microRNA regulates cytokinin induced parthenocarpy in cucumber (Cucumis sativus L.).

Parthenocarpy is one of the most important agronomic traits for fruit yield in cucumbers. However, the precise gene regulation and the posttranscriptional mechanism are elusive. In the presented study, one parthenocarpic line DDX and non-parthenocarpic line ZK were applied to identify the microRNAs (miRNAs) involved in parthenocarpic fruit formation. The differential expressed miRNAs among parthenocarpic fruit of forchlorfenuron (CPPU) treated ZK (ZK-CPPU), pollinated ZK (ZK-P), non-pollinated DDX (DDX-NP) were compared with the non-parthenocarpic fruits of non-pollinated ZK (ZK-NP). It indicated 98 miRNAs exhibited differential expression were identified. Notably, a significant proportion of these miRNAs were enriched in the signal transduction pathway of plant hormones, as identified by the KEGG pathway analysis. qRT-PCR validation indicated that CsmiR156 family was upregulated in the ZK-NP while downregulated in ZK-CPPU, ZK-P, and DDX-NP at 1 day after anthesis. Meanwhile, the opposite trend was observed for CsmiR164a. In ZK-CPPU, ZK-P, and DDX-NP, CsmiRNA156 genes (CsSPL16 and CsARR9-like) were upregulated while CsmiRNA164a genes (CsNAC6, CsCUC1, and CsNAC100) were downregulated. The GUS and dual luciferase assay validated that CsmiR156a inhibited while CsmiR164a induced their target genes' transcription. This study presents novel insights into the involvement of CsmiR156a and CsmiR164a in the CK-mediated posttranscriptional regulation of cucumber parthenocarpy, which will aid future breeding programs.

R2R3-MYB transcription factor CsMYB60 controls mature fruit skin color by regulating flavonoid accumulation in cucumber.

Skin color is an important trait that determines the cosmetic appearance and quality of fruits. In cucumber, the skin color ranges from white to brown in mature fruits. However, the genetic basis for this important trait remains unclear. We conducted a genome-wide association study of natural cucumber populations, along with map-based cloning techniques, on an F2 population resulting from a cross between Pepino (with yellow-brown fruit skin) and Zaoer-N (with creamy fruit skin). We identified CsMYB60 as a candidate gene responsible for skin coloration in mature cucumber fruits. In cucumber accessions with white to pale yellow skin color, a premature stop mutation (C to T) was found in the second exon region of CsMYB60, whereas light yellow cucumber accessions exhibited splicing premature termination caused by an intronic mutator-like element insertion in CsMYB60. Transgenic CsMYB60c cucumber plants displayed a yellow-brown skin color by promoting accumulation of flavonoids, especially hyperoside, a yellow-colored flavonol. CsMYB60c encodes a nuclear protein that primarily acts as a transcriptional activator through its C-terminal activation motif. RNA sequencing and DNA affinity purification sequencing assays revealed that CsMYB60c promotes skin coloration by directly binding to the YYTACCTAMYT motif in the promoter regions of flavonoid biosynthetic genes, including CsF3'H, which encodes flavonoid 3'-hydroxylase. The findings of our study not only offer insight into the function of CsMYB60 as dominantly controlling fruit coloration, but also highlight that intronic DNA mutations can have a similar phenotypic impact as exonic mutations, which may be valuable in future cucumber breeding programs.

RNA-seq-based comparative transcriptome analysis reveals the role of CsPrx73 in waterlogging-triggered adventitious root formation in cucumber.

Abiotic stressors like waterlogging are detrimental to cucumber development and growth. However, comprehension of the highly complex molecular mechanism underlying waterlogging can provide an opportunity to enhance cucumber tolerance under waterlogging stress. We examined the hypocotyl and stage-specific transcriptomes of the waterlogging-tolerant YZ026A and the waterlogging-sensitive YZ106A, which had different adventitious rooting ability under waterlogging. YZ026A performed better under waterlogging stress by altering its antioxidative machinery and demonstrated a greater superoxide ion (O 2-) scavenging ability. KEGG pathway enrichment analysis showed that a high number of differentially expressed genes (DEGs) were enriched in phenylpropanoid biosynthesis. By pairwise comparison and weighted gene co-expression network analysis analysis, 2616 DEGs were obtained which were categorized into 11 gene co-expression modules. Amongst the 11 modules, black was identified as the common module and yielded a novel key regulatory gene, CsPrx73. Transgenic cucumber plants overexpressing CsPrx73 enhance adventitious root (AR) formation under waterlogging conditions and increase reactive oxygen species (ROS) scavenging. Silencing of CsPrx73 expression by virus-induced gene silencing adversely affects AR formation under the waterlogging condition. Our results also indicated that CsERF7-3, a waterlogging-responsive ERF transcription factor, can directly bind to the ATCTA-box motif in the CsPrx73 promoter to initiate its expression. Overexpression of CsERF7-3 enhanced CsPrx73 expression and AR formation. On the contrary, CsERF7-3-silenced plants decreased CsPrx73 expression and rooting ability. In conclusion , our study demonstrates a novel CsERF7-3-CsPrx73 module that allows cucumbers to adapt more efficiently to waterlogging stress by promoting AR production and ROS scavenging.

Gold nanoclusters-based fluorescence resonance energy transfer for rapid and sensitive detection of Pb2.

Lead pollution has remained a significant global concern for several decades due to its detrimental effects on the brain, heart, kidneys, lungs, and immune system across all age groups. Addressing the demand for detecting trace amounts of lead in food samples, we have developed a novel biosensor based on fluorescence resonance energy transfer (FRET) from fluorescein R6G to gold nanoclusters (AuNCs-CCY). By utilizing polypeptides as a template, we successfully synthesized AuNCs-CCY with an excitation spectrum that overlaps with the emission spectrum of R6G. Exploiting the fact that Pb2+ induces the aggregation of gold nanoclusters, leading to the separation of R6G from AuNCs-CCY and subsequent fluorescence recovery, we achieved the quantitative detection of Pb2+. Within the concentration range of 0.002-0.20 µM, a linear relationship was observed between the fluorescence enhancement value (F-F0) and Pb2+ concentration, characterized by the linear equation y = 2398.69x + 87.87 (R2 = 0.996). The limit of detection (LOD) for Pb2+ was determined to be 0.00079 µM (3σ/K). The recovery rate ranged from 96 % to 104 %, with a relative standard deviation (RSD) below 10 %. These findings demonstrate the potential application value of our biosensor, which offers a promising approach to address the urgent need for sensitive detection of heavy metal ions, specifically Pb2+, in food samples.

Retrospective analysis of discordant results between histology and other clinical diagnostic tests on helicobacter pylori infection.

BACKGROUND: A reliable test is essential for diagnosing Helicobacter pylori (H. pylori) infection, and crucial for managing H. pylori-related diseases. Serving as an excellent method for detecting H. pylori infection, histologic examination is a test that clinicians heavily rely on, especially when complemented with immunohistochemistry (IHC). Additionally, other diagnostic tests for H. pylori, such as the rapid urease test (CLO test) and stool antigen test (SA), are also highly sensitive and specific. Typically, the results of histology and other tests align with each other. However, on rare occasions, discrepancy between histopathology and other H. pylori diagnostic tests occurs. AIM: To investigate the discordance between histology and other H. pylori tests, the underlying causes, and the impact on clinical management. METHODS: Pathology reports of gastric biopsies were retrieved spanning August 2013 and July 2018. Reports were included in the study only if there were other H. pylori tests within seven days of the biopsy. These additional tests include CLO test, SA, and H. pylori culture. Concordance between histopathology and other tests was determined based on the consistency of results. In instances where histology results were negative while other tests were positive, the slides were retrieved for re-assessment, and the clinical chart was reviewed. RESULTS: Of 1396 pathology reports were identified, each accompanied by one additional H. pylori test. The concordance rates in detecting H. pylori infection between biopsy and other tests did not exhibit significant differences based on the number of biopsy fragments. 117 discrepant cases were identified. Only 20 cases (9 with CLO test and 11 with SA) had negative biopsy but positive results in other tests. Four cases initially stained with Warthin-Starry turned out to be positive for H. pylori with subsequent IHC staining. Among the remaining 16 true discrepant cases, 10 patients were on proton pump inhibitors before the biopsy and/or other tests. Most patients underwent treatment, except for two who were untreated, and two patients who were lost to follow-up. CONCLUSION: There are rare discrepant cases with negative biopsy but positive in SA or CLO test. Various factors may contribute to this inconsistency. Most patients in such cases had undergone treatment.

Genetic liability between COVID-19 and pre-eclampsia/eclampsia: a Mendelian randomization study.

OBJECTIVE: The aim of this study was to investigate the possible causal relationship between COVID-19 and the risk of pre-eclampsia/eclampsia using a Mendelian randomized (MR) design. METHODS: We estimated their genetic correlations and then performed two-sample Mendelian randomization analyses using pooled statistics from the COVID-19 susceptibility/hospitalization genome-wide association study and the pre-eclampsia/eclampsia datasets. The main analyses were performed using the inverse variance weighting method, supplemented by the weighted median method and the MR-Egger method. RESULTS: We identified a significant and positive genetic correlation between COVID-19 susceptibility and pre-eclampsia/eclampsia [OR = 1.23 (1.01-1.51), p = 0.043]. Meanwhile, hospitalization of COVID-19 was significantly associated with a higher risk of pre-eclampsia/eclampsia [OR = 1.15 (1.02-1.30), p = 0.024]. Consistently, hospitalization of COVID-19 were nominally associated with higher risk of pre-eclampsia [OR = 1.14, (1.01-1.30), p = 0.040]. The results were robust under all sensitivity analyses. CONCLUSION: These results suggest that COVID-19 may increase the risk of pre-eclampsia/eclampsia. Future development of preventive or therapeutic interventions should emphasize this to mitigate the complications of COVID-19. [Figure: see text].

A review of recent progress in the application of Raman spectroscopy and SERS detection of microplastics and derivatives.

The global environmental concern surrounding microplastic (MP) pollution has raised alarms due to its potential health risks to animals, plants, and humans. Because of the complex structure and composition of microplastics (MPs), the detection methods are limited, resulting in restricted detection accuracy. Surface enhancement of Raman spectroscopy (SERS), a spectral technique, offers several advantages, such as high resolution and low detection limit. It has the potential to be extensively employed for sensitive detection and high-resolution imaging of microplastics. We have summarized the research conducted in recent years on the detection of microplastics using Raman and SERS. Here, we have reviewed qualitative and quantitative analyses of microplastics and their derivatives, as well as the latest progress, challenges, and potential applications.

Corrigendum: Analysis of a super-transmission of SARS-CoV-2 omicron variant BA.5.2 in the outdoor night market.

[This corrects the article DOI: 10.3389/fpubh.2023.1153303.].

A review on surfactin: molecular regulation of biosynthesis.

Surfactin has many biological activities, such as inhibiting plant diseases, resisting bacteria, fungi, viruses, tumors, mycoplasma, anti-adhesion, etc. It has great application potential in agricultural biological control, clinical medical treatment, environmental treatment and other fields. However, the low yield has been the bottleneck of its popularization and application. It is very important to understand the synthesis route and control strategy of surfactin to improve its yield and purity. In this paper, based on the biosynthetic pathway and regulatory factors of surfactin, its biosynthesis regulation strategy was comprehensively summarized, involving enhancement of endogenous and exogenous precursor supply, modification of the synthesis pathway of lipid chain and peptide chain, improvement of secretion and efflux, and manipulation some global regulatory factors, such as Spo0A, AbrB, ComQXP, phrCSF, etc. to directly or indirectly stimulate surfactin synthesis. And the current production and separation and purification process of surfactin are briefly described. This review also provides a scientific reference for promoting surfactin production and its applications in various fields.

Recurrent Primary Peritoneal Psammocarcinoma: A Case Report on a Diagnostic Challenge in Cytology.

Psammocarcinoma (PCa) is a rare variant of low-grade papillary serous carcinoma that can arise from the peritoneal as well as ovarian surfaces. When this tumor involves the extra-ovarian peritoneum significantly and the ovarian surface minimally or not at all, it is considered of peritoneal origin. PCa has a recurrent indolent clinical course. It is challenging to diagnose peritoneal PCa, particularly on cytological smears because of the bland cellular features of neoplastic cells. We report a case of recurrent metastatic primary peritoneal PCa in a 71-year-old female of Ashkenazi Jewish ancestry diagnosed on cytology of ascitic and cystic fluid.

Cucumber abscisic acid 8'-hydroxylase Csyf2 regulates yellow flesh by modulating carotenoid biosynthesis.

Cucumber (Cucumis sativus L.) flesh is typically colorless or pale green. Flesh with yellow or orange pigment, determined mainly by carotenoid content and composition, is mostly found in semi-wild Xishuangbanna cucumber, which has a very narrow genetic background. Here, we identified a spontaneous cucumber mutant with yellow flesh (yf-343), which accumulated more ß-cryptoxanthin and less lutein than regular cultivated European glasshouse-type cucumbers. Genetic analysis revealed that the yellow flesh phenotype was controlled by a single recessive gene. Through fine mapping and gene sequencing, we identified the candidate gene C. sativus yellow flesh 2 (Csyf2), encoding an abscisic acid (ABA) 8'-hydroxylase. Overexpression and RNAi-silencing of Csyf2 in cucumber hairy roots produced lower and higher ABA contents than in non-transgenic controls, respectively. Further, RNA-seq analysis suggested that genes related to ABA signal transduction were differentially expressed in fruit flesh between yf-343 and its wild type, BY, with white flesh. The carotenoid biosynthesis pathway was specifically enriched in fruit flesh at 30 days after pollination when yf-343 fruit flesh turns yellow. Our findings highlight a promising target for gene editing to increase carotenoid content, expanding our genetic resources for pigmented cucumber flesh breeding for improving the nutritional quality of cucumber.

Metabolomics and Transcription Profiling of Pumpkin Fruit Reveals Enhanced Bioactive Flavonoids and Coumarins in Giant Pumpkin (Cucurbita maxima).

Atlantic giant (AG, Cucurbita maxima) is a type of giant pumpkin in the Cucurbitaceae family and has the world's largest fruit. AG possesses excellent ornamental and economic value due to its well-known large fruit. However, giant pumpkins are usually thrown away after viewing, thus generating a waste of resources. To explore the additional value of giant pumpkins, a metabolome assay was performed between AG and Hubbard (a small fruit pumpkin) fruits. We found that bioactive compounds, especially flavonoids (including 8-prenylnaringenin, tetrahydrocurcumin, galangin, and acacetin) and coumarins (including coumarin, umbelliferone, 4-coumaryl alcohol, and coumaryl acetate), with extensive antioxidant and pharmacological functions, showed higher accumulation in AG fruit than in Hubbard fruits. Comparative transcriptomics of the two pumpkin fruits indicated that the differentially expressed genes (DEGs) encoding PAL, C4H, 4CL, CSE, HCT, CAD, and CCoAOMT were relatively highly expressed, which promoted an increased accumulation of the identified flavonoids and coumarins in giant pumpkins. In addition, the construction of a co-expression network and cis-element analysis of the promoter demonstrated that differentially expressed MYB, bHLH, AP2, and WRKY transcription factors might play vital roles in regulating the expression of DEGs involved in the biosynthesis of several flavonoids and coumarins. Our current results provide new insights into the accumulation of active compounds in giant pumpkins.

Analysis of a super-transmission of SARS-CoV-2 omicron variant BA.5.2 in the outdoor night market.

Introduction: The COVID-19 pandemic continues to ravage the world, and mutations of the SARS-CoV-2 continues. The new strain has become more transmissible. The role of aerosol transmission in the pandemic deserves great attention. Methods: In this observational study, we collected data from market customers and stallholders who had been exposed to the virus in the Qingkou night market on July 31 and were subsequently infected. We analyzed the possible infection zones of secondary cases and aerosol suspension time in ambient air. We described and analyzed the characteristics of the secondary cases and the transmission routes for customers. Results: The point source outbreak of COVID-19 in Qingkou night market contained a cluster of 131 secondary cases. In a less-enclosed place like the Qingkou night market, aerosols with BA.5.2 strain released by patients could suspend in ambient air up to 1 h 39 min and still be contagious. Conclusion: Aerosols with viruses can spread over a relatively long distance and stay in ambient air for a long time in a less enclosed space, but shorter than that under experimental conditions. Therefore, the aerosol suspension time must be considered when identifying and tracing close contact in outbreak investigations.

Synthesis of nanoparticles via microfluidic devices and integrated applications.

In recent years, nanomaterials have attracted the research intervention of experts in the fields of catalysis, energy, biomedical testing, and biomedicine with their unrivaled optical, chemical, and biological properties. From basic metal and oxide nanoparticles to complex quantum dots and MOFs, the stable preparation of various nanomaterials has always been a struggle for researchers. Microfluidics, as a paradigm of microscale control, is a remarkable platform for online stable synthesis of nanomaterials with efficient mass and heat transfer in microreactors, flexible blending of reactants, and precise control of reaction conditions. We describe the process of microfluidic preparation of nanoparticles in the last 5 years in terms of microfluidic techniques and the methods of microfluidic manipulation of fluids. Then, the ability of microfluidics to prepare different nanomaterials, such as metals, oxides, quantum dots, and biopolymer nanoparticles, is presented. The effective synthesis of some nanomaterials with complex structures and the cases of nanomaterials prepared by microfluidics under extreme conditions (high temperature and pressure), the compatibility of microfluidics as a superior platform for the preparation of nanoparticles is demonstrated. Microfluidics has a potent integration capability to combine nanoparticle synthesis with real-time monitoring and online detection, which significantly improves the quality and production efficiency of nanoparticles, and also provides a high-quality ultra-clean platform for some bioassays.

Changes in the Texture and Flavor of Lotus Root after Different Cooking Methods.

The changes in the texture and flavor of lotus root were determined before and after boiling, steaming and frying. Compared to fresh lotus root, all three kinds of cooking decreased the hardness and springiness, and frying significantly enhanced the gumminess, chewiness and cohesiveness. The flavor components, such as flavor amino acids, nucleotides and their taste character in lotus roots, were determined by liquid chromatography and electronic tongue. The amino acids and nucleotide contents of fresh lotus root were 20.9 and 0.07 µg/kg, respectively. The content of flavor substances in lotus roots decreased obviously, and the texture characteristics decreased after boiling and steaming. After deep-frying for 2 min, the free amino acids and nucleotide contents of lotus root were 32.09 and 0.85 µg/kg, respectively, which were the highest in all cooking methods. The contents of volatile flavor components and their smell character in lotus roots were determined by GC-MS and electronic nose. There were 58 kinds of flavor compounds identified in fresh lotus root, mainly alcohols, esters and olefins. The total amount of volatile flavor compounds decreased, and new compounds, such as benzene derivatives, were produced in lotus roots after boiling and steaming. After deep-frying, the content of volatile flavor compounds in lotus root increased significantly, especially the aldehyde volatile flavor compounds. The production of pyran, pyrazine and pyridine volatile flavor compounds made the lotus root flavor unique and delicious. The taste and smell character of lotus roots before and after cooking were effectively separated by an electronic tongue, nose and PCA analysis; the results suggested the boiled lotus root exhibited the most natural and characteristic taste and smell among the four groups.

Lignin Biosynthesis in Postharvest Water Bamboo (Zizania latifolia) Shoots during Cold Storage Is Regulated by RBOH-Mediated Reactive Oxygen Species Signaling.

Lignification is a major cause of senescence in fresh shoots of water bamboo (Zizania latifolia), which is a popular vegetable in southeast Asia; however, its physiological and molecular mechanisms is less understood. In the present study, lignin content and transcriptome change in postharvested water bamboo shoots under cold storage were investigated. We found that lignin significantly accumulated in the epidermis of the shoots with the increase of firmness. In the cold storage shoots, the major up-regulated genes were involved in phenylpropanoid biosynthesis, plant-pathogen interactions, and starch and sucrose metabolism. The lignin biosynthesis genes PAL, 4CL, C4H, CCoAOMT, CCR, F5H, CAD, and POD family were up-regulated during cold storage, while HCT and C3H were down-regulated. The MAPK signaling pathway was also up-regulated and respiratory burst oxidase homologue (RBOH) genes were strongly up-regulated. Therefore, we investigated the RBOH gene family and their expression profile in water bamboo shoots. The results indicated that 10 ZlRBOHs were up-regulated in cold storage shoots. Diphenyleneiodonium chloride (DPI), an inhibitor of RBOH oxidase, significantly inhibited the expression of genes involved in lignin deposition and biosynthesis, while H2O2 enhanced these processes. These results suggest that lignification of water bamboo shoots is regulated by RBOH-mediated ROS signaling.

Transcriptome analysis of the production enhancement mechanism of antimicrobial lipopeptides of Streptomyces bikiniensis HD-087 by co-culture with Magnaporthe oryzae Guy11.

The lipopeptides produced by Streptomyces bikiniensis have a significant inhibitory effect on Magnaporthe oryzae, but the low yield limits its application. In this study, the anti-M. oryzae activity of the broth of S. bikiniensis HD-087 co-cultured with M. oryzae Guy11 mycelium has risen by 41.22% compared with pure culture, and under induction conditions of adding Guy11-inducer (cell-free supernatant of M. oryzae Guy11), the activity of strain HD-087 improved 61.76%. The result proved that the enhancement effect of Guy11 on the antimicrobial activity of HD-087 was mainly related to metabolites but mycelium cells. Under optimum induction conditions, NRPS gene expression levels of HD-087 were significantly increased by induction with Guy11-inducer, the biomass of HD-087 had no significant change, but crude extract of lipopeptide (CEL) production was 107.4% higher than pure culture, and TLC result under acid hydrolysis showed that the induced culture has one component more than pure culture. To clarify the regulation mechanism of improving lipopeptide production of HD-087 with Guy11-inducer, transcriptomic analysis was performed using RNAseq to compare the induced culture and pure culture. In the induced culture, 943 genes were up-regulated, while 590 genes were down-regulated in DEGs (differentially expressed genes). KEGG results showed that the expression of genes related to amino acid synthesis, fatty acid metabolism, TCA cycle and pyruvate metabolism pathway were significantly increased. The increased expression of genes related to these metabolic pathways provided sufficient precursors for lipopeptide synthesis. Accordingly, key enzyme genes responsible for the synthesis of lipopeptides Srf and NRPS was significantly increased. Quorum sensing related genes OppA and MppA were significantly up-regulated, and then ComP was activated and promoted lipopeptide synthesis. These results provided a scientific basis for using M. oryzae to induce the increase of the production of Streptomyces lipopeptides, and also laid a foundation for further exploring the co-culture mechanisms among different genera.

One-pot fabrication of Mo1-xWxS2 alloy nanosheets as SERS substrates with highly Raman enhancement effect and long-term stability.

A new Mo1-xWxS2 two-dimensional nanosheets were prepared by the one-pot method. After certain Mo atoms in MoS2 were replaced by W ones in a hydrothermal reduction procedure, Mo1-xWxS2 was formed on the Mo foil. Well enhanced Mo1-xWxS2 nanosheets were prepared when the sodium tungstate concentration got under control. Various characterizations were carried out, which indicate that Mo1-xWxS2 nanosheets with good crystallinity. Compared with MoS2, the Raman intensity of Rhodamine 6G (10-6 M) was amplified by 1.7 times with Mo1-xWxS2 nanosheets as the substrate. The characteristic Raman peaks could still be clearly distinguished until the concentration of Rhodamine 6G (R6G), Methylene blue (MB) and Crystal violet (CV) down to 10-8, 10-8 and 10-7 M, respectively. With abundant edge active sites that facilitate charge transfer, Mo1-xWxS2 nanosheets could better enhance SERS signals of target detection molecules and get a good linear relationship exists within the concentration and Raman peak strength. In addition, R6G SERS detection also shows excellent reproducibility and long-term stability of this TMDs SERS substrate.

Nitric Oxide Extends the Postharvest Life of Water Bamboo Shoots Partly by Maintaining Mitochondrial Structure and Energy Metabolism.

Harvested water bamboo shoots can be stored for only a few days before they lose weight and become soft. Nitrogen oxide (NO) and modified atmosphere packaging (MAP) have previously been used to prolong horticultural crop storage. In the present study, we analyzed the joint effect of these two methods on extending the postharvest quality of water bamboo shoots. Water bamboo shoots were treated with (1) 30 µL L-1 NO, (2) MAP, and (3) a combination of NO and MAP. The NO treatment delayed the softness and weight loss through maintaining the integrity of the mitochondrial ultrastructure and enhancing the ATP level by activating the expressions and activities of succinic dehydrogenase, malic acid dehydrogenase, and cytochrome oxidase. MAP improved the effect of NO on the mitochondrial energy metabolism. These results indicate that NO and MAP treatments are effective at suppressing the quality deterioration of water bamboo shoots, MAP improves the effect of NO in extending postharvest life, and NO may be the main effective factor in the combination of NO and MAP.