleus-encoded mitochondria-targeted proteins, multiple organellar RNA editing factors (MORF3, MORF5, and MORF6), interact with Arabidopsis (Arabidopsis thaliana) PURPLE ACID PHOSPHATASE2 (AtPAP2) located on the chloroplast and mitochondrial outer membranes in a presequence-dependent manner. Phosphorylation of the presequence of the precursor MORF3 (pMORF3) by endogenous kinases in wheat germ translation lysate, leaf extracts, or STY kinases, but not in rabbit reticulocyte translation lysate, resulted in the inhibition of protein import into mitochondria. This inhibition of import could be overcome by altering threonine/serine residues to alanine on the presequence, thus preventing phosphorylation. Phosphorylated pMORF3, but not the phosphorylation-deficient pMORF3, can form a complex with 14-3-3 proteins and HEAT SHOCK PROTEIN70. The phosphorylation-deficient mutant of pMORF3 also displayed faster rates of import when translated in wheat germ lysates. Mitochondria isolated from plants with altered amounts of AtPAP2 displayed altered protein import kinetics. The import rate of pMORF3 synthesized in wheat germ translation lysate into pap2 mitochondria was slower than that into wild-type mitochondria, and this rate disparity was not seen for pMORF3 synthesized in rabbit reticulocyte translation lysate, the latter translation lysate largely deficient in kinase activity. Taken together, these results support a role for the phosphorylation and dephosphorylation of pMORF3 during the import into plant mitochondria. These results suggest that kinases, possibly STY kinases, and AtPAP2 are involved in the import of protein into both mitochondria and chloroplasts and provide a mechanism by which the import of proteins into both organelles may be coordinated.

Arabidopsis thaliana purple acid phosphatase 2 (AtPAP2) is the only phosphatase that is dual-targeted to both chloroplasts and mitochondria. Like Toc33/34 of the TOC and Tom 20 of the TOM, AtPAP2 is anchored to the outer membranes of chloroplasts and mitochondria via a hydrophobic C-terminal motif. AtPAP2 on the mitochondria was previously shown to recognize the presequences of several nuclear-encoded mitochondrial proteins and modulate the import of pMORF3 into the mitochondria. Here we show that AtPAP2 binds to the small subunit of Rubisco (pSSU) and that chloroplast import experiments demonstrated that pSSU was imported less efficiently into pap2 chloroplasts than into wild-type chloroplasts. We propose that AtPAP2 is an outer membrane-bound phosphatase receptor that facilitates the import of selected proteins into chloroplasts.

Role-Based Access Control (RBAC) is a kind of access control mechanism which is convenient and secure. To protect the resources of USB cryptographic card, a RBAC method is purposed and implemented in this paper, which meets the need of access control, least privilege, small computation and managed easily. Analyses the need of the USB cryptographic card, compares several main access control models, introduces the characteristic and application advantages of RBAC, finally demonstrates in detail how to apply RBAC in USB cryptographic card.