[Effects of Different Culture Systems on the Hematopoietic Differentiation Ability of iPS Cells].

OBJECTIVE: To investigate the in vitro effects of different culture systems on hematopoietic differentiation ability of induced pluripotent stem (iPS) cells. METHOD: Two culture systems including E8 and mTESR(freeder-free medium), and the classical ES culture medium were chosen for culture of iPS cells. The iPS cells maintaining in above mentioning culcure systems were co-cultured with OP9 cells(murine bone marrow stromal cells) in vitro to be induced to differentiate into hematopoietic stem/progenitor cells. Flow cytometry and real-time quantitative PCR were used to detect the expression of specific hematopoietic markers and the effects of different culture systems on the differentiation of iPS in vitro. RESULT: iPS cultured in the 3 selected medium could be differentiated into hematopoietic stem cells. Efficiency of hematopoietic differentiation was up to 28.4% in classical ES culture system, which was significantly higher than that in E8 and mTESR system. CONCLUSION: Under the co-culture with OP9, iPS can differentiate into hematopoietic stem/progenitor cells, which shows higher efficiency when iPS maintained in the ES medium.

Prenatal diagnosis of hemivertebrae--A likely association with 7q deletion.

OBJECTIVE: This study aims to investigate the possible cause of a prenatal case of hemivertebrae with a 7q terminal deletion. CASE REPORT: This case describes a fetus with hemivertebrae in thoracic vertebrae as the sole antenatal sonographic finding. Genetic testing was performed in order to find more information after the abnormal ultrasound finding. The array-based comparative genomic hybridization results showed that the fetus had approximately 6.4 Mb deletion of 7q36. We discussed the two genes (SHH and HLXB9) that may be associated with hemivertebrae in the deletion region and reviewed several literatures about 7q36 deletion. CONCLUSION: Our results suggest that the phenotype of hemivertebra in our case may be related to the deletion of 7q36.

Genetic Evaluation of Copy Number Variations, Loss of Heterozygosity, and Single-Nucleotide Variant Levels in Human Embryonic Stem Cells With or Without Skewed X Chromosome Inactivation.

Human embryonic stem cells (hESCs) exhibiting skewed X chromosome inactivation (XCI) have been reported. The copy number variations (CNVs), loss of heterozygosity (LOH), or single-nucleotide variant (SNV) events in those epigenetically distinct cells remain unknown, and whether such genetic abnormalities will influence the XCI status of hESCs is unclear. In this study, three hESCs with skewed XCI, three with random XCI, and two male hESC lines at different passages were analyzed for CNVs and LOH levels using a high-resolution genotyping microarray. Whole-exome sequencing was used to investigate the potentially damaging SNVs. On average, 17.6 CNVs and 5.3 cases of LOH were identified in the skewed hESCs, which were similar to the rates observed in random hESCs. Five recurrent CNV regions were uniquely identified in the skewed hESCs, but all of them were considered polymorphisms. With the exception of a nongenic CNV, no additional CNVs were detected on the X chromosome in the skewed hESCs. Although the XCI status in two hESC lines was observed to be changed from random to skewed, no significant CNV difference was identified before and after the XCI change. SNV analysis indicated that normal alleles are maintained for most genes within copy-neutral LOH regions. Three types of expression patterns were observed in heterozygous alleles, and the damaging SNVs in skewed hESCs favored the expression of the wild-type alleles. In conclusion, in the present study, we did not find genetic differences in the CNV and LOH levels between hESCs with and without skewed XCI. Wild-type allele expression in the presence of damaging SNVs on the X chromosome in skewed hESCs might alleviate adverse effects in those hESCs.

Analysis of expressed sequence tags and characterization of a novel gene, Slmg7, in the midgut of the common cutworm, Spodoptera litura.

BACKGROUND: Out of total 3,081 assembled expressed sequence tags (ESTs) sequences representing 6,815 high-quality ESTs identified in three cDNA libraries constructed with RNA isolated from the midgut of Spodoptera litura, 1,039 ESTs showed significant hits and 1,107 ESTs did not show significant hits in BLAST searches. It is of interest to clarify whether or not these ESTs that did not show hits function in S. Litura. RESULTS: Twenty "no-hit" ESTs containing at least one putative open reading frame were selected for further expression analysis. The results from northern blot analysis showed that six of the selected ESTs are expressed in the larval midgut of this insect at different levels, suggesting that these ESTs represent true mRNA products, whereas the other 14 ESTs could not be detected. Homologues of the four larval midgut-predominant genes (Slmg2, Slmg7, Slmg9 and Slmg17) were detected in the genomes of other lepidopteran insects but not in Drosophila melanogaster. A novel gene, Slmg7, is expressed at a high level specifically in the midgut during each of the larval stages. Slmg7 is a single copy gene and encodes a 143-amino acids protein. The SLMG7 protein was localized to the cytoplasm of Spli-221 cells. CONCLUSIONS: Six ESTs from the no hit list are transcribed into mRNA and are mainly expressed in the midgut of S. litura. Slmg7 is a novel gene that is localized to the cytoplasm.

Molecular characterization and expression analysis of elongation factors 1A and 2 from the Pacific white shrimp, Litopenaeus vannamei.

Elongation factors (EF) are abundant cell proteins that play important roles in the metabolism of all multicellular organisms. Here we describe a functional analysis of elongation factor 1-alpha (EF1A) and elongation factor 2 (EF2), from the Pacific white shrimp, Litopenaeus vannamei. Full-length cDNAs of genes corresponding to EF1A and EF2 were obtained that were 1547 and 2729 bp long, with open reading frames encoding 461 and 846 amino acids, respectively. The deduced amino acid sequences of L. vannamei EF1A and EF2 showed high similarity with those from mice, humans, chickens and other shrimps. RT-PCR analysis indicated that mRNA transcripts of EF1A and EF2 are strongly (but differentially) expressed in haemocytes and gill tissue, and at varying levels in other examined tissues, of the shrimps. Levels of both EF1A and EF2 transcripts increased when shrimps were challenged by pH and cadmium stress, but reached maximal levels after different exposure periods. These results indicate that EF1A and EF2 may play distinct, essential roles in the repair of cellular damage induced by pH and cadmium stress.

Expression of HSP60 and HSP70 in white shrimp, Litopenaeus vannamei in response to bacterial challenge.

In the present study, cDNA encoding a heat shock protein 60 (LvHSP60) gene in Litopenaeus vannamei was cloned using a combination of homology and rapid amplification of cDNA end (RACE) methods. The full length of the LvHSP60 cDNA was found to be 2379bp, with a 1737bp open reading frame. The translated amino acid sequence consisted of 579 residues with a calculated molecular mass of 60.8kD and an isoelectronic point (pI) of 5.97. Comparison of the deduced amino acid sequence showed that it has high identity (85-89%) with HSP60/chaperonins from insects and mammals. Quantitative real-time PCR and Western blot analysis were carried out to investigate the expression patterns and distribution profiles of LvHSP60 before and after stimulation with the Gram-positive bacterium Staphylococcus aureus and the Gram-negative bacterium Vibrio alginolyticus. LvHSP60 mRNA was found to be both constitutive and inducible, and was highly expressed in haemocytes and almost all tissues examined, including muscle, stomach, heart, hepatopancreas and gill tissue, but it was less strongly expressed in the intestine. The expression analysis revealed that LvHSP60 was significantly up-regulated in the gills, hepatopancreas and haemocytes after bacterial challenge. Transcription of LvHSP70 was also induced in haemocytes and the hepatopancreas after different bacteria injection. Subsequent flow cytometry analysis showed that the concentration of Ca(2+) ions increased significantly within bacteria-challenged haemocytes by 1.5h after injection. The results indicate that LvHSP60 and LvHSP70 may play important roles in mediating the immune responses of L. vannamei to bacterial challenge, and that the Ca(2+) signalling transduction pathway may be involved in the initiation of the shrimp's immune responses in early stages of infection.

Molecular cloning and characterization of an ATP-binding cassette (ABC) transmembrane transporter from the white shrimp Litopenaeusvannamei.

ATP-binding cassette transmembrane transporters (ABC transporters) have a potential role in drug and xenobiotic resistance. Here, we report for the first time the cloning of an ABC transporter from white shrimp Litopenaeusvannamei (designated LvABCG), along with a study of its phylogenetic relationships, and measurements of its expression in different shrimp tissues exposed to cadmium and pH stress (acidic and alkaline conditions). Sequence analysis showed that LvABCG shares many similarities with the white/ABC transmembrane transporter, including two conserved regions: a highly conserved ATP-binding cassette (ABC) and transmembrane domain (TMD). Spatial analyses of transcript levels for ABCG in shrimp tissues, using reverse transcript PCR, revealed the highest transcript level in the hepatopancreas, less in the intestine and stomach, and none in the other tissues examined. The ABC transporter mRNA transcript in the hepatopancreas of L.vannamei was significantly up-regulated after 1.5 h and 24 h of exposure to alkaline and acidic conditions, respectively. LvABCG was also induced in intestine, but was downregulated in the stomach under the alkaline treatment. Upon exposure to cadmium (4.25 micromol L(-1) and 8.5 micromol L(-1)) for 48 h, the mRNA expression of LvABCG was up-regulated 4.79-fold (at 6 h) and 2.09-fold (at 12 h) in the hepatopancreas. LvABCG was also induced in the stomach after exposure to 4.25 micromol L(-1) cadmium, but downregulated in the stomach and intestine after exposure to 8.5 micromol L(-1) cadmium. These findings indicate that LvABCG might play an important role in the physiological changes related to metabolism and cell detoxification that occur when Pacific white shrimp are exposed to cadmium and pH stress.

Glutathione S-transferase in the white shrimp Litopenaeus vannamei: Characterization and regulation under pH stress.

We first expressed a Mu-class GST from white shrimp Litopenaeus vannamei in Escherichia coli, and then characterized the purified recombinant enzyme with respect to the effects of pH, temperature on its catalytic (1-chloro-2, 4-dinitrobenzene-glutathione conjugation) activity. We also analyzed its expression profile in L. vannamei tissues, and assessed changes in Mu-GST expression, GST activity profiles and mortality rates following exposure of white shrimp to low and high pH (5.6 and 9.3, respectively). Realtime-PCR analysis showed that Mu-GST transcripts were expressed in all examined L. vannamei tissues, but were most abundant in the hepatopancreas. At low pH Mu-GST transcript levels in the hepatopancreas were highest after 12 h, and then declined to their original levels after 24 h. After 12 h they were also upregulated in haemocytes, but downregulated in the gills, and unchanged in the stomach following exposure to pH stress. Western blot analyses confirmed that the Mu-GST protein was strongly expressed in the hepatopancreas after 12 h at low pH and remain unchanged in the stomach after exposure to pH stress. pH-Related changes in GST activities in the shrimp hepatopancreas were similar to those displayed by the Mu-GST mRNA and protein profiles. In addition, the mortality of L. vannamei was higher at high pH than at low pH. These results suggest that L. vannamei Mu-GST expression is stimulated by acidic pH and that it may play important roles in detoxification of xenobiotics and antioxidant defenses.