Enhancing Leaf Area Index Estimation for Maize with Tower-Based Multi-Angular Spectral Observations.

The leaf area index (LAI) played a crucial role in ecological, hydrological, and climate models. The normalized difference vegetation index (NDVI) has been a widely used tool for LAI estimation. However, the NDVI quickly saturates in dense vegetation and is susceptible to soil background interference in sparse vegetation. We proposed a multi-angular NDVI (MAVI) to enhance LAI estimation using tower-based multi-angular observations, aiming to minimize the interference of soil background and saturation effects. Our methodology involved collecting continuous tower-based multi-angular reflectance and the LAI over a three-year period in maize cropland. Then we proposed the MAVI based on an analysis of how canopy reflectance varies with solar zenith angle (SZA). Finally, we quantitatively evaluated the MAVI's performance in LAI retrieval by comparing it to eight other vegetation indices (VIs). Statistical tests revealed that the MAVI exhibited an improved curvilinear relationship with the LAI when the NDVI is corrected using multi-angular observations (R2 = 0.945, RMSE = 0.345, rRMSE = 0.147). Furthermore, the MAVI-based model effectively mitigated soil background effects in sparse vegetation (R2 = 0.934, RMSE = 0.155, rRMSE = 0.157). Our findings demonstrated the utility of tower-based multi-angular spectral observations in LAI retrieval, having the potential to provide continuous data for validating space-borne LAI products. This research significantly expanded the potential applications of multi-angular observations.

Risk factors of disturbed sleep phases to posterior circulation cerebral infarctions: A single-center retrospective study.

Posterior circulation stroke differs from anterior circulation stroke in terms of etiological, clinical, and prognostic properties. Sleep architecture is impaired in patients with acute stroke, which may correlate with disease severity and outcome, and the correlation between the location of cerebral infarction (CI) and sleep phase disturbance remains unknown. This study aimed to assess the correlation between disturbed sleep phases in CI and posterior circulation cerebral infarction (PCCI). We retrospectively enrolled 192 patients with first-onset acute CI, who were assigned to the anterior circulation cerebral infarction (n = 101) and PCCI (n = 91) groups. The polysomnograms in both groups were analyzed by phase. The proportions of sleep phases were significantly different between the 2 groups (P < .05). The awake (W) and non-rapid eye movement 3 (N3) phases were independently associated with PCCI in multivariate analysis. The W phase may be a risk factor for PCCI (odds ratio = 1.60, 95% CI 1.30-1.97), while the N3 phase may be a protective factor for PCCI (odds ratio = 0.498, 95% CI 0.353-0.703). This study demonstrated that CI causes different degrees of sleep phase disturbances, and the percentages of W and N3 phase disturbances were independent factors associated with PCCI. The former was a risk factor, whereas the latter was a protective factor. This study demonstrated the correlation between cerebral infarction and sleep phase disturbances from a new perspective and suggested that cerebral infarcts may alter the structure of sleep.

Chromosome-level genome assembly of the Arctic fox (Vulpes lagopus) using PacBio sequencing and Hi-C technology.

The Arctic fox (Vulpes lagopus) is the only fox species occurring in the Arctic and has adapted to its extreme climatic conditions. Currently, the molecular basis of its adaptation to the extreme climate has not been characterized. Here, we applied PacBio sequencing and chromosome structure capture technique to assemble the first V. lagopus genome assembly, which is assembled into chromosome fragments. The genome assembly has a total length of 2.345 Gb with a contig N50 of 31.848 Mb and a scaffold N50 of 131.537 Mb, consisting of 25 pseudochromosomal scaffolds. The V. lagopus genome had approximately 32.33% repeat sequences. In total, 21,278 protein-coding genes were predicted, of which 99.14% were functionally annotated. Compared with 12 other mammals, V. lagopus was most closely related to V. Vulpes with an estimated divergence time of ~7.1 Ma. The expanded gene families and positively selected genes potentially play roles in the adaptation of V. lagopus to Arctic extreme environment. This high-quality assembled genome will not only promote future studies of genetic diversity and evolution in foxes and other canids but also provide important resources for conservation of Arctic species.

Ligand-based design of GLUT inhibitors as potential antitumor agents.

Glucose transporters (GLUTs) regulate glucose uptake and are often overexpressed in several human tumors. To identify new chemotypes targeting GLUT1, we built a pharmacophore model and searched against a NCI compound database. Sixteen hit molecules with good docking scores were screened for GLUT1 inhibition and antiproliferative activities. From these, we identified that compounds 2, 5, 6 and 13 inhibited the cell viability in a dose-dependent manner and that the IC50s of 2 and 6 are<10 µM concentration in the HCT116 colon cancer cell line. Lead compound 13 (NSC295720) was a GLUT1 inhibitor. Docking studies show that GLUT1 residues Phe291, Phe379, Glu380, Trp388, and Trp412 were important for inhibitor binding.

Carbon fluxes across alpine, oasis, and desert ecosystems in northwestern China: The importance of water availability.

Dryland regions cover >40% of the Earth's land surface, making these ecosystems the largest biome in the world. Ecosystems in these areas play an important role in determining the interannual variability of the global terrestrial carbon sink. Examining carbon fluxes of various types of dryland ecosystems and their responses to climatic variability is essential for improving projections of the carbon cycle in these regions. In this study, we made use of observations from a regional flux tower observation network in a typical arid endorheic basin, the Heihe river basin (HRB). As a representative area of both the arid region of China and the entire region of central Asia, the HRB includes the main ecosystems in arid regions. We compared the spatial variations of carbon fluxes of five terrestrial ecosystems (i.e., grassland, cropland, desert, wetland, and forest ecosystems) and explored the responses of ecosystem carbon fluxes to climatic factors across different ecosystems. We found that our region exhibits a carbon sink ranging from 85.9 to 508.7 gC/m2/yr for different ecosystems, and the water availability is critical to the spatial variability of carbon fluxes in arid regions. Carbon fluxes across all sites exhibited weak correlations with temperature and precipitation. Marked differences in precipitation effects were observed between the sites within oases and those outside of oases. Irrigation and groundwater recharge were of great importance to the variations in carbon fluxes for the sites within oases. Evapotranspiration (ET) exhibited strong relationships with carbon fluxes, indicating that ET was a better metric of soil water availability than was precipitation in driving the spatial variability of carbon fluxes in arid regions. This study has implications for better understanding the carbon budget of terrestrial ecosystems and informing ecological management in dryland regions.

Genome-wide differential expression of long noncoding RNAs and mRNAs in ovarian follicles of two different chicken breeds.

Bashang long-tail chickens are an indigenous breed with dual purpose in China (meat and eggs) but have low egg laying performance. To improve the low egg laying performance, a genome-wide analysis of mRNAs and long noncoding RNAs (lncRNAs) from Bashang long-tail chickens and Hy-Line brown layers was performed. A total of 16,354 mRNAs and 8691 lncRNAs were obtained from ovarian follicles. Between the breeds, 160 mRNAs and 550 lncRNAs were found to be significantly differentially expressed. Integrated network analysis suggested some differentially expressed genes were involved in ovarian follicular development through oocyte meiosis, progesterone-mediated oocyte maturation, and cell cycle. The impact of lncRNAs on cis and trans target genes, indicating some lncRNAs may play important roles in ovarian follicular development. The current results provided a catalog of chicken ovarian follicular lncRNAs and genes for further study to understand their roles in regulation of egg laying performance.

R-Spondin1/LGR5 Activates TGFß Signaling and Suppresses Colon Cancer Metastasis.

Leucine-rich repeat containing G-protein-coupled receptor 5 (LGR5), an intestinal stem cell marker, is known to exhibit tumor suppressor activity in colon cancer, the mechanism of which is not understood. Here we show that R-spondin 1 (RSPO1)/LGR5 directly activates TGFß signaling cooperatively with TGFß type II receptor in colon cancer cells, enhancing TGFß-mediated growth inhibition and stress-induced apoptosis. Knockdown of LGR5 attenuated downstream TGFß signaling and increased cell proliferation, survival, and metastasis in an orthotopic model of colon cancer in vivo Upon RSPO1 stimulation, LGR5 formed complexes with TGFß receptors. Studies of patient specimens indicate that LGR5 expression was reduced in advanced stages and positively correlated with markers of TGFß activation in colon cancer. Our study uncovers a novel cross-talk between LGR5 and TGFß signaling in colon cancer and identifies LGR5 as a new modulator of TGFß signaling able to suppress colon cancer metastasis. Cancer Res; 77(23); 6589-602. ©2017 AACR.

Transforming Growth Factor ß Mediates Drug Resistance by Regulating the Expression of Pyruvate Dehydrogenase Kinase 4 in Colorectal Cancer.

Drug resistance is one of the main causes of colon cancer recurrence. However, our understanding of the underlying mechanisms and availability of therapeutic options remains limited. Here we show that expression of pyruvate dehydrogenase kinase 4 (PDK4) is positively correlated with drug resistance of colon cancer cells and induced by 5-fluorouracil (5-FU) treatment in drug-resistant but not drug-sensitive cells. Knockdown of PDK4 expression sensitizes colon cancer cells to 5-FU or oxaliplatin-induced apoptosis in vitro and increases the effectiveness of 5-FU in the inhibition of tumor growth in a mouse xenograft model in vivo In addition, we demonstrate for the first time that TGFß mediates drug resistance by regulating PDK4 expression and that 5-FU induces PDK4 expression in a TGFß signaling-dependent manner. Mechanistically, knockdown or inhibition of PDK4 significantly increases the inhibitory effect of 5-FU on expression of the anti-apoptotic factors Bcl-2 and survivin. Importantly, studies of patient samples indicate that expression of PDK4 and phosphorylation of Smad2, an indicator of TGFß pathway activation, show a strong correlation and that both positively associate with chemoresistance in colorectal cancer. These findings indicate that the TGFß/PDK4 signaling axis plays an important role in the response of colorectal cancer to chemotherapy. A major implication of our studies is that inhibition of PDK4 may have considerable therapeutic potential to overcome drug resistance in colorectal cancer patients, which warrants the development of PDK4-specific inhibitors.

MicroRNA-520g confers drug resistance by regulating p21 expression in colorectal cancer.

Development of drug resistance is one of the major causes of colorectal cancer recurrence, yet mechanistic understanding and therapeutic options remain limited. Here, we show that expression of microRNA (miR)-520g is correlated with drug resistance of colon cancer cells. Ectopic expression of miR-520g conferred resistance to 5-fluorouracil (5-FU)- or oxaliplatin-induced apoptosis in vitro and reduced the effectiveness of 5-FU in the inhibition of tumor growth in a mouse xenograft model in vivo. Further studies indicated that miR-520g mediated drug resistance through down-regulation of p21 expression. Moreover, p53 suppressed miR-520g expression, and deletion of p53 up-regulated miR-520g expression. Inhibition of miR-520g in p53(-/-) cells increased their sensitivity to 5-FU treatment. Importantly, studies of patient samples indicated that expression of miR-520g correlated with chemoresistance in colorectal cancer. These findings indicate that the p53/miR-520g/p21 signaling axis plays an important role in the response of colorectal cancer to chemotherapy. A major implication of our studies is that inhibition of miR-520g or restoration of p21 expression may have considerable therapeutic potential to overcome drug resistance in colorectal cancer patients, especially in those with mutant p53.

Functional proteomic analysis reveals the involvement of KIAA1199 in breast cancer growth, motility and invasiveness.

BACKGROUND: KIAA1199 is a recently identified novel gene that is up-regulated in human cancer with poor survival. Our proteomic study on signaling polarity in chemotactic cells revealed KIAA1199 as a novel protein target that may be involved in cellular chemotaxis and motility. In the present study, we examined the functional significance of KIAA1199 expression in breast cancer growth, motility and invasiveness. METHODS: We validated the previous microarray observation by tissue microarray immunohistochemistry using a TMA slide containing 12 breast tumor tissue cores and 12 corresponding normal tissues. We performed the shRNA-mediated knockdown of KIAA1199 in MDA-MB-231 and HS578T cells to study the role of this protein in cell proliferation, migration and apoptosis in vitro. We studied the effects of KIAA1199 knockdown in vivo in two groups of mice (n = 5). We carried out the SILAC LC-MS/MS based proteomic studies on the involvement of KIAA1199 in breast cancer. RESULTS: KIAA1199 mRNA and protein was significantly overexpressed in breast tumor specimens and cell lines as compared with non-neoplastic breast tissues from large-scale microarray and studies of breast cancer cell lines and tumors. To gain deeper insights into the novel role of KIAA1199 in breast cancer, we modulated KIAA1199 expression using shRNA-mediated knockdown in two breast cancer cell lines (MDA-MB-231 and HS578T), expressing higher levels of KIAA1199. The KIAA1199 knockdown cells showed reduced motility and cell proliferation in vitro. Moreover, when the knockdown cells were injected into the mammary fat pads of female athymic nude mice, there was a significant decrease in tumor incidence and growth. In addition, quantitative proteomic analysis revealed that knockdown of KIAA1199 in breast cancer (MDA-MB-231) cells affected a broad range of cellular functions including apoptosis, metabolism and cell motility. CONCLUSIONS: Our findings indicate that KIAA1199 may play an important role in breast tumor growth and invasiveness, and that it may represent a novel target for biomarker development and a novel therapeutic target for breast cancer.

TGF-Beta suppresses VEGFA-mediated angiogenesis in colon cancer metastasis.

The FET cell line, derived from an early stage colon carcinoma, is non-tumorigenic in athymic nude mice. Engineered FET cells that express TGF-α (FETα) display constitutively active EGFR/ErbB signaling. These cells readily formed xenograft tumors in athymic nude mice. Importantly, FETα cells retained their response to TGF-beta-mediated growth inhibition, and, like the parental FET cells, expression of a dominant negative TGF-beta type II receptor (DNRII) in FETα cells (FETα/DNRII) abrogated responsiveness to TGF-beta-induced growth inhibition and apoptosis under stress conditions in vitro and increased metastatic potential in an orthotopic model in vivo, which indicates metastasis suppressor activity of TGF-beta signaling in this model. Cancer angiogenesis is widely regarded as a key attribute for tumor formation and progression. Here we show that TGF-beta signaling inhibits expression of vascular endothelial growth factor A (VEGFA) and that loss of autocrine TGF-beta in FETα/DNRII cells resulted in increased expression of VEGFA. Regulation of VEGFA expression by TGF-beta is not at the transcriptional level but at the post-transcriptional level. Our results indicate that TGF-beta decreases VEGFA protein stability through ubiquitination and degradation in a PKA- and Smad3-dependent and Smad2-independent pathway. Immunohistochemical (IHC) analyses of orthotopic tumors showed significantly reduced TGF-beta signaling, increased CD31 and VEGFA staining in tumors of FETα/DNRII cells as compared to those of vector control cells. These results indicate that inhibition of TGF-beta signaling increases VEGFA expression and angiogenesis, which could potentially contribute to enhanced metastasis of those cells in vivo. IHC studies performed on human colon adenocarcinoma specimens showed that TGF-beta signaling is inversely correlated with VEGFA expression, indicating that TGF-beta-mediated suppression of VEGFA expression exists in colon cancer patients.

Inhibition of phosphorylated c-Met in rhabdomyosarcoma cell lines by a small molecule inhibitor SU11274.

BACKGROUND: c-Met is a receptor tyrosine kinase (RTK) that is over-expressed in a variety of cancers and involved in cell growth, invasion, metastasis and angiogenesis. In this study, we investigated the role of c-Met in rhabdomyosarcoma (RMS) using its small molecule inhibitor SU11274, which has been hypothesized to be a potential therapeutic target for RMS. METHODS: The expression level of phosphorylated c-Met in RMS cell lines (RD, CW9019 and RH30) and tumor tissues was assessed by phospho-RTK array and immunohistochemistry, respectively. The inhibition effects of SU11274 on RMS cells were studied with regard to intracellular signaling, cell proliferation, cell cycle and cell migration. RESULTS: A high level of phosphorylated c-Met was detected in 2 alveolar RMS cell lines (CW9019 and RH30) and 14 out of 24 RMS tissue samples, whereas relatively low levels of phospho-c-Met were observed in the embryonic RMS cell line (RD). The small molecule SU11274 could significantly reduce the phosphorylation of c-Met, resulting in inhibition of cell proliferation, G1 phase arrest of cell cycle and blocking of cell migration in CW9019 and RH30 cell lines. CONCLUSION: These results might support the role of c-Met in the development and progression of RMS. Furthermore, the inhibitor of c-Met, SU11274, could be an effective targeting therapy reagent for RMS, especially alveolar RMS.

WITHDRAWN: Restoration of transforming growth factor-beta receptor II expression in colon cancer cells with microsatellite instability increases metastatic potential in vivo.

Microsatellite instability (MSI), which occurs in 15% of colorectal cancer, has been shown to have a lower incidence of metastasis and better patient survival rates compared with microsatellite stable colorectal cancer. However, a mechanistic understanding of the basis for this difference is very limited. Here, we show that restoration of TGFß signaling by re-expression of TGFß receptor II in MSI colon cancer cells increased PI3K/AKT activation, conferred resistance to growth factor deprivation stress-induced apoptosis, and promoted cell motility in vitro. Treatment with a potent PI3K inhibitor (LY294002) blocked the prosurvival and promotility effects of TGFß, indicating that TGFß-mediated promotion of cell survival and motility is dependent upon activation of the PI3K/AKT pathway. Analysis of apoptotic effectors that are affected by TGFß signaling indicated that Bim is an effector of TGFß-mediated survival. In addition, TGFß-induced down-regulation of E-cadherin contributed to the prosurvival effect of TGFß, and restoration of TGFß signaling in MSI colon cancer cells increased liver metastasis in an orthotopic model in vivo. Taken together, our results demonstrate that restoration of TGFß signaling promotes cell survival, motility, and metastatic progression in MSI colon cancer cells and indicate that TGFß receptor II mutations contribute to the favorable outcomes in colon cancer patients with MSI.

Phosphatase PRL-3 is a direct regulatory target of TGFbeta in colon cancer metastasis.

Metastasis causes most deaths from cancer yet mechanistic understanding and therapeutic options remain limited. Overexpression of the phosphatase PRL-3 (phosphatase of regenerating liver) is associated with metastasis of colon cancer. Here, we show that PRL-3 is a direct target of signaling by TGFß, which is broadly implicated in progression and metastasis. We found that suppression of PRL-3 expression by TGFß was mediated by Smad-dependent inhibition of PRL-3 transcription at the level of promoter activity. PRL-3 activation stimulated PI3K/AKT signaling that caused resistance to stress-induced apoptosis. PRL-3 overexpression promoted metastatic colonization in an orthotopic mouse model of colon cancer, whereas PRL-3 knockdown reduced metastatic potential. Altered metastatic phenotypes were not derivative of primary tumor development or local invasion but could be attributed to PRL-3-mediated cell survival. Our findings suggest that inhibiting PRL-3 expression might be an important mechanism through which TGFß suppresses metastasis in colon cancer. In addition, our findings suggest that loss of TGFß signaling, which occurs commonly during colon cancer progression, is sufficient to activate a PRL-3-mediated cell survival pathway that can selectively promote metastasis. Therefore, a major implication of our findings is that PRL-3 antagonists may offer significant value for antimetastatic therapy in patients with colon cancer.

Mycorrhizal synthesis of Tuber indicum with two indigenous hosts, Castanea mollissima and Pinus armandii.

Tuber indicum is one of the most renowned commercialized fungi in China. Mycorrhizal investigations, however, have been carried out mainly with exotic trees. Up to now there is no detailed description of morphology of the mycorrhizae formed with the indigenous hosts of T. indicum. Containerized seedlings of two indigenous hosts of the fungus in southwestern China, Pinus armandii and Castanea mollissima, were inoculated with aqueous spore suspension of T. indicum in two kinds of substrates. Mycorrhizae began to form 4 months after inoculation and were harvested at 9 months. The contributing fungus of the mycorrhizae was confirmed to be T. indicum by morphological and ITS-rDNA sequence analyses. The morphology of emanating hyphae and epidermoid-like mantle appearance was similar to the mycorrhizae obtained with some European trees. The high morphological variation and the similarity to that of Tuber melanosporum makes it difficult to distinguish the mycorrhizae of the two species by morphology alone. The synthesis of mycorrhizae of T. indicum with its indigenous hosts will be of great significance for planned cultivation of the Asian black truffles.

[Identification of SNPs located in putative microRNA tar-get region of six functional genes in chickens through bioinformatic analysis].

GDF-8, IGF-I, IGF-III, IGF2R, IGFBP2, and GHR are candidate genes affecting important economic trait in chicken. The putative microRNA target sites of 3' untranslated region of these genes were identified by means of specialized algorithms (miRanda and TargetScan), and the candidate SNPs located in the microRNA target region were also identified. Approximately 125 candidate SNPs were found throughout the 26 putative microRNA target regions in six gene 3'-UTR. Among the 125 SNPs, 47 were located in the microRNA targets, 44 and 35 were located in 5'and 3'flanking regions which equalled to the size of the given target. Twelve of the 47 candidates were located in the match of the microRNA seed. These SNPs, which were located in the match of the microRNA seed and 3 ' flanking regions may affect microRNA regulation and contribute to poultry phenotypic variation.

Effect of overexpression of inhibin alpha (1-32) fragment on bovine granulosa cell proliferation, apoptosis, steroidogenesis, and development of co-cultured oocytes.

The objective was to determine the effects of an inhibin alpha (1-32) fragment gene on proliferation, apoptosis, and steroidogenesis of bovine granulosa cells (GC) isolated from medium and small follicles (diameter >4-8 and 1-4mm, respectively), and the effect of GC, previously transfected with pEGISI, on oocyte maturation and in vitro embryo development. To enhance expression of the inhibin alpha (1-32) fragment, GC were transfected with pEGISI. Transfection inhibited (P<0.05) GC proliferation (88.8+/-2.1%; mean+/-S.E.M.) compared to the control and EGFP groups (100% and 97.5+/-2.1%) from medium follicles, with no significant effect on GC from small follicles. Apoptosis was higher (P<0.01) in transfected GC than in controls. Transfection increased (P<0.05) estradiol synthesis from both medium and small follicles (0.57+/-0.13 and 0.86+/-0.13 pg/mL vs. 0.19+/-0.05 and 0.35+/-0.09 pg/mL in controls) after culturing for 48 h, with suppression (P<0.05) in transfected GC after 96 h. Transfection reduced (P<0.05) progesterone synthesis in GC from both medium and small follicles (24.5+/-3.4 and 75.4+/-4.6 ng/mL vs. 45.42+/-5.33 and 117.32+/-11.99 ng/mL in controls) after culture for 48 h, with no significant difference after 96 h. Maturation rate of oocytes co-cultured with transfected GC from medium follicles was decreased relative to control (61.5+/-6.8% vs. 71.2+/-5.7%, P<0.05), with no significant effect on embryo development. In conclusion, overexpression of inhibin alpha (1-32) fragment regulated GC development; effects on subsequent oocyte maturation were both time- and stage-dependent.

Effects of iron and copper in culture medium on bovine oocyte maturation, preimplantation embryo development, and apoptosis of blastocysts in vitro.

The aim of this study was to investigate the effects of iron and copper on bovine oocyte maturation, preimplantation embryo development and apoptosis of blastocysts. The concentrations of iron in the culture media were 0 (control), 0.45, 0.81, 1.96 and 3.26 mg/l, and the concentrations of copper were 0 (control), 0.093, 0.27, 0.46 and 0.68 mg/l. The changes in the iron (1.96 mg/l) and copper concentrations (0.46 mg/l) in the culture media were measured after oocyte maturation for 22 h and after zygote culture for 48, 96, 144 and 192 h. The results showed that there were no significant differences in oocyte maturation and cleavage between media containing iron and the control, but the media containing iron had higher (P>0.05) rates of 8-cell embryos, morulae, and blastocysts than the control, and addition of 1.96 mg/l of iron increased the blastocyst rate (P>0.05). The effects of copper on oocyte maturation and cleavage were similar to iron, and addition of 0.46 and 0.68 mg/l of copper increased the rates of morulae and blastocysts (P>0.05). Addition of iron or copper significantly decreased the number of apoptotic blastomeres compared with the control (P>0.05). After oocyte maturation for 22 h and zygote culture for 48 h, the iron concentrations decreased by 3.6 and 9.2%, respectively, and the copper concentrations decreased by 6.5 and 10.9%, respectively. After zygote culture for 96, 144 and 192 h, the iron concentrations decreased by 21.4, 25.5 and 27.0%, respectively, the copper concentrations decreased by 23.9, 28.3 and 30.4%, respectively. In conclusion, iron and copper played an important role in the success of culture of 8-cell embryos, morulae, and blastocysts, and long-term lack of iron or copper increased the number of apoptotic blastomeres. Furthermore, transition of primary demand for trace amounts of iron or copper from the cytoplast to culture medium for utilization by zygotes may occur after in vitro zygote culture for 48 h.