WHITE STRIPE LEAF8, encoding a deoxyribonucleoside kinase, is involved in chloroplast development in rice.

KEY MESSAGE: WSL8 encoding a deoxyribonucleoside kinase (dNK) that catalyzes the first step in the salvage pathway of nucleotide synthesis plays an important role in early chloroplast development in rice. The chloroplast is an organelle that converts light energy into chemical energy; therefore, the normal differentiation and development of chloroplast are pivotal for plant survival. Deoxyribonucleoside kinases (dNKs) play an important role in the salvage pathway of nucleotides. However, the relationship between dNKs and chloroplast development remains elusive. Here, we identified a white stripe leaf 8 (wsl8) mutant that exhibited a white stripe leaf phenotype at seedling stage (before the four-leaf stage). The mutant showed a significantly lower chlorophyll content and defective chloroplast morphology, whereas higher reactive oxygen species than the wild type. As the leaf developed, the chlorotic mutant plants gradually turned green, accompanied by the restoration in chlorophyll accumulation and chloroplast ultrastructure. Map-based cloning revealed that WSL8 encodes a dNK on chromosome 5. Compared with the wild type, a C-to-G single base substitution occurred in the wsl8 mutant, which caused a missense mutation (Leu 349 Val) and significantly reduced dNK enzyme activity. A subcellular localization experiment showed the WSL8 protein was targeted in the chloroplast and its transcripts were expressed in various tissues, with more abundance in young leaves and nodes. Ribosome and RNA-sequencing analysis indicated that some components and genes related to ribosome biosynthesis were down-regulated in the mutant. An exogenous feeding experiment suggested that the WSL8 performed the enzymic activity of thymidine kinase, especially functioning in the salvage synthesis of thymidine monophosphate. Our results highlight that the salvage pathway mediated by the dNK is essential for early chloroplast development in rice.

Brassinosteroid (BR) biosynthetic gene lhdd10 controls late heading and plant height in rice (Oryza sativa L.).

KEY MESSAGE: A Brd2 allele suppresses heading date by altering the expression of heading date regulators such as OsMADS50 , and also negatively regulates chlorophyll biosynthesis. Heading date and plant height are important determinants of yield in rice (Oryza sativa L.). In this study, we characterized a late heading, dwarf mutant known as lhdd10 selected following ethyl methane sulfonate (EMS)-treatment of ssp. indica cultivar 93-11. lhdd10 showed late heading, dwarfness and slightly darker-green leaves than wild-type 93-11 under long-day and short-day conditions. We isolated lhdd10 by map-based cloning; it encoded a putative FAD-linked oxidoreductase protein (a brassinosteroid biosynthetic gene) that localized to the nucleus. LHDD10 was constitutively expressed in various tissues, but more so in shoot apices and panicles. Our data showed that lhdd10 influences heading date by controlling the expression of heading date regulators, such as OsMADS50 in both LD and SD conditions. lhdd10 also negatively regulated expression of chlorophyll biosynthetic genes to reduce the chlorophyll content. Our data indicated that BRs play important roles in regulating heading date and chlorophyll biosynthesis. This work provides material that will allow study of how BRs regulate heading date in rice.

Molecular analysis of an additional case of hybrid sterility in rice (Oryza sativa L.).

Hybrid sterility hinders the exploitation of the heterosis displayed by japonica × indica rice hybrids. The variation in pollen semi-sterility observed among hybrids between the japonica recipient cultivar and each of two sets of chromosome segment substitution lines involving introgression from an indica cultivar was due to a factor on chromosome 5 known to harbor the gene S24. S24 was fine mapped to a 42 kb segment by analyzing a large F(2) population bred from the cross S24-NIL × Asominori, while the semi-sterility shown by the F(1) hybrid was ascribable to mitotic failure at the early bicellular pollen stage. Interestingly, two other pollen sterility genes (f5-Du and Sb) map to the same region (Li et al. in Chin Sci Bull 51:675-680, 2006; Wang et al. in Theor Appl Genet 112:382-387, 2006), allowing a search for candidate genes in the 6.4 kb overlap between the three genes. By sequencing the overlapped fragment in wild rice, indica cultivars and japonica cultivars, a protein ankyrin-3 encoded by the ORF2 was identified as the molecular base for S24. A cultivar Dular was found to have a hybrid-sterility-neutral allele, S24-n, in which an insertion of 30 bp was confirmed. Thus, it was possible to add one more case of molecular bases for the hybrid sterility. No gamete abortion is caused on heterozygous maternal genotype with an impaired sequence from the hybrid-sterility-neutral genotype. This result will be useful in understanding of wide compatibility in rice breeding.

Heading date gene, dth3 controlled late flowering in O. Glaberrima Steud. by down-regulating Ehd1.

Heading date in rice is an important agronomic trait controlled by several genes. In this study, flowering time of variety Dianjingyou 1 (DJY1) was earlier than a near-isogenic line (named NIL) carried chromosome segment from African rice on chromosome 3S, when grown in both long-day (LD) and short-day (SD) conditions. By analyzing a large F2 population from NIL × DJY1, the locus DTH3 (QTL for days to heading on chromosome 3) controlling early heading date in DJY1 was fine mapped to a 64-kb segment which contained only one annotated gene, a MIKC-type MADS-box protein. We detected a 6-bp deletion and a single base substitution in the C-domain by sequencing DTH3 in DJY1 compared with dth3 in NIL, and overexpression of DTH3 caused early flowering in callus. Quantitative real-time PCR revealed that the transcript level of dth3 in NIL was lower than that DTH3 in DJY1 in both LD and SD conditions. The Early heading date 1 (Ehd1) which promotes the RFT1, was up-regulated by DTH3 in both LD and SD conditions. Based on Indel and dCAPs marker analysis, the dth3 allele was only present in African rice accessions. A phylogenetic analysis based on microsatellite genotyping suggested that African rice had a close genetic relationship to O. rufipogon and O. latifolia, and was similar to japonica cultivars. DTH3 affected flowering time and had no significant effect on the main agronomic traits.

Imaging of giant cell tumour of the tendon sheath.

Giant cell tumours of the tendon sheath (GCTTS) and pigmented villonodular synovitis (PVNS) are part of a spectrum of benign proliferative lesions of synovial origin that may affect the joints, bursae and tendon sheaths. This review article describes the clinicopathological features and imaging findings in patients with GCTTS. GCTTS usually presents as a soft tissue mass with pressure erosion of the underlying bone. Magnetic resonance (MR) imaging of GCTTS typically shows low to intermediate signal on T1- and T2-weighted spin-echo sequences due to the presence of haemosiderin, which exerts a paramagnetic effect. On gradient-echo sequences, the paramagnetic effect of haemosiderin is further exaggerated, resulting in areas of very low signal due to the blooming artefact. Ultrasonography shows a soft mass related to the tendon sheath that is hypervascular on colour or power Doppler imaging.

DS1/OsEMF1 interacts with OsARF11 to control rice architecture by regulation of brassinosteroid signaling.

BACKGROUND: Plant height and leaf angle are important determinants of yield in rice (Oryza sativa L.). Genes involved in regulating plant height and leaf angle were identified in previous studies; however, there are many remaining unknown factors that affect rice architecture. RESULTS: In this study, we characterized a dwarf mutant named ds1 with small grain size and decreased leaf angle,selected from an irradiated population of ssp. japonica variety Nanjing35. The ds1 mutant also showed abnormal floral organs. ds1 plants were insensitive to BL treatment and expression of genes related to BR signaling was changed. An F2 population from a cross between ds1 and indica cultivar 93-11 was used to fine map DS1 and to map-based clone the DS1 allele, which encoded an EMF1-like protein that acted as a transcriptional regulator. DS1 was constitutively expressed in various tissues, and especially highly expressed in young leaves, panicles and seeds. We showed that the DS1 protein interacted with auxin response factor 11 (OsARF11), a major transcriptional regulator of plant height and leaf angle, to co-regulate D61/OsBRI1 expression. These findings provide novel insights into understanding the molecular mechanisms by which DS1 integrates auxin and brassinosteroid signaling in rice. CONCLUSION: The DS1 gene encoded an EMF1-like protein in rice. The ds1 mutation altered the expression of genes related to BR signaling, and ds1 was insensitive to BL treatment. DS1 interacts with OsARF11 to co-regulate OsBRI1 expression.

Genetic dissection of silicon uptake ability in rice (Oryza sativa L.).

The adequate presence of silicon (Si) in rice plants can enhance their yield and improve their tolerance to various biotic and abiotic stresses. In this study Si uptake abilities were compared between the japonica rice cultivar (cv.) Kinmaze and the indica rice cv. DV85 under three Si concentrations (0.16, 0.4, and 1.6mM) at different time points from 1 to 12h. The results showed that the phenotypic values of two traits-Si uptake by individual plants (SP, Si uptake by all roots of a plant) and Si uptake per unit root dry weight (SR=SP/root dry weight)-of Kinmaze were significantly higher than those of DV85 (P<0.01). Meanwhile, a kinetic study indicated that the Si transporters in Kinmaze and DV85 had the same affinity for silicic acid, but with different Vmax values, indicating that Kinmaze had more Si transporters in the roots than DV85. This may be the main reason for the difference in Si uptake ability between Kinmaze and DV85. In addition, a mapping population consisting of 81 recombinant inbred lines (RILs) derived from the cross between Kinmaze and DV85 was used to detect quantitative trait loci (QTLs) underlying SP and SR. The RILs follow a continuous one-peak distribution and show transgressive segregation in both directions for SP, SR, and root dry weight (RDW). Three QTLs for SP, four for SR, and three for RDW were detected. This can explain 7.16-17.15% of the phenotypic variation (PVE). Thus, the results obtained in this study provide a better understanding of the mechanism of rice Si uptake ability and the basis for fine-mapping the genes involved.

Influence of interleukin-2 infusion on cell cycle kinetics in the Walker-256 carcinosarcoma.

The effect of recombinant human interleukin-2 (IL-2) on tumor cell cycle kinetics was evaluated in rats bearing the Walker-256 carcinosarcoma. Seven days after implantation, tumor-bearing rats were infused intravenously with IL-2 at a dose of 500 mg/kg body weight or 5% dextrose for 6 h. Tumor cell mean DNA synthesis time (TDNA), labeling index, potential doubling time (Tpot), and growth fraction (GF) were determined in vivo by use of bromodeoxyuridine (BrdUrd) pulse labeling and bivariate BrdUrd/DNA analysis using flow cytometry. IL-2 treatment significantly reduced the relative number of S-phase cells by 11.9% and increased the number of cells in the G0/G1 phase by 9.4%. The labeling index was reduced from 41.3 +/- 2.5% to 32.7 +/- 1.2% (P < .01). Estimates of TDNA derived from the change in BrdUrd movement relative to DNA content were not affected by IL-2 treatment. Based on these cytokinetic changes, IL-2 infusion significantly increased tumor Tpot from 15.3 +/- 0.3 h to 21.5 +/- 0.2 h (P < .001) and reduced GF from 1.01 +/- 0.07 to 0.83 +/- 0.01 (P < 0.001). The inhibitory effect of IL-2 infusion on tumor cell growth, which may be either direct or secondarily mediated by other factors, contrasts with other stimulatory effects of this cytokine on lymphoid cell proliferation.

A detailed analysis of the leaf rolling mutant sll2 reveals complex nature in regulation of bulliform cell development in rice (Oryza sativa L.).

Bulliform cells are large, thin-walled and highly vacuolated cells, and play an important role in controlling leaf rolling in response to drought and high temperature. However, the molecular mechanisms regulating bulliform cell development have not been well documented. Here, we report isolation and characterisation of a rice leaf-rolling mutant, named shallot-like 2 (sll2). The sll2 plants exhibit adaxially rolled leaves, starting from the sixth leaf stage, accompanied by increased photosynthesis and reduced plant height and tiller number. Histological analyses showed shrinkage of bulliform cells, resulting in inward-curved leaves. The mutant is recessive and revertible at a rate of 9%. The leaf rolling is caused by a T-DNA insertion. Cloning of the insertion using TAIL-PCR revealed that the T-DNA was inserted in the promoter region of LOC_Os07 g38664. Unexpectedly, the enhanced expression of LOC_Os07 g38664 by the 35S enhancer in the T-DNA is not responsible for the leaf rolling phenotype. Further, the enhancer also exerted a long-distance effect, including up-regulation of several bulliform cell-related genes. sll2 suppressed the outward leaf rolling of oul1 in the sll2oul1 double mutant. We conclude that leaf rolling in sll2 could be a result of the combined effect of multi-genes, implying a complex network in regulation of bulliform cell development.

Effect of low and high amounts of a structured lipid containing fish oil on protein metabolism in enterally fed burned rats.

To determine the optimal fat intake and source in nutritional support, we measured the protein-sparing effects of a structured lipid (SL) derived from 60% medium-chain triglyceride (MCT) and 40% fish oil and a 50:50 soybean to safflower oil emulsion (long-chain triglyceride, LCT). Male Sprague-Dawley rats received an enteral diet for 7 d with either all nonprotein energy as dextrose (control diet) or 10% or 35% nonprotein energy as SL or LCT. The rats were burned on day 3. Indirect calorimetry and nitrogen balance were measured on day 2 (preburn) and days 4 and 6 (postburn). Respiratory quotient decreased postburn. There was a significant increase in total energy expenditure postburn, particularly with 35% LCT. Nitrogen balance was best without fat and 10% fat compared with 35% fat and with SL compared with LCT. These results confirm previous studies that fish oil-containing SLs possess protein-sparing effects in burn injury and that 10% SL seems optimal for nutritional support in burn injury.

Induction of apoptosis in HL-60 cells by eicosapentaenoic acid (EPA) is associated with downregulation of bcl-2 expression.

Dietary polyunsaturated fatty acids (PUFAs) have been reported as a potential group of natural products which modulate tumor cell growth. In present study, eicosapentaenoic acid (EPA) was found to inhibit proliferation of human leukemic HL-60 and K-562 cells in vitro. EPA arrested cell cycle progression at G0/G1 phase, and induced necrosis in both HL-60 and K-562 cells. However, EPA induced apoptosis only in HL-60 but not K-562 cells. Also, bcl-2 protein expression was downregulated in much greater extent than that of bax showing that depression of bcl-2 might be an important step during the EPA-induced apoptosis in HL-60 cells.

DNA replication time accounts for tumor growth variation induced by dietary fat in a breast carcinoma model.

Female Fischer rats were pair-fed on diets containing either safflower oil (SO) or fish oil (FO) for 6 weeks. Implanted breast 13762 MAT tumors had a doubling times of 35.4 and 55.5 h in SO and FO rats, respectively (P < 0.001). Proliferation kinetics were measured in vivo by bromedeoxyuridine (BrdUrd) labeling and bivariate DNA/BrdUrd analysis by flow cytometry. After 1 h of pulsing, the labeling index was similar in both groups. However, 6 h later, tumor cells from FO rats had significantly lower relative movement of BrdUrd-labeled cells (0.78 vs. 0.91, P < 0.001). These results reflected a significantly longer S phase duration (15.0 vs. 9.1 h, P < 0.001) in FO rats and accounted for all the difference in tumor growth rates. This mechanism, which has not previously been reported, implies a significant role for fatty acids in DNA replication.

Induction of apoptosis by dietary polyunsaturated fatty acids in human leukemic cells is not associated with DNA fragmentation.

Apoptosis is important in anticancer strategy. In this study, bivariate annexin V/PI flow cytometry showed that dietary polyunsaturated fatty acids, arachidonic acid (AA) and eicosapentaenoic acid (EPA), induced apoptosis in human leukemic HL-60 but not K-562 cells. Results from DNA-PI flow cytometry and TUNEL flow cytometry illustrated that neither AA nor EPA induced DNA fragmentation in the leukemic cells. These findings suggested that the AA- and EPA-induced apoptosis might not associate with endonucleases activation, and DNA fragmentation could not be used as a sole criterion to identify apoptotic cells.

Eicosapentaenoic acid modulates cyclin expression and arrests cell cycle progression in human leukemic K-562 cells.

Eicosapentaenoic acid (EPA) is a polyunsaturated fatty acid (20:5omega3) found primarily in aquatic organisms. We have shown previously that EPA inhibits the growth but is not toxic to human leukemic K-562 cells. In this study, the anti-proliferative effect of EPA on the leukemic cells was further determined and its impacts on cell cycle progression and cyclin expression were investigated. EPA inhibited proliferation of K-562 cells, which was associated with accumulation of G0/G1 cells and down-regulation of cyclin E expression. Cyclin B1-expressing cells were also reduced showing that down-regulation of cyclin expression might be important in the anti-proliferation of EPA.

Influence of omega-3 fatty acids on splanchnic blood flow and lactate metabolism in an endotoxemic rat model.

Alteration in regional blood flow is important in the pathogenesis of organ failure during endotoxemia and sepsis. In particular, intestinal ischemia is thought to enhance the translocation of bacteria into the systemic circulation. We used radioactive microspheres to measure the influence of two intravenous (IV) dietary fats (vegetable oil containing high levels of omega-6 fatty acids, and fish oil containing high levels of omega-3 fatty acids) on regional blood flow during low-dose Escherichia coli endotoxin infusion (0.1 mg/100 g body weight [BW]) in a rat model. Despite absence of changes in the cardiac output, blood flow rates to the small and large intestines, stomach, and pancreas, and also to the skin and skeletal muscle were significantly reduced after 18 hours of endotoxin infusion in the rats fed standard vegetable oil. Short-term IV feeding during a period of 40 hours with an isonitrogenous, isocaloric nutrient solution containing fish oil as the only lipid source normalized intestinal perfusion and increased blood flow to the liver and spleen. Low-dose endotoxin infusion also resulted in significant increases in glucose, lactate, and pyruvate concentrations. In comparison to standard vegetable fat emulsion, fish oil significantly reduced these parameters. A second experiment was conducted to measure lactate kinetics. Based on the dilution of U-14C-lactate, fish oil feeding was associated with higher lactate clearance than standard vegetable oil feeding during the endotoxin infusion. We conclude that short-term IV feeding with fish oil improves intestinal perfusion and portal blood flow, improves glucose tolerance, and increases lactate clearance in a low-dose endotoxin rat model.

Comparative effects of omega-3 and omega-6 polyunsaturated fatty acids on protein metabolism in rats bearing the mammary adenocarcinoma.

The comparative effects of diets containing 20% (wt/wt) of either fish oil (FO) or safflower oil (SO) on protein synthesis and catabolism were determined in rats bearing the 7,12-dimethylbenz(a)anthracene (DMBA) 13762 mammary adenocarcinoma in vivo using a 6-hour constant infusion of L-(1-14C)-leucine. Tumor-bearing animals fed FO had significantly lower tumor growth rate (36 +/- 0.5 v 53 +/- 0.7%/d, P less than .05), total tumor protein synthesis (Ts) (1.25 +/- 0.1 v 1.85 +/- 0.1 mumol/h, P less than .05), and tumor protein concentration (12.0 +/- 0.5 v 14.0 +/- 0.7%/d, P less than 0.01). Tumor fractional synthetic rate and total protein breakdown rate of the tumor were unaffected by FO feeding. Both tumor-bearing and saline-control animals fed FO had significantly (P less than .01) lower liver fractional synthetic rate and total protein breakdown rate, and higher liver total protein compared with SO-fed rats. Muscle protein kinetics were unaffected by either treatment or diet. Whole body protein kinetics were not affected by dietary treatment, but the presence of tumor significantly (P less than .001) reduced whole body flux, synthesis, breakdown, and oxidation. Chronic FO feeding for 7 weeks significantly (P less than .001) lowered omega-6 polyunsaturated fatty acids (omega-6 PUFAs) and significantly elevated omega-3 polyunsaturated fatty acids (omega-3 PUFAs) (P less than .001) in both plasma phospholipid and triglycerides. The present study indicates that dietary FO can modulate mammary tumor growth in a manner that reflects changes in protein metabolism in both host and tumor tissues.

Long-term feeding with structured lipid composed of medium-chain and N-3 fatty acids ameliorates endotoxic shock in guinea pigs.

The metabolic and physiologic responses to 7-hour endotoxin infusion (5.0 mg/kg h) were evaluated in guinea pigs following 6 weeks of dietary enrichment with diets containing either chemically structured lipid (SL) composed of medium-chain triglycerides (MCT) and long-chain triglycerides (LCT) in the form of N-3 polyunsaturated fatty acids (PUFAs), or safflower oil (SO), which is high in N-6 fatty acids. Plasma phospholipid fatty acid profiles, arterial blood pH, PCO2, PO2, HCO3, lactate, blood pressure, oxygen consumption, and energy expenditure were examined. Plasma phospholipid fatty acids profiles reflected dietary intake with SL-fed animals demonstrating a significantly higher N-3 to N-6 fatty acid ratio compared with SO-fed animals. SL-fed animals responded to endotoxemia with a mild metabolic acidosis with respiratory compensation, which was associated with moderate lactatemia (3 mmol/L). SO-fed animals developed a severe metabolic acidosis with acidemia and respiratory compensation, which was associated with hyperlactatemia (8 mmol/L, P less than .05 v SL). No differences were observed in blood pressure, oxygen consumption, energy expenditure, or respiratory quotient during endotoxemia between dietary groups compared with controls. We conclude that diets enriched with structured lipid composed of medium-chain and N-3 fatty acids can attenuate the sequelae of endotoxemia.

Insulin-like growth factor-1 is not mitogenic for the Walker-256 carcinosarcoma.

This study was designed to determine whether intravenous infusion of recombinant human insulin-like growth factor 1 (IGF-1) stimulates tumor growth. In order to determine the potential interaction between nutrition and IGF-1 administration the study was conducted in fasting rats and during continuous feeding by total parenteral nutrition. Tumor cell cycle kinetics including labeling index, DNA synthesis time, cell cycle time in Go/G1, and G2/M in the total cell cycle, and potential doubling time were determined by flow cytometry after in vivo pulse labeling the rats bearing the Walker-256 Carcinosarcoma with 5'-bromo-2'-deoxyuridine (BrdUrd). The results show that IGF-1 treatment has no significant effects on the proliferative characteristics of the tumor model regardless of the feeding status of the animal. This study provides preliminary cell-cycle kinetics data on the short-term effect of IGF-1 on tumor growth. Failure to show a significant effect of IGF-1 on the proliferative characteristics of the tumor suggests that IGF-1 may be given to cancer patients in amounts sufficient to promote weight gain without deleterious stimulation of tumor proliferation.

Nutrition and tumor promotion: in vivo methods for measurement of cellular proliferation and protein metabolism.

The notion that tumors act as "nitrogen traps" has led to the belief that nutrition support of the cancer-bearing patient can enhance tumor growth. Proponents of this theory consider the provision of energy and essential nutrients as well as the influence of hormones and growth factors as responsible for this effect. On the other hand, nutrition administration in the debilitated cancer patient may improve antitumor host defense mechanisms and reduce tumor growth. This paper reviews methodologic issues related to the study of nutrition and cancer growth with emphasis on in vivo methods for measuring tumor protein turnover and cytokinetics. Using this combined approach, we previously demonstrated that dietary fat may significantly regulate tumor growth during chronic feeding as well as with short-term intravenous nutrition support in experimental models. Although the mechanism of this effect remains unclear, we have reasoned that by altering arachidonic acid metabolism and prostaglandin synthesis, omega-3 fatty acids could change tumor protein breakdown rates and inhibit the proliferation potential of these tumors. Acknowledging alternative hypotheses, we now present cytokinetic evidence that intracellular protein degradation may regulate tumor cell proliferation. Additional studies relating dietary fat, tumor protein metabolism and tumor proliferation potential are currently in progress. We propose that the effect of nutrition administration on tumor growth is complex and involves several regulatory systems. Thus, based on available evidence, an a priori tumor-enhancing effect for nutrition support is clearly not warranted. Intracellular protein breakdown and host defense mechanisms, both of which are energy dependent, are important loci at which nutrition and tumor growth regulation could interact.(ABSTRACT TRUNCATED AT 250 WORDS)

Invited comment: lipids and the development of immune dysfunction and infection.

Excessive W-6 PUFA metabolism due to high levels of dietary fat intake can encourage infection via prolonged inflammation, enhanced Gram negative survival, reticuloendothelial blockage, immunosuppression, and monokine depression. Lipids can influence host immunity by altering eicosanoid metabolism and membrane structure and function. Further investigations are essential to answer questions regarding the levels and properties of various essential fatty acids in TPN lipid emulsions. Combining the features of LCT in the form of W-3 PUFA (fish oil) and MCT in the form of medium-chain triglyceride in a "structured lipid" may decrease infection and may improve survival rates by producing fewer inflammatory eicosanoids of the two- and four-series, and serving as a more "efficient fuel." The introduction of W-3 polyunsaturated fatty acids into the TPN emulsions as well as into normal diets may provide an important therapeutic advance in the pathogenesis of disease. Such unique antiinflammatory properties of W-3 PUFA require intensive research.