Recombinant Arginine Deiminase from Levilactobacillus brevis Inhibits the Growth of Stomach Cancer Cells, Possibly by Activating the Intrinsic Apoptosis Pathway.

The anticancer potential of Levilactobacillus brevis KU15176 against the stomach cancer cell line AGS has been reported previously. In this study, we aimed to analyze the genome of L. brevis KU15176 and identify key genes that may have potential anticancer properties. Among potential anticancer molecules, the role of arginine deiminase (ADI) in conferring an antiproliferative functionality was confirmed. In vitro assay against AGS cell line confirmed that recombinant ADI from L. brevis KU15176 (ADI_br, 5 µg/mL), overexpressed in E. coli BL21 (DE3), exerted an inhibitory effect on AGS cell growth, resulting in a 65.32% reduction in cell viability. Moreover, the expression of apoptosis-related genes, such as bax, bad, caspase-7, and caspase-3, as well as the activity of caspase-9 in ADI_br-treated AGS cells, was higher than those in untreated (culture medium-only) cells. The cell-scattering behavior of ADI_br-treated cells showed characteristics of apoptosis. Flow cytometry analyses of AGS cells treated with ADI_br for 24 and 28 h revealed apoptotic rates of 11.87 and 24.09, respectively, indicating the progression of apoptosis in AGS cells after ADI_br treatment. This study highlights the potential of ADI_br as an effective enzyme for anticancer applications.

Construction of SARS-CoV-2 spike-pseudotyped retroviral vector inducing syncytia formation.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is handled in biosafety level 3 (BSL-3) facilities, whereas the antiviral screening of pseudotype virus is conducted in BSL-2 facilities. In this study, we developed a SARS-CoV-2 spike-pseudotyped virus based on a semi-replication-competent retroviral (s-RCR) vector system. The s-RCR vector system was divided into two packageable vectors, each with gag-pol and env genes. For env vector construction, SARS-CoV-2 SΔ19 env was inserted into the pCLXSN-IRES-EGFP retroviral vector to generate pCLXSN-SΔ19 env-EGFP. When pCLXSN-gag-pol and pCLXSN-SΔ19env-EGFP were co-transfected into HEK293 T cells to generate an s-RCR virus, titers of the s-RCR virus were consistently low in this transient transfection system (1 × 104 TU/mL). However, a three-fold higher amounts of MLV-based SARS-CoV-2 pseudotyped viruses (3 × 104 TU/mL) were released from stable producer cells, and the spike proteins induced syncytia formation in HEK293-hACE2 cells. Furthermore, s-RCR stocks collected from stable producer cells induced more substantial syncytia formation in the Vero E6-TMPRSS2 cell line than in the Vero E6 cell line. Therefore, a combination of the s-RCR vector and the two cell lines (HEK293-hACE2 or Vero E6-TMPRSS2) that induce syncytia formation can be useful for the rapid screening of novel fusion inhibitor drugs.

Construction of a replication-competent retroviral vector for expression of the VSV-G envelope glycoprotein for cancer gene therapy.

Gibbon ape leukemia virus (GALV) can infect a wide variety of cells but fails to infect most cells derived from laboratory mice. Transduction of human hematopoietic stem cells with GALV retroviral vectors is more efficient than with amphotropic vectors. In this study, a Moloney murine leukemia virus-gibbon ape leukemia virus (MoMLV-GALV) vector was constructed by replacing the natural env gene of the full-length Moloney MLV genome with the GALV env gene. To monitor viral transmission by green fluorescent protein (GFP) expression, internal ribosomal entry site-enhanced GFP (IRES-EGFP) was positioned between the GALV env gene and the 3' untranslated region (3' UTR) to obtain pMoMLV-GALV-EGFP. The MoMLV-GALV-EGFP vector was able to replicate with high titer in TE671 human rhabdomyosarcoma cells and U-87 human glioma cells. To evaluate the potential of the MoMLV-GALV vector as a therapeutic agent, the gene for the fusogenic envelope G glycoprotein of vesicular stomatitis virus (VSV-G) was incorporated into the vector. Infection with the resulting MoMLV-GALV-VSV-G vector resulted in lysis of the U-87 cells due to syncytium formation. Syncytium formation was also observed in the transfected human prostate cancer cell line LNCaP after extended cultivation of cells. In addition, we deleted the GALV env gene from the MoMLV-GALV-VSV-G vector to improve viral genome stability. This MoMLV-VSV-G vector is also replication competent and induces syncytium formation in 293T, HT1080, TE671 and U-87 cells. These results suggest that replication of the MoMLV-GALV-VSV-G vector or MoMLV-VSV-G vector may directly lead to cytotoxicity. Therefore, the vectors developed in this study are potentially useful tools for cancer gene therapy.

Human-APOBEC3G-dependent restriction of porcine endogenous retrovirus replication is mediated by cytidine deamination and inhibition of DNA strand transfer during reverse transcription.

Although human apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G, hA3G)-mediated deamination is the major mechanism used to restrict the infectivity of a broad range of retroviruses, it is unclear whether porcine endogenous retrovirus (PERV) is affected by hA3G or porcine A3F (poA3F). To determine whether DNA deamination is required for hA3G- and poA3F-dependent inhibition of PERV transmission, we developed VSV-pseudotype PERV-B expressing hA3G, mutant hA3G-E67Q (encapsidation and RNA binding activity-deficient), mutant hA3G-E259Q (deaminase-deficient), or poA3F. hA3G-E67Q decreased virus infectivity by ~ 93% compared to the ~ 99% decrease of viral infectivity by wild-type hA3G, while hA3G-E259Q decreased the infectivity of PERV-B by ~ 35%. These data suggest that cytidine deamination activity is crucial for efficient restriction of PERV by hA3G, but cytidine deamination cannot fully explain the inactivation of PERV by hA3G. Furthermore, differential DNA denaturation PCR (3D-PCR) products from 293T cells infected with PERV-B expressing hA3G mutants were sequenced. G-to-A hypermutation was detected at a frequency of 4.1% for hA3G, 3.4% for hA3G-E67Q, and 4.7% for poA3F. These results also suggest that hA3G and poA3F inhibit PERV by a deamination-dependent mechanism. To examine the effect of hA3G on the production of PERV DNA, genomic DNA was extracted from 293T cells 12 h after infection with PERV expressing hA3G, and this DNA was used as template for real-time PCR. A 50% decrease in minus strand strong stop (-sss) DNA synthesis/transfer was observed in the presence of hA3G. Based on these results, we conclude that hA3G may restrict PERV by both deamination-dependent mechanisms and inhibition of DNA strand transfer during PERV reverse transcription.

Differential sensitivity of porcine endogenous retrovirus to APOBEC3-mediated inhibition.

Pigs are considered to be suitable xenotransplantation organ donors. However, the risk of pathogen transmission from pigs to humans is a major concern in the transplantation of porcine tissues. The porcine endogenous retroviruses (PERVs) PERV-A, PERV-A/C, and PERV-B can infect human cells, but PERV-C is an ecotropic virus infecting only pig cells. Thus, several strategies have been proposed to reduce PERV transmission in xenograft recipients. Human APOBEC3G (huA3G) is a single-strand DNA cytosine deaminase, which inactivates the coding capacity of the virus by deamination of cDNA cytosines to uracils. This reaction occurs within the (-) DNA strand during reverse transcription, resulting in a G-to-A mutation in the (+) strand. While recent data have shown that PERV-B is severely inhibited by huA3G and porcine A3Z2-Z3 (poA3F) in a pseudotype assay, little is known about PERV-C. Here, we compare the antiretroviral activities of huA3G, huA3F and poA3Z2-Z3 against PERV-C. Our data show that APOBEC3 was packaged into PERV-C particles and inhibited PERV-C replication in a dose-dependent manner. PERV-C infectivity was strongly inhibited by poA3Z2-Z3, but it did not markedly reduce PERV-B infectivity. This suggests that PERV-C Gag interacts efficiently with poA3Z2-Z3. In addition, we constructed stably huA3G- and poA3Z2-Z3-expressing 293-PERV-PK-CIRCE cells (human 293 cells infected with PK15-derived PERVs) to examine whether PERV is resistant to poA3Z2-Z3 in a virus-spreading assay. The stably expressed huA3G and poA3Z2-Z3 were more packaging-competent than transiently expressed APOBEC3 proteins. These results suggest that poA3Z2-Z3 can inhibit PERV replication in a pseudotype assay as well as in a virus-spreading assay.

A Replication-Competent Retroviral Vector Expressing the HERV-W Envelope Glycoprotein is a Potential Tool for Cancer Gene Therapy.

The fusogenic membrane glycoprotein (FMG) derived from the human endogenous retrovirus-W (HERV-W) exhibits fusogenic properties, making it a promising candidate for cancer gene therapy. When cells are transfected with HERV-W FMG, they can fuse with neighboring cells expressing the receptor, resulting in the formation of syncytia. These syncytia eventually undergo cell death within a few days. In addition, it has been observed that an HERV-W env mutant, which is truncated after amino acid 483, displays increased fusogenicity compared to the wild-type HERV-W env. In this study, we observed syncytium formation upon transfection of HeLa and TE671 human cancer cells with plasmids containing the HERV-W 483 gene. To explore the potential of a semi-replication-competent retroviral (s-RCR) vector encoding HERV-W 483 for FMG-mediated cancer gene therapy, we developed two replication-defective retroviral vectors: a gag-pol vector encoding HERV-W 483 (MoMLV-HERV-W 483) and an env vector encoding VSV-G (pCLXSN-VSV-G-EGFP). When MoMLV-HERV-W 483 and pCLXSN-VSV-G-EGFP were co-transfected into HEK293T cells to produce the s-RCR vector, gradual syncytium formation was observed. However, the titers of the s-RCR virus remained consistently low. To enhance gene transfer efficiency, we constructed an RCR vector encoding HERV-W 483 (MoMLV-10A1-HERV-W 483), which demonstrated replication ability in HEK293T cells. Infection of A549 and HT1080 human cancer cell lines with this RCR vector induced syncytium formation and subsequent cell death. Consequently, both the s-RCR vector and RCR encoding HERV-W 483 hold promise as valuable tools for cancer gene therapy.

Production of a replicating retroviral vector expressing Reovirus fast protein for cancer gene therapy.

Reovirus fusion-associated small transmembrane (FAST) proteins induce syncytium formation. Recently, several studies have shown that the use of recombinant vectors engineered to express fusion proteins is becoming attractive for the development of enhanced oncolytic viruses. In this study, we investigated the cytotoxic effect of four different FAST proteins (p10 FAST of Avian reovirus [ARV], p10 FAST of Pulau virus [PuV], p13 FAST of Broome virus [BroV], and p14 FAST of reptilian reovirus [RRV]). Plasmids encoding FASTs were transfected into Vero cells. All FAST proteins induced syncytium formation at varying intensities. To achieve high levels of FAST expression, four different FAST genes were inserted into the murine leukemia virus (MLV)-based replication-competent retroviral (RCR) vector. Two days after transfection in 293 T cells, only the MoMLV-10A1-p10(PuV) RCR vector showed syncytia formation. Based on these results, p10(Puv) was selected from the four FASTs. Next, we investigated the cytotoxicity of p10(PuV) on HeLa cervical carcinoma cells, HT1080 human fibrosarcoma cells, and U87 human glioma cells. Although three human cancer cell lines induced syncytium formation, U87 cells were highly susceptible to syncytia formation by transfection with p10(PuV). In addition, the viral supernatants from MoMLV-10A-p10(PuV) RCR vector-transfected 293 T cells also induced syncytium formation in HT1080, TE671, and U87 cells. This RCR vector encoding p10(PuV) is a promising candidate for cancer gene therapy.

Reactive-oxygen-species-mediated mechanism for photoinduced antibacterial and antiviral activities of Ag3PO4.

Cubic-shaped Ag3PO4 crystals with a mean size of 1 µm were synthesized by a precipitation method from a mixed solution of AgNO3, Na2HPO4, and triethanolamine. The antibacterial activities against Escherichia coli, Listeria innocua, and Pseudomonas syringae DC3000 in both the absence and presence of Ag3PO4 under dark conditions and in the presence of Ag3PO4 under red-light (625 nm) and blue-light (460 nm) irradiation were examined. The concentrations of reactive oxygen species (ROS) were also measured in the antibacterial action of the Ag3PO4 against Escherichia coli. The photoinduced enhancement of the Ag3PO4 antibacterial activity under blue-light irradiation is explained by the formation of ROS during the antibacterial action of the Ag3PO4. Moreover, the antiviral activity of Ag3PO4 against amphotropic 10A1 murine leukemia virus enhanced under blue-light irradiation via ROS production. These results provide an insight into extended bio-applications of Ag3PO4.

Construction of replication-competent oncolytic retroviral vectors expressing R peptide-truncated 10A1 envelope glycoprotein.

Replication-deficient retroviral (RDR) vectors have been generally used for gene therapy, but clinically beneficial transduction efficiency is difficult to achieve with these vectors. In recent times, attention has been focused on the use of murine leukemia virus (MLV)-based replication-competent retroviral (RCR) vectors. RCR vectors have been shown to achieve efficient tumor reduction in a wide variety of cancer models. Most RCR vectors have been developed from amphotropic 4070 A MLV env, which is broadly applied in basic research. In this study, we generated RCR vectors based on Moloney MLV by replacing the native env gene in a full-length viral genome with the 10A1 env gene. 10A1 MLV can infect a wide variety of cells. Unlike amphotropic MLV, the 10A1 MLV can use amphotropic MLV receptor Pit2 or gibbon ape leukemia virus (GaLV) receptor Pit1. The resulting construct MoMLV-10A1-EGFP was able to replicate in 293 T, NIH3T3, and Mus dunni cells. To evaluate the potential of MoMLV-10A1 vector as a therapeutic agent, we incorporated the yeast cytosine deaminase (CD) suicide gene into vectors. The resulting vector MoMLV-10A1-CD could inhibit the growth of human 293T cells upon 5-fluorocytosine (5-FC) administration. In addition, to lyse tumor cells by syncytium, MoMLV-10A1-R(-)-EGFP was generated by replacing wild-type 10A1 env with the 16-amino acid R peptide-truncated 10A1 env gene. Syncytium formation was observed in the TE671 human tumor cells, 293 T and PG13 cells upon transfection of the MoMLV-10A1-R(-)-EGFP vector. This result suggests that replication of this vector could be oncolytic in itself. We also found that syncytium could contribute to enhance cell-to-cell transmission of the retroviral vectors. Our results thus show that the MoMLV-10A1 vectors can be potentially useful for cancer gene therapy.

Generation and characterization of a stable full-length ecotropic murine leukemia virus molecular clone that produces novel phenotypes to Fv1 restriction.

Retrovirus tropism can be restricted by host cell factors such as Fv1, TRIM5alpha, and Lv1 that inhibit infection by targeting the incoming viral capsid. The Fv1 gene inhibits murine leukemia virus infection in mice, but the precise mechanism of Fv1-mediated restriction is poorly understood. Our previous studies had demonstrated that Fv1-mediated viral tropism can be determined within the capsid protein at position 114 (Jung and Kozak. 2000. J. Virol. 74: 5385-7). To study the interaction between Fv1 and CA, we introduced amino acid substitution and deletion at this site in the N-tropic AKV capsid gene. The mutated two-LTR proviral DNAs were introduced into SC-1 cells by transfection. After transfection, cell supernatants collected from transfected cells were tested for host range susceptibility. The result indicated that substitution of amino acids did not alter tropism, but the deletion of 114His produced a virus with unusual tropism. The novel phenotype produced here failed to replicate in Fv1-expressing cells. This mutant virus showing such an extreme restriction pattern would be useful for studying the mechanism of Fv1- mediated restriction.

Isolation and characterization of PERV-C env from domestic pig in Korea.

Clone PERV-C (A3) env was isolated from the genomic DNA of domestic pig (Sus scrofa domesticus) in Korea to investigate the molecular properties of PERV-C. The nucleic acid homologies between the PERV-MSL (type C) reference and the PERV-C(A3) clone was 99% for env, but a single base pair deletion was found in the transmembrane (TM) region of the env open reading frame. To examine the functional characteristics of truncated PERV-C env, we constructed a replication-incompetent retroviral vector by replacing the env gene of the pCL-Eco retrovirus vector with PERV-C env. A retroviral vector bearing PERV-C/A chimeric envelopes was also created to complement the TM defect. Our results indicated that truncated PERV-C env was not infectious in human cells as expected. Interestingly, however, the vector with the PERV-C/A envelope was able to infect 293 cells. This observation suggests that recombination within PERV-C TM could render PERV-C infectious in humans. To further characterize PERV-C/A envelopes, we constructed an infectious molecular clone by using a PCR-based technique. This infectious molecular clone will be useful to examine more specific regions that are critical for human cell tropism.

Thoracolumbar Paraspinal Myonecrosis after Aortic Dissection.

Thoracolumbar paraspinal myonecrosis can be developed with various etiologies. It can induce compartment syndrome of spinal muscles and cause elevated pressure on back muscles, resulting in severe back pain. Thoracolumbar paraspinal myonecrosis is a very rare disease. There are only a few studies about paraspinal myonecrosis. Here we report a case of a spontaneous thoracolumbar paraspinal myonecrosis in a patient who had asymptomatic abdominal aortic dissection. Through this case, etiologies, clinical features, radiologic findings, and treatment options for thoracolumbar paraspinal myonecrosis are discussed.

Role of a third extracellular domain of an ecotropic receptor in Moloney murine leukemia virus infection.

The murine ecotropic retroviral receptor has been demonstrated to function as a mouse cationic amino acid transporter 1 (mCAT1), and is comprised of multiple membranespanning domains. Feral mouse (Mus dunni) cells are not susceptible to infection by the ecotropic Moloney murine leukemia virus (MoMLV), although they can be infected by other ecotropic murine leukemia viruses, including Friend MLV and Rauscher MLV. The relative inability of MoMLV to replicate in M. dunni cells has been attributed to two amino acids (V214 and G236) located within the third extracellular loop of the M. dunni CAT1 receptor (dCAT1). Via the exchange of the third extracellular loop of the mCAT1 cDNA encoding receptor from the permissive mouse and the corresponding portion of cDNA encoding for the nonpermissive M. dunni receptor, we have identified the most critical amino acid residue, which is a glycine located at position 236 within the third extracellular loop of dCAT1. We also attempted to determine the role of the third extracellular loop of the M. dunni CAT1 receptor with regard to the formation of the syncytium. The relationship between dCAT1 and virus-induced syncytia was suggested initially by our previous identification of two MLV isolates (S82F in Moloney and S84A in Friend MLV), both of which are uniquely cytopathic in M. dunni cells. In an attempt to determine the relationship existing between dCAT1 and the virally-induced syncytia, we infected 293-dCAT1 or chimeric dCAT1 cells with the S82F pseudotype virus. The S82F pseudotype virus did not induce the formation of syncytia, but did show increased susceptibility to 293 cells expressing dCAT1. The results of our study indicate that S82F-induced syncytium formation may be the result of cell-cell fusion, but not virus-cell fusion.

Gamma knife surgery for intracranial cavernous hemangioma.

OBJECT: The purpose of this study was to assess the benefits of radiosurgery for cavernous hemangioma. METHODS: Sixty-five cavernous hemangiomas were treated with gamma knife surgery (GKS) between October 1994 and December 2002. Forty-two patients attended follow up. The mean patient age was 37.6 years (range 7-60 years). The lesions were located in the frontal lobe in 12 cases, deep in the parietal lobe in five, in the basal ganglia in five, in the temporal in three, in the cerebellum in three, in the pons/midbrain in six, and in multiple locations in eight cases. The presenting symptoms were seizure in 12, hemorrhage in 11, and other in 19. The maximum dose was 26.78 Gy, and the mean margin dose was 14.55 Gy. The mean follow-up period after radiosurgery was 29.6 months (range 5-93 months). The tumor decreased in size in 29 cases, was unchanged in 12, and increased in size in one. In the seizure group, seizures were controlled without anticonvulsant medication in nine cases (81.8%) after 31.3 months (range 12-80 months). After 93 months, one patient developed a cyst, which was resected. Rebleeding occurred in one case (2.3%). On T2-weighted imaging changes were seen in 11 cases (26.2%), in three (7.1 %) of which neurological deterioration was correlated with imaging changes. In other cases these deficits were temporary. CONCLUSIONS: The authors found that GKS was an effective treatment modality for cavernous hemangiomas, especially for those located within the brainstem, basal ganglia, or deep portions of the brain. It can reduce seizure frequency significantly although this takes time. In the group receiving a marginal dose below 15 Gy the patients fared better than when the dose exceeded 15 Gy.

Inhibition of herpesvirus-6B RNA replication by short interference RNAs.

RNA interference (RNAi) is a process of sequence-specific gene silencing, which is initiated by double-stranded RNA (dsRNA). RNAi may also serve as an antiviral system in vertebrates. This study describes the inhibition of herpesvirus-6B (HHV-6B) replication by short interference RNAs (siRNAs) that are targeted to the U38 sequence that encodes DNA polymerase. When virus-infected SupT1 cells were treated by siRNA, these cells blocked the cytopathic effect (CPE) and detected the HHV-6B antibody-negative in indirect immunofluorescence assays (IFA). Our result suggests that RNAi can efficiently block Herpesvirus-6B replication.

Characteristics of HIV-Tat protein transduction domain.

The human immunodeficiency virus type 1 (HIV-1) Tat protein transduction domain (PTD), which contains rich arginine and lysine residues, is responsible for the highly efficient transduction of protein through the plasma membrane. In addition, it can be secreted from infected cells and has the ability to enter neighboring cells. When the PTD of Tat is fused to proteins and exogenously added to cells, the fusion protein can cross plasma membranes. Recent reports indicate that the endogenously expressed Tat fusion protein can demonstrate biodistribution of several proteins. However, intercellular transport and protein transduction have not been observed in some studies. Therefore, this study examined the intercellular transport and protein transduction of the Tat protein. The results showed no evidence of intercellular transport (biodistribution) in a cell culture. Instead, the Tat fusion peptides were found to have a significant effect on the transduction and intercellular localization properties. This suggests that the HIV-1 PTD passes through the plasma membrane in one direction.

Comparison of the effects of retroviral restriction factors involved in resistance to porcine endogenous retrovirus.

Three major classes of retroviral restriction factors (APOBEC3G, Tetherin, and TRIM5α) have been identified in mammals. Restriction factors are cellular proteins that are able to limit viral replication by targeting specific steps of the viral life cycle. To evaluate which restriction factor is the most effective to inhibit the replication of porcine endogenous retroviruses (PERVs), the antiviral activity of each restriction factor was compared. In pseudotype assay, the antiviral activity of human tetherin against PERV pseudotype was slightly weaker than that of human APOBEC3G (hA3G). A combination of tetherin and hA3G was more potent than each individual restriction factor. We questioned whether a combination of tetherin and hA3G could also inhibit the spreading replication of PERV. In agreement with the pseudotype assay, two restriction factors inhibit infectious PERV replication in a spreading infection. In this study, hA3G could strongly inhibit the replication of PERV, but tetherin modestly restricted it. Based on these results, we concluded that a combination of tetherin and hA3G is the most effective way to restrict PERV. A combination of different restriction factors will encourage the development of a new approach to treat retroviral disease.

The Effectiveness of Early Tracheostomy (within at least 10 Days) in Cervical Spinal Cord Injury Patients.

OBJECTIVE: This study aimed to determine the optimal time for tracheostomy by evaluating the benefits and safety of early versus late tracheostomy in spinal cord injury (SCI) patients. METHODS: We retrospectively reviewed a total of 254 patients with spinal cord injury. Of them, we selected 21 spinal cord injury patients who required tracheostomy due to long-term mechanical ventilation and analyzed their medical records. The patients were categorized into two groups. Early tracheostomy was performed day 1-10 from intubation in 10 patients and the late tracheostomy was performed after day 10 in 11 cases. We also evaluated the duration of mechanical ventilation, stay in the ICU and complications related to tracheostomy, the injury level of and clinical severity. All data was analyzed using SPSS 18.0/WIN. RESULTS: The early tracheostomy offered clear advantages for shortening the total ICU stay (20.8 day vs. 38.0 day, p=0.010). There was also statistically significant reduction in the total length of time on mechanical ventilation (5.2 day vs. 29.2 day, p=0.009). However, the reductions in the incidence of pneumonia (40% vs. 82%) and the length of ICU stay post to tracheostomy (6 day vs. 15 day) were found to be statistically not significant. There were also no statistically significant differences in the injury level and clinical severity between the groups. CONCLUSION: We concluded that the early tracheostomy (at least 10 days) is beneficial for SCI patients who are likely to require prolonged mechanical ventilation.

Development of an indirect ELISA and immunocapture rt-PCR for Lily virus detection.

Multiple viruses such as Lily symptomless virus (LSV), Lily mottle virus (LMoV), and cucumber mosaic virus (CMV) are the most prevalent viruses infecting lilies in Korea. Leaf samples and bulbs showing characteristic symptoms of virus infection were collected from Gangwon, Chungnam, and Jeju provinces of Korea in 2008-2011. Coat protein (CP) genes of LSV and LMoV were amplified from collected samples by reverse transcription-polymerase chain reaction (RT-PCR) and cloned into a pET21d(+) expression vector to generate recombinant CPs. The resulting carboxy-terminal His-tagged CPs were expressed in Escherichia coli strain BL21(DE3) by isopropyl-1-thio- beta-D-galactoside induction. The recombinant proteins were purified using Ni-NTA agarose beads, and the purified proteins were used as an immunogen to produce polyclonal antibodies in rabbits. The resulting polyclonal antisera recognized specifically LSV and LMoV from infected plant tissues in Western blotting assays. Indirect enzyme-linked immunosorbent assay and immunocapture RTPCR using these polyclonal antisera were developed for the sensitive, efficient, economic, and rapid detection of Lily viruses. These results suggest that large-scale bulb tests and economic detection of Lily viruses in epidemiological studies can be performed routinely using these polyclonal antisera.

Recurrent spinal meningioma: a case report.

Meningiomas are the second most common intradural spinal tumors accounting for 25% of all spinal tumors. Being a slow growing and invariably benign tumor, it responds favorably to surgical excision. In addition, spinal meningioma has low recurrence rates. However, we experienced a case of intradural extramedullary spinal meningioma which recurred 16 years after the initial surgery on a 64-year-old woman. She presented with progressive neurological symptoms and had a surgical history of removal of thoracic spinal meningioma 16 years ago due to bilateral low leg weakness. She underwent a second operation at the same site and a pale yellowish tumor was excised, which was histopathologically confirmed as meningothelial meningioma, compared with previously transitional type. she showed neurological recovery after the operation. We, therefore, report the good results of this recurrent intradural spinal meningioma case developed after 16 years with literature review.