Establishment and characterization of a canine chondrosarcoma cell line:Mango

In the global progress of bone tumor research,established stable and long-lasting transgenic chondrosarcoma(CSA)cell lines are rare,mainly of murine and human origin,while the establishment of canine CSA cell lines has yet to be reported.This study established a canine CSA cell line to facilitate the basic clinical study of canine CSA.Fifty fve cases of canine osteolytic disease were collected,and more than 10 bone tumor samples from dogs with typical clinical signs were used for primary cell culture.A cell line with stable passaging for more than 100 generations and mouse tumorigenic ability was successfully cultured.According to the clinical characteristics of the dog and the histopathological results of the primary tumor,CSA was diagnosed,and the CSA cell line was designated Mango.Immunohistochemical(IHC)results showed that the immunoreactivity of bone gamma-carboxyglutamate protein(BGLAP),secreted protein acidic and rich in cysteine(SPARC),alkaline phosphatase(ALPL),vimentin(VIM)and S100 were positive.However,the immunoreactivity of pan-cytokeratin(PCK),chromogranin A(CGA),and platelet endothelial cell adhesion molecule-1(CD31)was negative.Immunofuorescence(IF)results showed that the protein expressions in the Mango cell line were consistent with the IHC identifcation of the primary tumor.The Mango cell line’s doubling time was 43.92 h,and the cell formation rate exceeded 20%.There were abnormal chromosome numbers,hetero staining with toluidine blue,and certain calcifcation abilities.It could be passaged stably and continuously without changing the cell morphology and characteristics.In vivo,the cells were successfully injected into the nude mice model with a tumorigenic rate of 100%.The immunophenotype of the xenograft tumor was consistent with that of the primary tumor.Therefore,we efectively established a canine CSA cell line.As a promising cell material,this cell line can be used to construct a tumor-bearing model conducive to the subsequent basic research of canine CSA.Moreover,because of its similarity to human CSA,the animal model of CSA is also indispensable for investigating human CSA.

First report on establishment and characterization of the extrahepatic cholangiocarcinoma sarcoma cell line CBC2T-2

BACKGROUND Extrahepatic cholangiocarcinoma sarcoma is extremely rare in clinical practice.These cells consist of both epithelial and mesenchymal cells.Patient-derived cell lines that maintain tumor characteristics are valuable tools for studying the molecular mechanisms associated with carcinosarcoma.However,cholangiocarcinoma sarcoma cell lines are not available in cell banks.AIM To establish and characterize a new extrahepatic cholangiocarcinoma sarcoma cell line,namely CBC2T-2.METHODS We conducted a short tandem repeat(STR)test to confirm the identity of the CBC2T-2 cell line.Furthermore,we assessed the migratory and invasive properties of the cells and performed clonogenicity assay to evaluate the ability of individual cells to form colonies.The tumorigenic potential of CBC2T-2 cells was tested in vivo using nonobese diabetic/severe combined immunodeficient(NOD/SCID)mice.The cells were injected subcutaneously and tumor formation was observed.In addition,immunohistochemical analysis was carried out to examine the expression of epithelial marker CK19 and mesenchymal marker vimentin in both CBC2T-2 cells and xenografts.The CBC2T-2 cell line was used to screen the potential therapeutic effects of various clinical agents in patients with cholangiocarcinoma sarcoma.Lastly,whole-exome sequencing was performed to identify genetic alterations and screen for somatic mutations in the CBC2T-2 cell line.RESULTS The STR test showed that there was no cross-contamination and the results were identical to those of the original tissue.The cells showed round or oval-shaped epithelioid cells and mesenchymal cells with spindle-shaped or elongated morphology.The cells exhibited a high proliferation ratio with a doubling time of 47.11 h.This cell line has migratory,invasive,and clonogenic abilities.The chromosomes in the CBC2T-2 cells were polyploidy,with numbers ranging from 69 to 79.The subcutaneous tumorigenic assay confirmed the in vivo tumorigenic ability of CBC2T-2 cells in NOD/SCID mice.CBC2T-2 cells and xenografts were positive for both the epithelial marker,CK19,and the mesenchymal marker,vimentin.These results suggest that CBC2T-2 cells may have both epithelial and mesenchymal characteristics.The cells were also used to screen clinical agents in patients with cholangiocarcinoma sarcoma,and a combination of paclitaxel and gemcitabine was found to be the most effective treatment option.CONCLUSION We established the first human cholangiocarcinoma sarcoma cell line,CBC2T-2,with stable biogenetic traits.This cell line,as a research model,has a high clinical value and would facilitate the understanding of the pathogenesis of cholangiocarcinoma sarcoma.

Chemical simulation teaching system based on virtual reality and gesture interaction

Background Most existing chemical experiment teaching systems lack solid immersive experiences,making it difficult to engage students.To address these challenges,we propose a chemical simulation teaching system based on virtual reality and gesture interaction.Methods The parameters of the models were obtained through actual investigation,whereby Blender and 3DS MAX were used to model and import these parameters into a physics engine.By establishing an interface for the physics engine,gesture interaction hardware,and virtual reality(VR)helmet,a highly realistic chemical experiment environment was created.Using code script logic,particle systems,as well as other systems,chemical phenomena were simulated.Furthermore,we created an online teaching platform using streaming media and databases to address the problems of distance teaching.Results The proposed system was evaluated against two mainstream products in the market.In the experiments,the proposed system outperformed the other products in terms of fidelity and practicality.Conclusions The proposed system which offers realistic simulations and practicability,can help improve the high school chemistry experimental education.

Principle for the Installation of Accommodation for Sick guards in Public Health Establishments in Brazzaville in the Republic of Congo

The results of scientific studies of human social facts in the field of health show that the management of a patient should involve the patient’s entourage,whatever the status or size of the health establishment.In healthcare establishments in the Congo,the following are recognised as being responsible for medical care:specialist doctors,doctors,midwives,nurses and care assistants.The patient’s family and close friends are responsible for looking after the patient and financing care.The hospital infrastructure does not provide any space for the patient warden who accompany the patient during reception and hospitalisation.This makes Congolese hospitals inefficient for patient care.How can we integrate the function of the Sick guard and the assistance of the family,in order to reduce the mortality rate and repair the harm caused to patients requiring the presence of relatives during their stay in hospital,which is considered to be a dangerous place?This article examines the functional principles for configuring the space that patient warden would occupy in the patient care system.On the basis of a documentary analysis of sociological and architectural studies of existing facilities,this article proposes a typical accommodation model with the spaces needed to ensure the well-being and effectiveness of the patient warden with the patient.These are rooms with minimum space for 2 to 4 individual beds,equipped with toilets and showers.The accommodation has a dining area,kitchen and laundry facilities.In the future,this accommodation will become part of the hospital estate and may be occupied by orderlies and patient warden recruited by the hospital administration.

Keeping Commitments,Meeting Expectations

How well is FOCAC meeting Africa’s aspirations for development?The idea of establishing the Forum on China-Africa Cooperation(FOCAC)in 2000 initially came from African countries,eager to coordinate and manage their relationship with China.

Establishment and Optimization of ISSR-PCR Reaction System for Picea crassifolia kom.

[Objective] The experiment aimed to determine the optimum ISSR-PCR reaction system of Picea crassifolia kom. [Method] Picea crassifolia kom. was used as material to select and optimize influencing factors of ISSR-PCR such as Mg2+, dNTPs, Taq DNA polymerase, template DNA, primers, annealing temperature. [Result] The optimum ISSR-PCR reaction system in 20 μl reaction system was consisted of 1 μl 10×buffer, 1.5 mmol/L Mg2+, 0.2 mmol/L dNTPs, 1.0 U Taq DNA polymerase, 40 ng template DNA, 0.6 μmol/L primers. According to gradient test of annealing temperature in optimum ISSR-PCR reaction system of Picea crassifolia kom, it was found that the optimum annealing temperature of UBC 818 was 54.2 ℃ and the annealing temperature was different for different primers.[Conclusion]The construction of ISSR-PCR reaction system provided technical basis for classification of germplasm resources, construction of genetic map, gene mapping of Picea crassifolia kom. through using ISSR technology.

Stand establishment,root development and yield of winter wheat as affected by tillage and straw mulch in the water deficit hilly region of southwestern China

Good crop stand establishment and root system development are essential for optimum grain yield of dryland wheat （Triti-cum aestivumL.）. At present, little is known about the effect of tilage and straw mulch on the root system of wheat under dryland areas in southwestern China. The aim of this study was to evaluate the effect of three tilage treatments （no-til, NT; rotary til, RT; conventional til, CT） and two crop residue management practices （straw mulch, ML; non-straw mulch, NML） on stand establishment, root growth and grain yield of wheat. NT resulted in lower soil cover thickness for the wheat seed, higher number of uncovered seeds, lower percentage of seedling-less ridges and lower tiler density compared to RT and CT; ML resulted in higher tiler density compared to NML. Straw mulching resulted in more soil water content and root length density （RLD） at most of the growth stages and soil depths. The maximum RLD, root surface area density and root dry matter density were obtained under NT. In the topmost 10 cm soil layer, higher RLD values were found under NT than those under RT and CT. There were no signiifcant differences in the yield or yield components of wheat among the tilage treatments in 2011–2012, but NT resulted in a signiifcant higher yield compared to RT and CT in 2012–2013. Grain yield was signiifcantly higher in ML compared to in NML. A strong relationship was observed between the water-use efifciency and the grain yield. Both NT and ML proved beneifcial for wheat in term of maintaining higher tiler density, better soil water status and root growth, leading to a higher grain yield and enhanced water-use efifciency, especialy in a low rainfal year.

Establishment and characterization of a cholangiocarcinoma cell line (RMCCA-1) from a Thai patient

AIM: To establish and characterize a new cell line derived from peripheral cholangiocarcinoma of a Thai patient.METHODS: The peripheral cholangiocarcinoma specimen surgically obtained from the patient was aseptically processed by washing and mincing before culturing in Ham’s F12 medium containing 10% fetal bovine serum. After 3 mo, when the cell line has become homogeneous and stabilized, several features were investigated, including growth characteristics, immunofluorescence staining for cytokeratins, expression of tumor markers, chromosomal analysis by G-banding and multicolour fluorescence in situ hybridization (mFISH), in vitro migration and invasion characteristics. RESULTS: The RMCCA-1 cell line has been established. These cells proliferated as a monolayer with a population doubling time of 48 h. Immunofluorescence staining showed positive staining for human cytokeratin 7 and 19 verifying the biliary epithelial origin. RMCCA-1 secreted carbohydrate antigen 19-9 (CA19-9), but insignificant levels of carcinoembryonic antigen (CEA) and α-fetoprotein (AFP). Chromosome analysis identified aneuploidy karyotypes with a modal chromosome number of 59. RMCCA-1 exhibited a low level of in vitro invasiveness, but a high degree of motility. The cell line exhibited a significant number of chromosomal aberrations as shown by mFISH and G-banding methods.CONCLUSION: A new cell line derived from peripheral cholangiocarcinoma of a Thai patient has been established. This cell line shows a low level of in vitro invasiveness, but a high degree of motility. It will serve as a valuable tool for further studies on tumor biology, molecular pathogenesis, metastatic mechanism and response to therapeutic drugs of cholangiocarcinoma.

Establishing Submersed Macrophytes via Sinking and Colonization of Shoot Fragments Clipped off Manually

In this paper, sinking and growth of apexes and mid-stems of Myriophyllum spicatum L., Hydrilla verticillata （L.f.） Royle and Ceratophyllum demersum L. in concrete ponds containing eutrophic water and sediment were investigated. Sinking rates of apexes and mid-stems reached 34.8% and 4.4% at the 6 th day and 91.1% and 66.7% at the 22 nd day for M. spicatum, 57.8% and 55.6% at the 6 th day and 100% and 97.8% at the 22 nd day for H. vertieillata, 18.9% and 86.7% at the 6 th day and 95.6% and 100% at the 22 nd day for C. demersum, respectively. Most sunken fragments established themselves successfully with significant growth. Total shoot length ofplantlets developed from apexes and mid-stems increased by 399% and 61% for M. spicatum, 593% and 256% for H. vertieillata and 114% and 104% for C. demersum, respectively. The results showed that it was feasible to establish submersed macrophytes via sinking and colonization of shoot fragments clipped off manually.

Yield and tillering response of super hybrid rice Liangyoupeijiu to tillage and establishment methods

Tillering is an important agronomic trait for rice grain production. To evaluate yield and tillering response, Liangyoupeijiu(super hybrid rice) was grown in Hunan, China during 2011–2012 under different methods of tillage(conventional and no-tillage system) and crop establishment methods(transplanting at a spacing of 20 cm × 20 cm with one seedling per hill and direct seeding at a seeding rate of 22.5 kg ha-1). Our results revealed that, at maximum tillering(Max.) and at maturity(MA) stages, direct seeding(DS) resulted in 22% more tillers than transplanting(TP) irrespective of tillage system. Tiller mortality reached a peak between panicle initiation(PI) and booting(BT) stages, and was 16% higher under conventional tillage(CT) than under no-tillage(NT). Transplanting required 29% more time for the completion of tillering and less for DS. Tillering rate was 43% higher in DS than TP under either CT or NT. There was a positive correlation between panicle number per m2and maximum tiller number per m2, but not panicle-bearing tiller rate. The panicle bearing tiller rate was higher under DS than TP and higher under NT than CT. Tiller dry weight gradually increased up to heading(HD) stage, and was 14% higher under TP than DS. Leaf area(cm2tiller-1) gradually increased from Max. to HD stage and then decreased by 34% in conventional tillage transplanting(CTTP) and 45% in no-tillage transplanting(NTTP) from 12DAH–24DAH(days after heading), but was similar(35%) under DS under either CT or NT. Grain yield was higher under CTTP owing to the larger sink size(heavier panicle, more spikelets in per cm length of panicle) than under DS.

Rehabilitation of bauxite residue to support soil development and grassland establishment

Rehabilitation(amendment and vegetation establishment)on bauxite residue is viewed as a promising strategy to stabilize the surface and initiate soil development.However,such approaches are inhibited by high pH,high exchangeable sodium(ESP)and poor nutrient status.Amendment with gypsum is effective in improving residue physical and chemical properties and promoting seed establishment and growth.Application of organics(e.g.compost)can address nutrient deficiencies but supplemental fertilizer additions may be required.A series of germination bioassays were performed on residue to determine candidate species and optimum rehabilitation application rates.Subsequent field trials assessed establishment of grassland species Holcus lanatus and Trifolium pratense as well as physical and chemical properties of amended residue.Follow up monitoring over five years assessed elemental content in grassland and species dynamics.With co-application of the amendments several grassland species can grow on the residue.Over time other plant species can invade the restored area and fast growing nutrient demanding grasses are replaced.Scrub species can establish within a 5 Yr period and there is evidence of nutrient cycling.High pH,sodicity and nutrient deficiencies are the major limiting factors to establishing grassland on residue.Following restoration several plant species can grow on amended residue.

Establishment and characterization of an opisthorchiasis-associated cholangiocarcinoma cell line (KKU-100)

AM: To establish and characterize a new cholangiocarcinoma cell line from a patient living in the Opisthorchis viverrini (O. viverrini) endemic area of Northeast Thailand. METHODS: Fresh liver biopsy and bile specimens were obtained from a 65-year-old Thai woman with cholangiocarcinoma of the ports hepatis. After digestion, the cells were cultured in Ham's F12 media. The established cell line was then characterized for growth kinetics, cell morphology, imm-unocytochemistry and cytogenetics. Tumorigenicity of the cell line was determined by heterotransplanting in nude mice. RESULTS: The primary tumor was a poorly differentiated tubular adenocarcinoma. Examination of the bile revealed malignant cells with O. viverrin eggs. The cholangiocarcinoma cell line KKU-100 was established 4 mo after the primary culture-population doubling time was 72 h. KKU-100 possesses compact and polygonal-shaped epithelial cells. Immunocytochemically, this cell line exhibited cytokeratin, EMA, CEA, and CA125, but not a-fetoprotein (AFP), CA19-9, desmin, c-met, or p53. Such protein expressions parallel those of the primary tumor. Cytogenetic analysis identified aneuploidy karyotypes with a modal chromosome number of 78 and marked chromosomal structural changes. Inoculation of KKU-100 cells into nude mice produced a transplantable, poorly differentiated aden-ocarcinoma, similar to the original tumor. CONCLUSION: KKU-100 is the first egg-proven, Opisthorchis- associated cholangiocarcinoma cell line, which should prove useful for further investigations of the tumor biology of this cancer.

High-throughput screening of mouse gene knockouts identifies established and novel skeletal phenotypes

Screening gene function in vivo is a powerful approach to discover novel drug targets. We present high-throughput screening （HTS） data for 3 762 distinct global gene knockout （KO） mouse lines with viable adult homozygous mice generated using either gene-trap or homologous recombination technologies. Bone mass was determined from DEXA scans of male and female mice at 14 weeks of age and by microCT analyses of bones from male mice at 16 weeks of age. Wild-type （WT） cagemates/littermates were examined for each gene KO. Lethality was observed in an additional 850 KO lines. Since primary HTS are susceptible to false positive findings, additional cohorts of mice from KO lines with intriguing HTS bone data were examined. Aging, ovariectomy, histomorphometry and bone strength studies were performed and possible non-skeletal phenotypes were explored. Together, these screens identified multiple genes affecting bone mass： 23 previously reported genes （Calcr, Cebpb, Crtap, Dcstamp, Dkkl, Duoxa2, Enppl, Fgf23, Kissl/Kisslr, Kl （Klotho）, Lrp5, Mstn, Neol, Npr2, Ostml, Postn, Sfrp4, S1c30a5, Sic39a13, Sost, Sumf1, Src, Wnt10b）, five novel genes extensively characterized （Cldn18, Fam20c, Lrrkl, Sgpll, Wnt16）, five novel genes with preliminary characterization （Agpat2, RassfS, Slc10a7, Stc26a7, Slc30a10） and three novel undisclosed genes coding for potential osteoporosis drug targets.

PHENOMENON OF ESTABLISHING COENOSIUM IN DESERT AND THE ECOLOGICAL RESTORATION IN THE AREA AROUND THE OASIS

The area around the oasis, lying between the oasis and the natural desert and encirclesthe oasis is different from ecotone of the oasis and the desert, because it is not the trans-forming area betwen them, but it is ecological rift valley or ecological burst zone. The oasisbase will be shaked and the nature desert will be degenerative too. It is the crux of the envi-

T_(BB) DATA-REVEALED FEATURES OF ASIAN-AUSTRALIAN MONSOON SEASONAL TRANSITION AND ASIAN SUMMER MONSOOM ESTABLISHMENT

Based on TBB data from Meteorological Institute Research of Japan, study is carried out of the features of seasonal transition of Asian-Australian monsoons and Asian summer monsoon establishment,indicating that the transition begins as early as in April, followed by abrupt change in May-June; the Asian summer monsoon situation is fully established in June. The winter convective center in Sumatra moved steadily northwestward across the "land bridge" of the maritime continent and the Indo-China Peninsula as time goes from winter to summer, thus giving rise to the change in large scale circulations that is responsible for the summer monsoon establishment over SE Asia and India; the South China Sea to the western Pacific summer monsoon onset bears a close relation to the active convection in the Indo China Peninsula and steady eastward retreat of the subtropical TBB high-value band,corresponding to the western Pacific subtropical high.

Establishment and assessment of rodent models of medication-related osteonecrosis of the jaw(MRONJ)

Medication-related osteonecrosis of the jaw(MRONJ)is primarily associated with administering antiresorptive or antiangiogenic drugs.Despite significant research on MRONJ,its pathogenesis and effective treatments are still not fully understood.Animal models can be used to simulate the pathophysiological features of MRONJ,serving as standardized in vivo experimental platforms to explore the pathogenesis and therapies of MRONJ.Rodent models exhibit excellent effectiveness and high reproducibility in mimicking human MRONJ,but classical methods cannot achieve a complete replica of the pathogenesis of MRONJ.Modified rodent models have been reported with improvements for better mimicking of MRONJ onset in clinic.This review summarizes representative classical and modified rodent models of MRONJ created through various combinations of systemic drug induction and local stimulation and discusses their effectiveness and efficiency.Currently,there is a lack of a unified assessment system for MRONJ models,which hinders a standard definition of MRONJ-like lesions in rodents.Therefore,this review comprehensively summarizes assessment systems based on published peer-review articles,including new approaches in gross observation,histological assessments,radiographic assessments,and serological assessments.This review can serve as a reference for model establishment and evaluation in future preclinical studies on MRONJ.

Establishment and identification of induced pluripotent stem cells in liver cancer patients

Objective:To induce pluripotent stem(IPS)cells from fibrocytes that are separated from liver cancer patients.Methods:The fibrocytes were reprogrammed to IPS cells by lentiviral vector,stained and identified by immunohistochemistry.Results:The IPS cells were successfully established from fibrocytes after infection,and IPS cell clones formed in round shape under a microscopy.The induction rate was 0.013%±0.007%.No tumor formed at the back of nude mice within 8 weeks after the inoculation of cell clone.However,tetatoma appeared in nude mice within 1 week after IPS inoculation.A few tumors formed in nude mice within 4 weeks after the inoculation of cell clones.However,subcutaneous tumors formed within 1 week after IPS inoculation.The induced IPS cells showed three germ layers in tetatoma.Nanog and OCT4 in the induced IPS cells showed hypomethylation.SSEA-A,TRA-1-6-,TRA-1-81 and Nanog were highly expressed in the induced IPS cells,indicating the IPS cells possessed the similar ability as the stem cells.Conclusion:The IPS cells of liver cancer patients can be established effectively from fibrocytes and can be cultured stably in vitro,which provides an approach for the treatment of intermediate or advanced stage liver cancer.

Effects of aggregating forests, establishing forest road networks, and mechanization on operational efficiency and costs in a mountainous region in Japan

We investigated forest road networks and forestry operations before and after mechanization on aggregated forestry operation sites. We developed equations to estimate densities of road networks with average slope angles, operational efficiency of bunching operations with road network density, and average forwarding distances with operation site areas. Subsequently, we analyzed the effects of aggregating forests, establishing forest road networks, and mechanization on operational efficiency and costs. Six ha proved to be an appropriate operation site area with minimum operation expenses. The operation site areas of the forest owners＇ cooperative in this region aggregated approximately 6 ha and the cooperative conducted forestry operations on aggregated sites. Therefore, 6 ha would be an appropriate operation site area in this region. Regarding road network density, higher-density road networks increased operational expenses due to the higher direct operational expenses of strip road establishment. Therefore, road network density should be reduced to approximately 200 m.

Experiences with Rice Grown on Permanent Raised Beds: Effect of Crop Establishment Techniques on Water Use, Productivity, Profitability and Soil Physical Properties

In recent years, conventional rice production technologies have been leading to deterioration of soil health and declining farm profitability due to high inputs of water and labor. Conservation agriculture （CA） based resource-conserving technologies i.e. zero-tillage （ZT）, raised-bed planting and direct-seeded rice （DSR） have shown promise as alternatives to conventional production technologies to overcome these problems. Present study was undertaken during 2009-2012 to establish an understanding of how permanent raised bed cropping system could be practiced to save water at the field application level to improve water productivity and also have the capability to enhance productivity, profitability and soil physical quality. The results showed that among different crop establishment techniques, conventional-tilled puddle transplanted rice （CT-TPR） required 14%-25% more water than other techniques. Compared with the CT-TPR system, zero till direct-seeded rice （ZT-DSR） consumed 6%-10%less water with almost equal system productivity and demonstrated higher water productivity. Wide raised beds saved about 15%-24% water and grain yield decrease of about 8%. Direct-seeded rice after ZT or reduced tillage or on unpuddled soil provided more net income than CT-TPR. The CT-TPR system had higher bulk density and penetration resistance due to compaction caused by the repeated wet tillage in rice. The steady-state infiltration rate and soil aggregation （〉 0.25 mm） were higher under permanent beds and ZT and lower in the CT-TPR system. Under CT-TPR, soil aggregation was static across seasons, whereas it improved under no-till and permanent beds. Similarly, mean weight diameter of aggregates was higher under ZT and permanent beds and increased over time. The study reveals that to sustain the rice productivity, CA-based planting techniques can be more viable options. However, the long-term effects of these alternative technologies need to be studied under varying agro-ecologies in western Uttar Pradesh, India.