Modal Chromosome Number Research Articles

Cytogenetic and <i>ISSR</i>-Markers Polymorphism in the Population of Local Ukrainian Lebedyn Cows

Preservation of the fund of local breeds of agricultural animals, which are breeding material for the creation of new ones and the improvement of existing ones, meets the requirements of the FAO - the protection of biological diversity. The study of the genetic structure of cows of the local Lebedyn breed, which was bred in Ukraine, with the use of cytogenetic and molecular genetic methods, is aimed at establishing information about the structure of the gene pool of these animals and the uniqueness of their genotype. Cytogenetic monitoring showed that the modal number of chromosomes corresponded to the species norm and was 2n=60, no constitutional abnormalities in the form of Robertsonian translocations were detected, the average frequency of genomic disorders (aneuploidy) was 10,2±2,10%, polyploidy was not manifested. structural disorders (chromosomal breaks) accounted for 3,1±1,88% and asynchronous separation of the centromeric regions of chromosomes – 0,8±0,10%. The average share of cytogenetic parameters of cells was determined: lymphocytes with micronucleus – 3,6±0,61‰, binucleus lymphocytes – 4,3±0,97‰ and mitotic index – 3,7±1,20‰. An analysis of the genetic structure of the local, small-numbered Lebedyn cattle breed of Ukraine was carried out using 8 ISSR-systems. All 8 microsatellite primers showed high efficiency. ISSR labeling revealed 88 amplified DNA fragments, of which only 18 were polymorphic. The total share of polymorphic loci was 25,45%. 11 species-specific loci for (ACC)6G, 5 loci for (CTC)6C, 10 for (GAG)6C, 7 for (GA)6CC, 10 for (AG)8CG, 9 for (AG)8CA, 13 for (GA)9C and 14 for (AG)9C. It was established that the studied animals were characterized by karyotype stability, reduced sensitivity to mutagenic factors of various nature, absence of inbreeding depression and reproductive isolation.

Occurrence of high hyperdiploid karyotypes in childhood Acute Lymphoblastic Leukemia

Abstract Introduction/Objective High hyperdiploidy is defined as the non-random gain of chromosomes, increasing the modal chromosome number from 46 to between 51 and 65 or 67. This condition is the most common finding in childhood acute lymphoblastic leukemia, with a good outcome. It is generally characterized by the gain of chromosomes, typically X, 4, 6, 10, 14, 17, 18, and 21. The objective of this study is to determine the frequency of high hyperdiploidy in childhood ALL. Methods/Case Report Cytogenetic results of 435 patients, reported between October 2020 and September 2022 were reviewed retrospectively. All individuals were suspected for ALL between ages 1-15 years. Chromosomal analysis was performed in the Cytogenetics Lab of the Indus hospital and health network. Fluorescent in-situ hybridization (FISH) analysis were also carried out with probes for co-relating the gain of chromosomes. Results (if a Case Study enter NA) A total of 435 individuals were successfully karyotyped, 82 karyotypes showed hyperploidy without any additional abnormality, 213 patients showed hyperploidy along with additional structural aberrations, and 143 individuals showed normal karyotype. Among all the hyperploid karyotypes, chromosomes X, 4, 6, 10, 14, 17, 18, 21 were all gained in approximately 70% of the cases. Whereas, chromosome 21 was gained in 100% of all cases. FISH results also showed the gain of signals for particular probes including chromosomes 8, 9, 11, 12, 21 and 22. Conclusion In summary, this study leads to the fact that high hyperdiploidy is the most common abnormality in childhood acute lymphoblastic leukemia. It is assured that the occurrence of constituent trisomies of certain chromosomes is linked with the prognosis of the disease and also helps in determining the differences in treatment of the disease. Furthermore, correlation of karyotypes with the FISH results have also played a vital role in increasing the accuracy of results in this study.

Development and characterization of a spleen cell line from yellowfin seabream Acanthopagrus latus and its susceptibility to Mandarinfish ranavirus.

Yellowfin seabream (Acanthopagrus latus) is one of the most commercially important marine fish in China. In this study, a new continuous cell line, named ALS cells, was developed from the spleen tissue of A. latus. The cell line was maintained in Dulbecco's modified Eagle medium/Nutrient Mixture F-12 Ham (DMEM/F-12) supplemented with 10% fetal bovine serum (FBS) and successfully cultured up to 50 passages. The cell line was authenticated by amplifying and sequencing mitochondrial cytochrome C oxidase subunit-I (coi-I) gene. The ALS cell line had the maximum growth rate in DMEM/F-12 medium containing 20% FBS at 27°C. Chromosome number analysis showed that the ALS cells have a modal diploid chromosome number of 34. The ALS cell line was transfected with the pEGFP-N1 plasmid, and green fluorescence was observed. The ALS cell line was used for testing Mandarinfish ranavirus (MRV) susceptibility, and the cytopathic effects in the cell line were observed at 4 days post-infection (dpi). Furthermore, the susceptibility of the ALS cell line to MRV and the levels of MRV mRNA and viral loads were found to be significantly increased at 1-7 dpi. This study revealed that the ALS cell line could be useful for molecular, virological, and biotechnological studies on yellowfin seabream.

Establishment and characterization of a brain tissue cell line from spotted knifejaw (Oplegnathus punctatus) and its susceptibility to several fish viruses.

Cells are important in the study of virus isolation and identification, viral pathogenic mechanisms and antiviral immunity. The spotted knifejaw (Oplegnathus punctatus) is a significant farmed fish in China that has been greatly affected by diseases in recent years. In this study, a new cell line derived from the spotted knifejaw brain (SKB) was established and characterized. SKB cells multiplied well in Leibovitz's L-15 medium supplemented with 10% fetal bovine serum at 28°C. Chromosome analysis revealed that modal chromosome number was 48 for SKB. SKB cells exhibit susceptibility to several fish viruses, such as a largemouth bass virus, red grouper nervous necrosis virus (RGNNV), infectious spleen and kidney necrosis virus (ISKNV), Singapore grouper iridovirus (SGIV) and spotted knifejaw iridovirus isolate (SKIV-TJ), as shown by cytopathic effect and increased viral titers. Electron microscopy results showed that the cytoplasm contained a large number of vacuoles, and many virus particles existed at the edge of the vacuoles in RGNNV-infected cells and numerous viral particles were scattered throughout the cytoplasm in both ISKNV- and SKIV-TJ-infected cells. These results suggest that SKB is an ideal tool for studying host-virus interactions and potential vaccine development.

Development, characterization and in vitro applications of a thymus cell line from Pangasianodon hypophthalmus (Sauvage 1878)

Pangasianodon hypophthalmus is an economically important catfish species cultured in India. In the present study, a continuous cell line from the thymus of P. hypophthalmus (PHT) was established using the explant method and subcultured 52 times since development. PHT cells showed optimal growth in L-15 medium containing 20% fetal bovine serum at 28°C. The species of the cell line from striped catfish was confirmed through PCR amplification and sequencing of 16S, and COI genes. The cell line was found to be Mycoplasma free. The modal chromosome number of PHT cells was 60 (2n). Immunophenotyping using different antibodies showed the epithelial nature of the cells. Cytotoxicity of arsenic and mercury was assessed using Neutral red and MTT assay, which revealed reduced cell survival with an increase in toxicant concentration. Cells transfected successfully with the GFP reporter gene using lipofectamine reagent indicated the suitability of the cell line for expression studies. The cell line has been submitted to NRFC, Lucknow with accession no. NRFC-078 at ICAR-NBFGR, Lucknow. The developed cell line will have applications in suspected viral disease investigation, transgenic, and immunological studies.

Germplasm Rescue of Postmortem Critically Endangered Yangtze Sturgeon (Acipenser dabryanus) by Cell Preservation

With the growing sophistication of cloning technology, rescuing cell resources is of great significance for the protection of endangered animals. The Yangtze sturgeon (Acipenser dabryanus), one of the three Acipenseriformes species in the Yangtze River, is critically endangered. Natural reproduction of the Yangtze sturgeon has not been detected since 2000. Less than 20 wild individuals are kept in husbandry, and all are too old to breed. Therefore, it is urgent to rescue the genetic resources of every wild Yangtze sturgeon. Here, we isolated and preserved viable cells from critically endangered postmortem Yangtze sturgeon for the first time. Attempts to rescue and preserve cell resources were carried out from 8 tissues, brain, kidney, gonad, fin, liver, skin, spleen, and muscle of an over 35-year-old female wild Yangtze sturgeon between 11 and 14 hours after death at 19.8°C in an outdoor concrete pond, and only muscle tissue cells could be successfully subcultured and preserved. Furthermore, the cultured cells showed high post-thaw cell viability and normal growth with a population doubling time of 52.98 h. Moreover, they were fibronectin- and desmin-positive, characterizing them as fibroblastic confirmed muscle cells with fibroblastic properties and myogenic origin. Tests for microbial contamination of the cell lines were negative. Chromosome analysis demonstrated that muscle cells possess a modal polyploid chromosome number of 264. The mitochondrial sequence data of COI genes and 12S rRNA confirmed that the developed cell line originated from A. dabryanus. Furthermore, transfection results indicate that muscle cells could be used for gene manipulation and functional studies. These results suggest that viable muscle cells could also be successfully isolated and cryopreserved from the wild Yangtze sturgeon in a short time after death. This report is not only of great significance for the germplasm rescue of critically endangered Yangtze sturgeon but also provides some scientific reference for the germplasm preservation of other endangered fish.

Derivation and characterization of new cell line from intestine of turbot (Scophthalmus maximus)

A continuous intestine cell line from turbot (Scophthalmus maximus) designated as SMI was established utilizing the tissue explant technique. Primary SMI cell was cultured at 24 °C in a medium with 20% fetal bovine serum (FBS), then subcultured in 10% FBS after 10 passages. Impacts of medium or temperature on the growth of SMI were examined and the results indicated it grew well in DMEM supplemented with 10% FBS at 24 °C. The SMI cell line was subcultured more than 60 times. Karyotyping, chromosome number, and ribosomal RNA genotyping analysis revealed that SMI had a modal diploid chromosome number of 44 and originated from turbot. After being transfected with pEGFP-N1 and FAM-siRNA, a large number of green fluorescence signals were observed in SMI, indicating that SMI could be used as an ideal platform to explore gene function in vitro. In addition, the expression of epithelium-associated genes such as itga6, itgb4, gja1, claudin1, zo-1, and E-cadherin in SMI suggested the SMI had some characteristics of epidermal cells. The upregulation of immune-associated genes such as TNF-β, NF-κB, and IL-1β in SMI after stimulation with pathogen-associated molecular patterns suggested the SMI might exhibit immune functions similar to the intestinal epithelium in vivo.

Establishment of a new intestinal cell line from the grouper (Epinephelus coioides) for aquatic virus pathogenesis

A new cell line (ECGI-21) was developed from the intestine of grouper (Epinephelus coioides). The cell line was routinely grown in Leibovitz's L-15 medium supplemented with 15% fetal bovine serum (FBS) at 28 °C. ECGI-21 cells had an epithelioid morphology. At present, the cell line has been stably passed over 80 generations and has been successfully cryopreserved. The origin of grouper was confirmed by the sequence of 18S ribosomal RNA (18S rRNA). Chromosome analysis indicated that the modal chromosome number was 48. The green fluorescence signals were detected when pEGFP-N3 was transfected into ECGI-21 cells. Moreover, the ECGI-21 cells were susceptible to Singapore grouper iridovirus (SGIV) and Red-spotted grouper nervous necrosis virus (RGNNV), which was demonstrated by the presence of a significant cytopathic effect (CPE), increased expression of viral proteins after viral infection. Meantime, abundant viral particles were observed in the cells infected with SGIV and RGNNV by electron microscopy. Furthermore, virus replication significantly increased the level of interferon (IFN) related signaling molecules and pro-inflammatory cytokine expression in cells. In summary, ECGI-21 cell line was established and become the first stable intestinal cell line from marine fish. It provides a useful tool for studying problems of gastrointestinal health, viral pathogenesis and virus-host interaction in vitro.

Stable CDK12 Knock-Out Ovarian Cancer Cells Do Not Show Increased Sensitivity to Cisplatin and PARP Inhibitor Treatment.

Cyclin-dependent kinase 12 (CDK12) is a serine/threonine kinase involved in the regulation of RNA polymerase II and in the transcription of a subset of genes involved in the DNA damage response. CDK12 is one of the most mutated genes in ovarian carcinoma. These mutations result in loss-of-function and can predict the responses to PARP1/2 inhibitor and platinum. To investigate the role of CDK12 in ovarian cancer, CRISPR/Cas9 technology was used to generate a stable CDK12 knockout (KO) clone in A2780 ovarian carcinoma cells. This is the first report on a CDK12 null cell line. The clone had slower cell growth and was less clonogenic than parental cells. These data were confirmed in vivo, where CDK12 KO transplanted cells had a much longer time lag and slightly slower growth rate than CDK12-expressing cells. The slower growth was associated with a higher basal level of apoptosis, but there were no differences in the basal level of autophagy and senescence. While cell cycle distribution was similar in parental and knockout cells, there was a doubling in DNA content, with an almost double modal number of chromosomes in the CDK12 KO clone which, however did not display any increase in γH2AX, a marker of DNA damage. We found partial down-regulation of the expression of DNA repair genes at the mRNA level and, among the down-regulated genes, an enrichment in the G2/M checkpoint genes. Although the biological features of CDK12 KO cells are compatible with the function of CDK12, contrary to some reports, we could not find any difference in the sensitivity to cisplatin and olaparib between wild-type and CDK12 KO cells.

Establishment and characterization of a liver cell line from black porgy, Acanthopagrus schlegelii

In this study, a liver cell line (ASL) derived from black porgy (Acanthopagrus schlegelii) was established and characterised. After the initial primary culture, the cell line has been cultured for over 40 passages at 28 °C in Dulbecco's modified Eagle's medium (DMEM) with 15 % foetal bovine serum (FBS). Immunostaining results indicated that ASL cells contained at least two cytotypes in the initial culture stage; however, epithelioid cells dominated and replaced the other type during passage 6–20. ASL cells grow in temperature range 20–28 °C in DMEM containing 15–20 % FBS. Cytogenetic analysis showed that the modal chromosome number in ASL cells was 48. Partial amplification of the cytochrome c oxidase subunit I gene indicates that ASL originates from black porgy. ASL cells showed a transfection efficiency of approximately 15 % after transfection with the pEGFP-N3 plasmid. Cytopathic effects were observed in ASL cells after red-spotted grouper nervous necrosis virus (RGNNV) infection, and viral replication in RGNNV-infected ASL cells was confirmed using quantitative reverse transcription polymerase chain reaction, virus titre assays, western blot, and transmission electron microscopy assays. Furthermore, several antiviral genes were induced to varying degrees in RGNNV-infected ASL cells, indicating that the immune response was activated in ASL cells post RGNNV infection. In conclusion, the newly established ASL cell line is an ideal in vitro tool for studying NNV-black porgy interactions.

Establishment and characterization of a continuous cell line from caudal fin of Labeo calbasu (Hamilton, 1822).

Labeo calbasu is an important food fish and candidate species for diversification of carp aquaculture. In the present study, we have established a continuous cell line, designated as L. calbasu fin(LCF), from caudal fin of L. calbasu using explant method. The cell line has been subcultured for over 73 passages and the LCF cells show optimal growth in Leibovitz's L-15 medium supplemented with 20% fetal bovine serum at a temperature of 28°C. In karyotype analysis, the modal chromosome number of LCF cells at 35th passage was found to be 50. The amplification and sequencing of partial fragments of mitochondrial genes, namely 16S rRNA and COI from LCF cells confirmed the origin of cell line from L. calbasu. The LCF cells could be successfully transfected with GFP reporter gene, indicating suitability of these cells for expression of foreign genes. Further, following inoculation with supernatant from Tilapia lake virus (TiLV) infected cell line, no cytopathic effects were observed in the LCF cells and cell pellet was negative for TiLV in RT-PCR, indicating that LCF cells were not susceptible to TiLV. The developed cell line has been submitted to National Repository of Fish Cell Lines being maintained at ICAR-National Bureau of Fish Genetic Resources, Lucknow (accession no. NRFC063). The newly developed LCF cell line would be helpful in investigating diseases affecting this candidate species particularly the ones suspected to be of viral etiology, and for cytotoxicity and transgenic studies.

Construction of a human hTERT RPE-1 cell line with inducible Cre for editing of endogenous genes.

ABSTRACTThe human retinal pigment epithelial RPE-1 cell line immortalized with hTERT retains a stable karyotype with a modal chromosome number of 46 and has been widely used to study physiological events in human cell culture systems. To facilitate inducible knock-out or knock-in experiments in this cell line, we have modified the AAVS1 locus to harbour a DNA fragment encoding ERT2-Cre-ERT2 fusion protein under regulation of a Tet-On expression system. In the generated cell line, active Cre recombinase was induced by simple addition of doxycycline and tamoxifen to the culture medium. As proof of concept, we successfully introduced an oncogenic point mutation to the endogenous KRAS gene locus of this cell line. The cell line will serve as a powerful tool to conduct functional analyses of human genes.

Bionomics and Karyotypic analysis of two species of Caccobius (Scarabaeinae: Scarabaeidae: Coleoptera) detected by standard staining, C-banding and AgNOR-banding

Out of 16 species of caccobius available in India, 9 are found in Haryana and 6 in Kurukshetra. The Karyotypic characterisation of two species of Caccobius, C. ultor and C. vulcanus belonging to subfamily Scarabaeinae was undertaken using standard staining, silver staining and C-banding. Both of the species were comprised of 20 chromosomes in karyotype with meioformula, 9AA+Xyp. Modal chromosome number and meta-, submeta- centric nature revealed the conservative nature of the subfamily Scarabaeinae. The analysis of constitutive heterochromatin (CH) revealed small centromeric bands in spermatogonial metaphase and small blocks located in the terminal regions of dumb bell shaped bivalents in metaphase I. At the pachytene stage of prophase I, silver staining revealed dense spots associated with nucleolar organizer regions. In addition to this, the Chiasma frequency and terminalisation coefficient were also calculated, illustrating the close relationship of Caccobius ultor and C. vulcanus with other Scrabaeinae species. In beetles as well as in other insects, the chromosome structure, pattern of constitutive heterochromatin distribution, and position of the nucleolar organizer region are all key factors in speciation.

Flow Cytometric DNA Ploidy Analysis in Haemato-Lymphoid Neoplasms: An Analysis of 132 Cases.

Background: FxCycleTM Violet (FCV) based flow cytometric (FCM) DNA ploidy analysis is a rapid and simple tool that can substantiate in characterizing the biological behaviour across the spectrum of haematological malignancies and correlates with cytogenetic studies. Materials and Methods: In this prospective study, we performed simultaneous immunophenotyping with FCV based on ploidy analysis in n=132 consecutive new samples, comprising n=110 samples of haemato-lymphoid neoplasms, including acute leukemias (n=67, 60.9%), CML with myeloid blast crisis (n=1, 0.9%), MDS with excess blasts (n=2, 1.8%), mature B cell/ T cell neoplasms (n=37, 33.7%), multiple myeloma (n=3, 2.7%) along with n=22 normal samples. The FCM DNA data was compared with corresponding conventional karyotyping results, wherever available. Results: In FCM ploidy analysis (n=110), the overall DNA index (DI) ranged from 0.81 to 2.17 and S-Phase fraction (SPF) from 0.1-31.6%. Diploidy was seen in n = 90 (81.8%), low-hyperdiploidy in n = 10 (9.1%), high-hyperdiploidy in n = 7 (6.4%) with one case each (0.9% each) having near-tetraploidy, high-hypodiploidy and low-hypodiploidy. The DI of all viable cell populations in normal samples ranged from 0.96-1.05. Conventional karyotyping was performed in n=76/110 cases (70%) with n= 11/76 (15%) culture failures. The modal chromosome number ranged from 45 to 63. A concordance of 95.4% (n=62/65) was noted with corresponding FCM DI. Conclusion: FCV-based ploidy is a sensitive technique that provides complementary information and ascertains a strong correlation with conventional cytogenetics across all haemato-lymphoid neoplasms. It can detect aneuploidy in all B-ALL and myeloma cases, even in hemodiluted samples with cytogenetic culture failure; supplement the diagnoses of erythroleukemia, and provide a useful screen for a higher grade lymph node disease in lymphoma cases with SPF > 3%.

Near-Haploidy and Low-Hypodiploidy in B-Cell Acute Lymphoblastic Leukemia: When Less Is Too Much.

Simple SummaryB-cell acute lymphoblastic leukemia (B-ALL) is characterized by an uncontrolled proliferation of blood cells in the bone marrow. A small fraction of B-ALL patients shows abnormally low chromosome numbers, defined as hypodiploidy, in leukemic cells. Hypodiploidy with less than 40 chromosomes is a rare genetic abnormality in B-ALL and is associated to an extremely poor outcome, with low survival rates both in pediatric and adult cases. In this review, we describe the main clinical and genetic features of hypodiploid B-ALL subtypes with less than 40 chromosomes, the current treatment protocols and their clinical outcomes. Additionally, we discuss the potential cellular mechanisms involved on the origin of hypodiploidy, as well as its leukemogenic impact. Studies aiming to decipher the biological mechanisms involved in hypodiploid subtypes of B-ALL with less than 40 chromosomes are crucial to improve the poor survival rates in these patients. Hypodiploidy with less than 40 chromosomes is a rare genetic abnormality in B-cell acute lymphoblastic leukemia (B-ALL). This condition can be classified based on modal chromosome number as low-hypodiploidy (30–39 chromosomes) and near-haploidy (24–29 chromosomes), with unique cytogenetic and mutational landscapes. Hypodiploid B-ALL with <40 chromosomes has an extremely poor outcome, with 5-year overall survival rates below 50% and 20% in childhood and adult B-ALL, respectively. Accordingly, this genetic feature represents an adverse prognostic factor in B-ALL and is associated with early relapse and therapy refractoriness. Notably, half of all patients with hypodiploid B-ALL with <40 chromosomes cases ultimately exhibit chromosome doubling of the hypodiploid clone, resulting in clones with 50–78 chromosomes. Doubled clones are often the major clones at diagnosis, leading to “masked hypodiploidy”, which is clinically challenging as patients can be erroneously classified as hyperdiploid B-ALL. Here, we summarize the main cytogenetic and molecular features of hypodiploid B-ALL subtypes, and provide a brief overview of the diagnostic methods, standard-of-care treatments and overall clinical outcome. Finally, we discuss molecular mechanisms that may underlie the origin and leukemogenic impact of hypodiploidy and may open new therapeutic avenues to improve survival rates in these patients.

Susceptibility of a cell line derived from the kidney of Chinese rice-field eel, Monopterus albus to the infection of rhabdovirus, CrERV.

Chinese rice-field eels rhabdovirus (CrERV), belonging to the genus Perhabdovirus in the family Rhabdoviridae, is the causative agent of the haemorrhagic disease of Chinese rice-field eels, Monopterus albus. The present study aims to establish a cell line derived from the kidney of Chinese rice-field eel (CrEK) for the further study of the pathogenic virus. CrEK cells were epithelioid-like and grew well in M199 medium supplemented with 10% foetal bovine serum at 28°C, and the cell line has been subcultured for more than 80 times. Karyotyping analysis of CrEK cells at 25th passage indicated a modal chromosome number of 24. Significant cytopathic effect (CPE) was observed in CrEK cells after infection with CrERV, and the virus titre reached 107.8±0.45 TCID50 /mL. The transmission electron microscopy revealed that there were a large number of virus particles in the cytoplasm of cells. The virus infection in cells was also assayed by using indirect immunofluorescence assay (IFA), fluorescence in situ hybridization (FISH), reverse transcription PCR (RT-PCR) and quantitative real-time reverse transcription-PCR (qRT-PCR). In experimental infection, CrERV cultured by cells could cause over 90% mortality in fish. CrEK represents the first kidney cell line originated from Chinese rice-field eels and be a potential material for investigating the mechanism of virus infection in this fish and the control methods for the disease.

Establishment of a cell line from the head kidney of giant grouper (Epinephelus lanceolatus) and its susceptibility to fish viruses

Grouper is an important marine fish mainly cultured in China and Southeast Asia. Here, a continuous cell line, named as ELHK, was established from head kidney of giant grouper (Epinephelus lanceolatus). The origin of cell line was confirmed by sequencing of 18s rRNA genes. ELHK cells consisted predominantly of fibroblast-like cells, and multiplied well in Leibovitz’s L-15 medium supplemented with 10% fetal bovine serum at 28 °C. Karyotyping analysis indicated that the modal chromosome number of ELHK cell was 60 at 50 passages. The cells were successfully transfected with GFP reporter gene suggesting that ELHK cell could be used for exogenous gene expression in vitro. Moreover, ELHK cells was susceptible to Singapore grouper iridovirus (SGIV) and red-spotted grouper nervous necrosis virus (RGNNV), demonstrated by the occurrence of cytopathic effects (CPEs) and increased transcription of the viral genes during virus infection. Furthermore, numerous viral particles were observed in SGIV- or RGNNV-infected ELHK cells using electron microscopy, which further confirmed that ELHK cells could be used for fish virus propagation in vitro. In addition, quantitative PCR analysis indicated that SGIV and RGNNV infection in ELHK cells significantly increased the transcripts of several interferon- or pro-inflammation-related cytokines, suggested that both of them up-regulated host interferon and inflammatory immune response in vitro. Taken together, our established ELHK cell line could be used as an in vitro tool for gene expression, as well as virus propagation and virus-host interaction studies.

Establishment and characterization of a liver cell line, ALL, derived from yellowfin sea bream, Acanthopagrus latus, and its application to fish virology.

Yellowfin sea bream (Acanthopagrus latus) is an important economic fish, which is seriously threatened by various fish viruses. In this study, a cell line designated as ALL derived from the liver of yellowfin sea bream was developed and characterized. The cell line grew well in Dulbecco's modified Eagle's medium containing 10%-20% foetal bovine serum at 28°C. Amplification of the cytochrome B gene indicated that ALL cells originated from yellowfin sea bream. The modal chromosome number of ALL cells was 48. ALL cells were efficiently transfected with pEGFP-N3 plasmids, indicating the potential application of ALL cells in exogenous gene manipulation studies. ALL cells were susceptive to three main fish viruses, including viral haemorrhagic septicaemia virus (VHSV), red-spotted grouper nervous necrosis virus (RGNNV) and largemouth bass virus (LMBV). The replication of VHSV, RGNNV and LMBV in ALL cells was confirmed by quantitative real-time polymerase chain reaction, virus titre and transmission electron microscopy assays. Moreover, ALL cells could respond to VHSV, RGNNV and LMBV infections, as indicated by the differential expression of antiviral genes involving in the innate immune response. In conclusion, the newly established ALL cell line will be an excellent in vitro platform for the study of the virus-yellowfin sea bream interaction.

Establishment of a new fish cell line from the brain of humpback grouper (Cromileptes altivelis) and its application in toxicology and bacterial susceptibility.

Cromileptes altivelis, humpback grouper, belongs to the family Epinephelidae and is one popular farmed fish species because of its high economic value and ornamental value. However, more and more diseases outbreaks have been reported with C. altivelis aquaculture. Today, a new brain cell line of C. altivelis (named CAB) was established and characterized. Our results showed that CAB cells were suitable for growth at 26°C in L-15 medium supplemented with 15% fetal bovine serum (FBS). The results of 18S rRNA gene sequencing confirmed that CAB cell line was derived from C. altivelis. Moreover, chromosomal aneuploidy was observed in CAB cells, and the modal chromosome number of CAB cells was 48 by chromosome analysis. In addition, CAB cells could transfect pEGFP-N3 plasmid with high transfection efficiency, indicating that CAB cell line has the potential to investigate the function of exogenous genes in vitro. Furthermore, the bacterial susceptibility results suggested that CAB cells were susceptive to Vibrio harveyi and Edwardsiella tarda. And, heavy metals (Hg, Cd, and Cu) were toxic to the CAB cells, and the toxic effect was dose-dependent. In summary, the CAB cell line could be a powerful tool in vitro to study functional genes and has the potential application in bacterial susceptibility and toxicology.

Nationwide Survey of Pediatric Hypodiploid Acute Lymphoblastic Leukemia in Japan

BackgroundPloidy is a highly significant prognostic factor in childhood acute lymphoblastic leukemia (ALL). Children with hypodiploid ALL continue to have inferior outcomes despite current intensive chemotherapy treatment. Hypodiploid ALL is divided into two groups based on genetic alterations. Near-haploid ALL with 24-31 chromosomes harbors alterations targeting receptor tyrosine kinase signaling, RAS signaling, and the lymphoid transcription factor gene IKZF3 and low-hypodiploid ALL with 32-39 chromosomes is characterized by alterations in TP53 that are commonly present in non-tumor cells, IKZF2 and RB1 . There is little information about children with hypodiploid ALL in Asia including Japan, although ethnic differences seem to exist. The aim of this study was to clarify the clinical characteristics and outcome of pediatric hypodiploid ALL in Japanese patients.MethodsPatients with fewer than 46 chromosomes were defined as hypodiploid ALL in this study. All reports of chromosomal analysis were re-reviewed by two independent investigators and 117 cases of hypodiploid ALL were identified out of 4261 patients who were registered in clinical trials between 1997 and 2012. A total of 64 patients were excluded due to the lack of follow-up data (n = 39), only a single cell exhibiting a chromosome number less than 46 in G-banding experiments (n = 18), incorrect diagnosis (n = 4), or congenital chromosomal aberrations such as Down syndrome (n = 1) and Robertson translocations (n = 2). Seventeen patients whose karyotypes at remission were not evaluable were also excluded.ResultsIn total 66 male (56.4%) and 51 female (43.6%) patients' data was evaluated with a median age of 6 years (range 1 to 15 years). There were 101, 8, 2, and 3 patients with chromosome numbers of 45, 44, 43, and 36, respectively. One patient exhibited each with 27, 28, and 29 chromosomes. No patients had a modal chromosome number of 30-35 or 37-42. Among the 117, 110 (94%) had B-precursor ALL and 105 (90%) had CD10 positivity. All T-ALL patients (n = 7) had 45 chromosomes. Out of 117 patients, 22 were TEL-AML1 fusion positive of which 19 had 45 chromosomes, 2 had 44 chromosomes, and 1 had 43 chromosomes. There were no patients with central nervous system (CNS) infiltration at the time of diagnosis. All but one patient who was diagnosed with T-ALL achieved a complete remission (CR) with a CR rate of 98.3%. Overall survival at 5 years varied significantly at 86.0%, 87.5%, and 62.5% in patients with 45, 44, and fewer than 44 chromosomes, respectively (P=0.037). There were no patients with CNS relapse in both the 44 and fewer than 44 chromosomes groups. Five patients out of 8 with fewer than 44 chromosomes had bone marrow relapse, even though 1 patient had undergone hematopoietic stem cell transplantation (HSCT) during his first CR. Four patients underwent HSCT after relapse, but only 1 was cured. In the 44 chromosome group, 2 of 8 patients underwent HSCT during their first CR and did not show relapse. However, one of them developed secondary renal cell carcinoma 10 years after treatment. One patient with 44 chromosomes underwent HSCT after relapse and failed to be salvaged. We also analyzed some variables that influenced event-free and overall survival in the 45 chromosome group: monosomy 7, sex chromosome abnormality, and 9p21.3 deletion. We were not able to identify any significant prognostic factor among patients with 45 chromosomes.ConclusionsAlthough the number of patients was small, patients with 44 chromosomes without HSCT showed better prognosis than those reported previously. However, the outcome of Japanese patients with fewer than 44 chromosomes was as poor as previously reported in other countries. The effect of HSCT in patients with hypodiploid ALL needs to be the subject of future studies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.