[A Case of Hepatic Undifferentiated Embryonal Sarcoma in an Adult].

A 20-year-old woman was admitted with abdominal pain and a cystic liver tumor. A hemorrhagic cyst was suspected. Contrast-enhanced computed tomography(CT)and magnetic resonance imaging(MRI)revealed a space-occupying solid mass in the right lobule. Positron emission tomography(PET)-CT revealed 18F-fluorodeoxyglucose uptake in the tumor. We performed a right hepatic lobectomy. Histopathological evaluation of the resected tumor revealed an undifferentiated embryonal sarcoma of the liver(UESL). The patient refused adjuvant chemotherapy but showed no recurrence 30 months postoperatively. UESL is a rare malignant mesenchymal tumor that occurs in infants and children. It is extremely rare and is associated with poor prognosis in adults. In this report, we described a case of adult UESL.

Development of multiplexing gene silencing system using conditionally induced polycistronic synthetic antisense RNAs in Escherichia coli.

Although efficient methods of gene silencing have been established in eukaryotes, many different techniques are still used in bacteria due to the lack of a standardized tool. Here, we developed a convenient and efficient method to downregulate the expression of a specific gene using ∼140 nucleotide RNA with a 24-nucleotide antisense region from an arabinose-inducible expression plasmid by taking Escherichia coli lacZ and phoA genes encoding ß-galactosidase and alkaline phosphatase, respectively, as target genes to evaluate the model. We examined the antisense RNA (asRNA) design, including targeting position, uORF stability elements at the 5'-end, and Hfq-binding module at the 3'-end, and inducer amount required to obtain effective experimental conditions for gene silencing. Furthermore, we constructed multiplexed dual-acting asRNA genes in the plasmid, which were transcribed as polycistronic RNA and were able to knockdown multiple target genes simultaneously. We observed the highest inhibition level of 98.6% when lacZ was targeted using the pMKN104 asRNA expression plasmid, containing a five times stronger PBAD -10 promoter sequence with no requirement of the Hfq protein for repression. These features allow the system to be utilized as an asRNA expression platform in many bacteria, besides E. coli, for gene regulation.

Antimicrobial antisense RNA delivery to F-pili producing multidrug-resistant bacteria via a genetically engineered bacteriophage.

Multidrug-resistant bacteria are a growing issue worldwide. This study developed a convenient and effective method to downregulate the expression of a specific gene to produce a novel antimicrobial tool using a small (140 nucleotide) RNA with a 24-nucleotide antisense (as) region from an arabinose-inducible expression phagemid vector in Escherichia coli. Knockdown effects of rpoS encoding RNA polymerase sigma factor were observed using this inducible artificial asRNA approach. asRNAs targeting several essential E. coli genes produced significant growth defects, especially when targeted to acpP and ribosomal protein coding genes rplN, rplL, and rpsM. Growth inhibited phenotypes were facilitated in hfq- conditions. Phage lysates were prepared from cells harboring phagemids as a lethal-agent delivery tool. Targeting the rpsM gene by phagemid-derived M13 phage infection of E. coli containing a carbapenem-producing F-plasmid and multidrug-resistant Klebsiella pneumoniae containing an F-plasmid resulted in the death of over 99.99% of infected bacteria. This study provides a possible strategy for treating bacterial infection and can be applied to any F-pilus producing bacterial species.

Artificial small RNA-mediated growth inhibition in Escherichia coli and Salmonella enterica serovar Typhimurium.

We developed a synthetic RNA approach to identify growth inhibition sequences by cloning random 24-nucleotide (nt) sequences into an arabinose-inducible expression vector. This vector expressed a small RNA (sRNA) of ∼140 nt containing a 24 nt random sequence insert. After transforming Escherichia coli with the vector, 10 out of 954 transformants showed strong growth defect phenotypes and two clones caused cell lysis. We then examined growth inhibition phenotypes in the Salmonella Typhimurium LT2 strain using the twelve sRNAs that exerted an inhibitory effect on E. coli growth. Three of these clones showed strong growth inhibition phenotypes in S. Typhimurium LT2. The most effective sRNA contained the same insert (N1) in both bacteria. The 24 nt random sequence insert of N1 was abundant in guanine residues (ten out of 24 nt), and other random sequences causing growth defects were also highly enriched for guanine (G) nucleotides. We, therefore, generated clones that express sRNAs containing a stretch of 16 to 24 continuous guanine sequences (poly-G16, -G18, -G20, -G22, and -G24). All of these clones induced growth inhibition in both liquid and agar plate media and the poly-G20 clone showed the strongest effect in E. coli. These results demonstrate that our sRNA expression system can be used to identify nucleotide sequences that are potential candidates for oligonucleotide antimicrobial drugs.