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Transforming growth factor β (TGF-β) is a multifunctional cytokine that plays crucial pathophysiological roles in various diseases, such as cancer and fibrosis. However, the disease modulation by targeting TGF-β1 isoform remains to be established, regardless of several studies employed with limited antibodies. Here, we developed an RNA aptamer to human active TGF-β1, named APT-β1, and characterized its properties

Background Bowel herniation through a defect in the broad ligament of the uterus is a rare disease and few cases of recurrence have been reported. We report herein a recurrence case of a patient with broad ligament hernia (BLH), along with a review of the literature. Case presentation A 53-year-old woman complaining of abdominal pain was transported to our hospital. She had a history of laparotomy for small-bowel obstruction associated with hernia in the broad ligament of the uterus 10 years ago at a local hospital. Abdominal pelvic contrast-enhanced computed tomography revealed that the mesentery of the dilated bowels converged at a thick band in the pelvis, suggesting closed loop obstruction of the small bowel. The patient underwent urgent laparotomy and was diagnosed with bowel herniation through an opening in the broad ligament of the uterus on the right side, which was ipsilateral with the previous surgery. The hernia orifice was widened by incision and incarcerated bowel segments were released and preserved because ischemia was reversible. The membranous defect of BLH was closed by suture with braded silk strings. Conclusions Although BLH is a rare disease, patients face a significant risk of disease recurrence. Nonabsorbable suture may be advisable for closure of the hernia orifice in BLH.

Purpose Small bowel obstruction (SBO) remains a common complication after pelvic or abdominal surgery. However, the risk factors for SBO in ulcerative colitis (UC) surgery are not well known. The aim of the present study was to clarify the risk factors associated with SBO after ileal pouch-anal anastomosis (IPAA) with a loop ileostomy for patients with UC. Methods The medical records of 96 patients who underwent IPAA for UC between 1999 and 2011 were reviewed. SBO was confirmed based on the presence of clinical symptoms and radiographic findings. The patients were divided into 2 groups: the SBO group and the non-SBO group. We also analyzed the relationship between SBO and computed tomography (CT) scan image parameters. Results The study included 49 male and 47 female patients. The median age was 35.5 years (range, 14-72 years). We performed a 2- or 3-stage procedure as a total proctocolectomy and IPAA for patients with UC. SBO in the pretakedown of the loop ileostomy after IPAA occurred in 22 patients (22.9%). Moreover, surgical intervention for SBO was required for 11 patients. In brief, closure of the loop ileostomy was performed earlier than expected. A multivariate logistic regression analysis revealed that the 2-stage procedure (odds ratio, 2.850; 95% confidence interval, 1.009-8.044; P = 0.048) was a significant independent risk factor associated with SBO. CT scan image parameters were not significant risk factors of SBO. Conclusion The present study suggests that a 2-stage procedure is a significant risk factor associated with SBO after IPAA in patients with UC.

When translating mRNAs are cleaved in protein-coding regions, 5' fragments of mRNAs are detached from stop codons (i.e., nonstop mRNAs) and protected from 3'-5' exonucleases by ribosomes stalled at the 3' termini. It has been shown in yeast that the nonstop mRNA decay (NSD) machinery triggers nonstop mRNA degradation by removing stalled ribosomes in the artificial reporter mRNAs. However, it is not known well whether NSD is involved in the degradation of endogenous nonstop mRNAs in higher eukaryotes. In this work, we addressed the question of whether 5'-nonstop-mRNA fragments generated by siRNA cleavage or nonsense-mediated-mRNA decay (NMD) are degraded by the NSD pathway in Drosophila melanogaster cells by knocking down three NSD components, Pelota (a yeast Dom34 homolog), Hbs1 and ABCE1 (a ribosome-recycling factor). We found that double, but not single, knockdown of any two of these three factors efficiently stabilized nonstop reporter mRNAs and triple knockdown of Pelota, Hbs1 and ABCE1 further stabilized nonstop mRNAs in highly ribosome-associated state. These findings demonstrated that Pelota, Hbs1 and ABCE1 are crucial for NSD in Drosophila cells as in yeast for rescuing stalled ribosomes and degrading nonstop mRNAs. To our knowledge, this is the first comprehensive report to show the involvement of the NSD machinery in the clearance of mRNA 5'-fragments produced by RNAi and NMD in eukaryotes.

Solinviviridae is a family of picorna/calici-like viruses with non-segmented, linear, positive-sense RNA genomes of approximately 10-11 kb. Unusually, their capsid proteins are encoded towards the 3'-end of the genome where they can be expressed both from a subgenomic RNA and as an extension of the replication (picorna-like helicase-protease-polymerase) polyprotein. Members of two species within the family infect ants, but related unclassified virus sequences derive from a large variety of insects and other arthropods. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the Solinviviridae, which is available at www.ictv.global/report/solinviviridae.

A46 -year-old male presented with bloody stool and a descending colon tumor, as identified using colon fiberscopy. The patient did not complain of any remarkable abdominal symptoms. Computed tomography revealed descending colon tumor intussusception. We performed partial resection of the descending colon and D2 lymphadenectomy without intraoperative reduction. The descending colon was barely attached to the retroperitoneum and was mobile. The underlying tumor was type 1 and measured 8.3×5.8 cm. The pathology report indicated a mucinous adenocarcinoma with extension through the submucosa into the subserosa, and metastasis in 6 nearby lymph nodes(n2). Intussusception is relatively rare in adults, particularly in portions of the colon fixed to the retroperitoneum, such as the descending colon. In contrast to previous reports of descending colon intussusception caused by age-related tissue dysfunction, we report our experience with a young patient and present the results obtained.

Polypeptide chain release factor eRF3 plays pivotal roles in translation termination and post-termination events including ribosome recycling and mRNA decay. It is not clear, however, if eRF3 is targeted for the regulation of gene expression. Here we show that DNA-damaging agents (UV and etoposide) induce the immediate cleavage and degradation of eRF3 in a caspase-dependent manner. The effect is selective since the binding partners of eRF3, eRF1 and PABP, and an unrelated control, GAPDH, were not affected. Point mutations of aspartate residues within overlapping DXXD motifs near the amino terminus of eRF3 prevented the appearance of the UV-induced cleavage product, identifying D32 as the major cleavage site. The cleavage and degradation occurred in a similar time-dependent manner to those of eIF4G, a previously established caspase-3 target involved in the inhibition of translation during apoptosis. siRNA-mediated knockdown of eRF3 led to inhibition of cellular protein synthesis, supporting the idea that the decrease in the amount of eRF3 caused by the caspase-mediated degradation contributes to the inhibition of translation during apoptosis. This is the first report showing that eRF3 could serve as a target in the regulation of gene expression.

Iflaviridae is a family of small non-enveloped viruses with monopartite, positive-stranded RNA genomes of approximately 9–11 kilobases. Viruses of all classified species infect arthropod hosts, with the majority infecting insects. Both beneficial and pest insects serve as hosts, and infections can be symptomless (Nilaparvatalugens honeydew virus 1) or cause developmental abnormalities (deformed wing virus), behavioural changes (sacbrood virus) and premature mortality (infectious flacherie virus). The host range has not been examined for most members. The most common route of infection for iflaviruses is the ingestion of virus-contaminated food sources. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Iflaviridae, which is available at www.ictv.global/report/iflaviridae.

Dicistroviridae is a family of small non-enveloped viruses with monopartite, linear, positive-sense RNA genomes of approximately 8–10 kb. Viruses of all classified species infect arthropod hosts, with some having devastating economic consequences, such as acute bee paralysis virus in domesticated honeybees and taura syndrome virus in shrimp farming. Conversely, the host specificity and other desirable traits exhibited by several members of this group make them potential natural enemies for intentional use against arthropod pests, such as triatoma virus against triatomine bugs that vector Chagas disease. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Dicistroviridae which is available at www.ictv.global/report/dicistroviridae.

Lepidopteran heat-tolerant (ht) cell lines have been obtained with sf-9, sf-21 and several Bombyx cells. They have a distinct karyotype, membrane lipid composition, morphology and growth kinetics from the parental cell lines. In this paper, we report the development of ht cell lines from other insect species and examination of their growth characteristics and virus susceptibility. Adaptation of cell lines sf-9, BTI-TN-5B1-4 (High5) and BTI-TN-MG1 (MG1) to 33°C and 35°C was carried out by shifting the culture temperature between 28°C and higher temperatures by a gradual stepwise increase in temperature. The process of adaption to a higher culture temperature was accomplished over a period of 2 months. The cell lines with the temperature adaption were designated as sf9-ht33, sf9-ht35, High5-ht33, High5-ht35, MG1-ht33, MG1-ht35. These cell lines have been subcultured over 70 passages. Adaption to high temperatures was confirmed by a constant population doubling time with individual cell lines. The population doubling time of heat adapted cell lines were 1–4 h less than these of parental cell lines. Cell shapes did not show obvious change, however, the cell size of sf9-ht cells was enlarged and those of High5 and MG1 ht cells were reduced after heat adaption. When the cell lines were infected with Autographa californica nuclear polyhedrosis virus (AcMNPV) at 28°C, 33°C, 35°C and 37°C, production of budded virus and occlusion bodies in each cell line was optimum at its own adapted temperature.

We report the discovery of a new virus from the red imported fire ant, Solenopsis invicta . Solenopsis invicta virus 3 (SINV-3) represents the third virus discovered from this ant species using the metagenomics approach. The single (positive)-strand RNA, monopartite, bicistronic genome of SINV-3 was sequenced in entirety (GenBank accession number FJ528584 ), comprised of 10,386 nucleotides, and polyadenylated at the 3′ terminus. This genome size was confirmed by Northern analysis. The genome revealed 2 large open reading frames (ORFs) in the sense orientation with an untranslated region (UTR) at each end and between the two ORFs. The 5′ proximal ORF (ORF 1) encoded a predicted protein of 299.1 kDa (2580 amino acids). The 3′ proximal ORF (ORF 2) encoded a predicted protein of 73.2 kDa (651 amino acids). RNA-dependent RNA polymerase (RdRp), helicase, and protease domains were recognized in ORF 1. SDS-PAGE separation of purified SINV-3 particles yielded 2 bands (ostensibly capsid proteins) with a combined molecular mass of 77.3 kDa which was similar to the mass predicted by ORF 2 (73.2 kDa). Phylogenetic analysis of the conserved amino acid sequences containing domains I to VIII of the RdRp from dicistroviruses, iflaviruses, plant small RNA viruses, picornaviruses, and 4 unassigned positive-strand RNA viruses revealed a trichotomous phenogram with SINV-3 and Kelp fly virus comprising a unique cluster. Electron microscopic examination of negatively stained samples of SINV-3 revealed isometric particles with apparent projections and a diameter of 27.3 ± 1.3 nm. SINV-3 was successfully transmitted to uninfected workers by feeding. The minus (replicative) strand of SINV-3 was detected in worker ants indicating replication of the virus. The possibility of using SINV-3 as a microbial control agent for fire ants is discussed.

Solenopsis invicta virus 2 (SINV-2) is the second virus identified from the fire ant, S. invicta Buren. SINV-2 is unique among positive-strand RNA viruses from insects by possessing four cistrons in a monopartite genome. Fire ant colonies testing positive for SINV-2 by RT-PCR did not exhibit any discernable symptoms. RT-PCR-based surveys for SINV-2 among 688 fire ant mounds in Alachua County, Florida, sampled during the period January, 2006 through December, 2007 showed that the prevalence of SINV-2 among nests ranged from 1.6% to 16.4%. Unlike S. invicta virus 1, no seasonal-associated prevalence was observed with regard to SINV-2 infection among fire ant colonies. No social form specificity was evident; SINV-2 was found in both monogyne and polygyne S. invicta ants. Real-time quantitative PCR experiments showed that SINV-2 genome equivalents per individual ant ranged from 1.9x10(7)in. pupae to 4.3x10(11)in. inseminated queens. The SINV-2 infection was detected in all ant stages examined (eggs, larvae, pupae, workers, and queens). Tissue tropism studies indicated that the alimentary canal (specifically the midgut) is most likely the susceptible tissue. SINV-2 was successfully transmitted to uninfected S. invicta ants by feeding a partially purified homogenate of SINV-2-infected ants. The SINV-2 transmission rate ranged from 30% to 80%, and both positive (genomic) and negative (replicative) SINV-2 RNA strands accumulated in recipient ants over the course of the experiment. These results indicated that SINV-2 replicates within S. invicta.

Quantitative real-time PCR was employed to measure the Solenopsis invicta virus 1 (SINV-1) load in tissues, individuals, and among colonies of the red imported fire ant, S. invicta Buren. Among tissues examined from SINV-1-infected adults and larvae, the alimentary canal (specifically the midgut) consistently had the highest number of SINV-1 genome copies (91.1 and 99.9%, respectively). Negative staining of a supernatant of the gut homogenate demonstrated the presence of spherical virus particles with a diameter of 30-35 nm, consistent with SINV-1. The number of SINV-1 genome copies in infected larvae and workers from the same queenright colonies were similar to each other. In other words, the infection rate was consistent among both developmental stages. No significant differences were observed in SINV-1 genome copy number among infected colonies sampled during the winter and summer. Although the SINV-1 infection rate of summer-collected mounds was previously shown to be six-times higher than winter-collected mounds, the intra-colony infection rate appears to be unaffected by season. Perhaps less inter-mound interaction during the winter months among S. invicta restricts spread of the virus. A positive correlation between intra-colony infection rate and mean SINV-1 genome copy number per ant was also observed. Based on these results, it is likely that SINV-1 replicates in gut epithelia of S. invicta and virus is shed into the gut lumen where it may be transmitted to nestmates by trophallaxis.

We report the discovery of a new virus with unique genome characteristics from the red imported fire ant, Solenopsis invicta. This virus represents the second identified from this ant species. It is provisionally named Solenopsis invicta virus 2 (SINV-2). The SINV-2 genome was constructed by compiling sequences from successive 5′ RACE reactions, a 3′ RACE reaction, and expressed sequence tag, c246 (accession number EH413675), from a fire ant expression library. The SINV-2 genome structure was monopartite, polycistronic and RNA-based. The genome consensus sequence (EF428566) was 11,303 nucleotides in length, excluding the poly(A) tail present on the 3′ end. Analysis of the genome revealed 4 major open reading frames (ORFs; comprised of ≥100 codons) and 5 minor ORFs (comprised of 50–99 codons) in the sense orientation. No large ORFs were found in the inverse orientation suggesting that the SINV-2 genome was from a positive-strand RNA virus. Further evidence for this conclusion includes abolished RT–PCR amplification by RNase treatment of SINV-2 nucleic acid template, and failure to amplify without first conducting cDNA synthesis. Blastp analysis indicated that ORF 4 contained conserved domains of an RNA-dependent RNA polymerase, helicase, and protease, characteristic of positive-strand RNA viruses. However, the protease domain and putative structural proteins (ORFs 1, 2, and 3) were less well conserved. Phylogenetic analysis of the RdRp, helicase, and ORF 1 indicate unique placement of SINV-2 exclusive from the Dicistroviridae, iflaviruses, Picornaviridae, and plant small RNA viruses.

Studies were conducted to examine the phenology, geographic distribution, and host specificity of the Solenopsis invicta virus-1 (SINV-1). Two genotypes examined, SINV-1 and -1A, exhibited similar seasonal prevalence patterns. Infection rates among colonies of S. invicta in Gainesville, Florida, were lowest from early winter (December) to early spring (April) increasing rapidly in late spring (May) and remaining high through August before declining again in the fall (September/October). Correlation analysis revealed a significant relationship between mean monthly temperature and SINV-1 (p0.0005, r=0.82) and SINV-1A (p0.0001, r=0.86) infection rates in S. invicta colonies. SINV-1 was widely distributed among S. invicta populations. The virus was detected in S. invicta from Argentina and from all U.S. states examined, with the exception of New Mexico. SINV-1 and -1A were also detected in other Solenopsis species. SINV-1 was detected in Solenopsis richteri and the S. invicta/richteri hybrid collected from northern Alabama and Solenopsis geminata from Florida. SINV-1A was detected in S. geminata and Solenopsis carolinensis in Florida and the S. invicta/richteri hybrid in Alabama. Of the 1989 arthropods collected from 6 pitfall trap experiments from Gainesville and Williston, Florida, none except S. invicta tested positive for SINV-1 or SINV-1A. SINV-1 did not appear to infect or replicate within Sf9 or Dm-2 cells in vitro. The number of SINV-1 genome copies did not significantly increase over the course of the experiment, nor were any cytopathic effects observed. Phylogenetic analyses of SINV-1/-1A nucleotide sequences indicated significant divergence between viruses collected from Argentina and the U.S.

Magnetic ‘nanofactories’, for localized manufacture and signal-guided delivery of small molecules to targeted cell surfaces, are demonstrated. They recruit nearby raw materials for synthesis, employ magnetic mobility for capture and localization of target cells, and deliver molecules to cells triggering their native phenotypic response, but with user-specified control. Our nanofactories, which synthesize and deliver the “universal” bacterial quorum-sensing signal molecule, autoinducer AI-2, to the surface of Escherichia coli , are assembled by first co-precipitating nanoparticles of iron salts and the biopolymer chitosan. E. coli AI-2 synthases, Pfs and LuxS, constructed with enzymatically activatable “pro-tags”, are then covalently tethered onto the chitosan. These enzymes synthesize AI-2 from metabolite S-adenosylhomocysteine. Chitosan serves as a molecular scaffold and provides cell capture ability; magnetite provides stimuli responsiveness. These magnetic nanofactories are shown to modulate the natural progression of quorum-sensing activity. New prospects for small molecule delivery, based on localized synthesis, are envisioned.

In the age of IoT (Internet of Things), tens of billions sensors or devices will be connected to cloud services. Although cloud environment is suitable for incredibly growing IoT services because of elasticity, scalability and efficiency for collecting and analyzing such data, protecting privacy on public cloud is a big issue. We proposed m-cloud, a distributed computation mechanism on multiple and independent public cloud environment. The mechanism enables to calculate various statistical indices from a large number of sensor data while it reduces the risk of revealing privacy information. We also propose an extension of the m-cloud computation mechanism so that it provides high scalability for infinitely increasing number of IoT devices by applying DHT (Distributed Hash Table) technique.

An 81-year-old man presented with chief complaints of abdominal pain and vomiting. Intestinal obstruction was found at the time of admission to a local hospital in October 2011. Conservative treatment provided symptomatic relief; however, he was readmitted with similar symptoms in December 2011. Small-intestinal wall thickening was detected by abdominal and pelvic computed tomography, and he was referred to our hospital. Small-bowel endoscopy revealed an elevated subcircumferential tumor in the jejunum. Biopsy revealed well to moderately differentiated adenocarcinoma diagnosed as jejunal cancer, which caused narrowing of the jejunum. Single-incision laparoscopy-assisted small-bowel resection was performed. The intraoperative findings were a tumor with inflammatory changes in the jejunum and enlarged surrounding lymph nodes. We performed regional lymph node dissection. Histopathological analysis showed moderately differentiated small-intestinal tubular adenocarcinoma and 2 of 5 lymph nodes positive for metastatic cancer cells. After an uneventful postoperative course, he was discharged on day 7. He preferred not to undergo postoperative adjuvant chemotherapy and quickly recovered his activities of daily living postoperatively. He stayed home until he developed abdominal distention resulting from peritoneal recurrence 1 year and 6 months postoperatively and died 1 month later.

A 57-year-old woman was referred to our hospital because of descending colon cancer with multiple liver metastases. Abdominal magneticresonanc e imaging (MRI) revealed 13 liver metastases across the lobes. We started combination che- motherapy with capecitabine/oxaliplatin (CapeOX) and bevacizumab. After 9 courses of the treatment, the number and size of the liver metastases were remarkably reduced on MRI. Left colectomy and partial hepatectomy were performed. Histopathological examination revealed no residual cancer cells in the colon but revealed a few cancer cells in 4 of 7 resected liver specimens. At 11 postoperative months, 1 liver metastasis reappeared, for which we performed laparoscopy-assisted partial hepatectomy. At 21 months after the second operation, the patient was well without any signs of recurrence. Thus, the combination chemotherapy with CapeOX and bevacizumab allowed for the successful resection of the tumor and metastasis in our patient who initially had unresectable colon cancer and multiple liver metastases.

The cross-species bacterial communication signal autoinducer 2 (AI-2), produced by the purified enzymes Pfs (nucleosidase) and LuxS (terminal synthase) from S- adenosylhomocysteine, directly increased Escherichia coli biofilm mass 30-fold. Continuous-flow cells coupled with confocal microscopy corroborated these results by showing the addition of AI-2 significantly increased both biofilm mass and thickness and reduced the interstitial space between microcolonies. As expected, the addition of AI-2 to cells which lack the ability to transport AI-2 ( lsr null mutant) failed to stimulate biofilm formation. Since the addition of AI-2 increased cell motility through enhanced transcription of five motility genes, we propose that AI-2 stimulates biofilm formation and alters its architecture by stimulating flagellar motion and motility. It was also found that the uncharacterized protein B3022 regulates this AI-2-mediated motility and biofilm phenotype through the two-component motility regulatory system QseBC. Deletion of b3022 abolished motility, which was restored by expressing b3022 in trans . Deletion of b3022 also decreased biofilm formation significantly, relative to the wild-type strain in three media (46 to 74%) in 96-well plates, as well as decreased biomass (8-fold) and substratum coverage (19-fold) in continuous-flow cells with minimal medium (growth rate not altered and biofilm restored by expressing b3022 in trans ). Deleting b3022 changed the wild-type biofilm architecture from a thick (54-μm) complex structure to one that contained only a few microcolonies. B3022 positively regulates expression of qseBC , flhD , fliA , and motA , since deleting b3022 decreased their transcription by 61-, 25-, 2.4-, and 18-fold, respectively. Transcriptome analysis also revealed that B3022 induces crl (26-fold) and flhCD (8- to 27-fold). Adding AI-2 (6.4 μM) increased biofilm formation of wild-type K-12 MG1655 but not that of the isogenic b3022, qseBC , fliA , and motA mutants. Adding AI-2 also increased motA transcription for the wild-type strain but did not stimulate motA transcription for the b3022 and qseB mutants. Together, these results indicate AI-2 induces biofilm formation in E. coli through B3022, which then regulates QseBC and motility; hence, b3022 has been renamed the motility quorum-sensing regulator gene (the mqsR gene).

Transgenic plants that produce anti-insect substances are vital in improving crop yields and in reducing the environmental risks of chemical insecticides. Enhancin is a metalloprotease produced in occlusion bodies of the Trichoplusia ni granulovirus (TnGV). It is a key substance that enhances infection of the nucleopolyhedrovirus in lepidopteran insects. Rice (Oryza sativa L. cv. Nipponbare) protoplasts were cotransformed with pREXΦVEF and pLTHyg, which respectively bear the chimeric enhancin gene and the hygromycin-resistance gene. Hygromycin-resistant regeneration plants were examined by genomic polymerase chain reaction and genomic Southern and northern blotting analyses to confirm the presence and expression of the enhancin gene. Fourteen transgenic plant lines harboring the enhancin gene were obtained, and stable inheritance and expression of the enhancin gene were confirmed in the second, third, and fourth plant generations. Feeding Spodoptera exigua larvae leaves of enhancin-expressing rice plants in the presence of S. exigua nucleopolyhedrovirus occlusion bodies enhanced infection of the virus. Further, the development of Pseudaletia separata, S. exigua, and S. litura, none of which are host insects of TnGV, was inhibited when these larvae were fed enhancin-expressing rice leaves. This indicates that expression of the enhancin gene confers resistance to lepidopteran insect pests in rice.

Bacterial autoinducer 2 (AI-2) is proposed to be an interspecies mediator of cell-cell communication that enables cells to operate at the multicellular level. Many environmental stimuli have been shown to affect the extracellular AI-2 levels, carbon sources being among the most important. In this report, we show that both AI-2 synthesis and uptake in Escherichia coli are subject to catabolite repression through the cyclic AMP (cAMP)-CRP complex, which directly stimulates transcription of the lsr (for “ luxS regulated”) operon and indirectly represses luxS expression. Specifically, cAMP-CRP is shown to bind to a CRP binding site located in the upstream region of the lsr promoter and works with the LsrR repressor to regulate AI-2 uptake. The functions of the lsr operon and its regulators, LsrR and LsrK, previously reported in Salmonella enterica serovar Typhimurium, are confirmed here for E. coli. The elucidation of cAMP-CRP involvement in E. coli autoinduction impacts many areas, including the growth of E. coli in fermentation processes.

Transgenic tobacco transformed with the Trichoplusia ni granulovirus enhancin gene has been demonstrated to enhance baculovirus infection in larvae. In this paper we describe the effect of the long-term feeding of lyophilized transgenic tobacco material to Pseudaletia separata and Spodoptera exigua larvae. Our results demonstrated that the baculovirus enhancin gene products have potential for use in insect pest management.

The Twister &! RapidPlate Integrated System by TANabe, or TRISTAN, consists of a 96-channel dispenser (Rapid-Plate 96), a plate reader (V-MAX), and a simple robot arm (Twister). We developed TRISTAN for effectively conducting a homogeneous assay. Although this system accommodates fewer than 20 microplates, it has several advantages over conventional robotic systems for high-throughput screening in the following aspects: parameter setting, running time, hardware errors, manpower, and cost-effectiveness. The system proved to be effective and efficient for homogeneous assays.

A putative novel rhabdovirus (SfRV) was previously identified in a Spodoptera frugiperda cell line (Sf9 cells [ATCC CRL-1711 lot 58078522]) by next generation sequencing and extensive bioinformatic analysis. We performed an extensive analysis of our Sf9 cell bank (ATCC CRL-1711 lot 5814 [Sf9L5814]) to determine whether this virus was already present in cells obtained from ATCC in 1987. Inverse PCR of DNA isolated from Sf9 L5814 cellular DNA revealed integration of SfRV sequences in the cellular genome. RT-PCR of total RNA showed a deletion of 320 nucleotides in the SfRV RNA that includes the transcriptional motifs for genes X and L. Concentrated cell culture supernatant was analyzed by sucrose density gradient centrifugation and revealed a single band at a density of 1.14 g/ml. This fraction was further analysed by electron microscopy and showed amorphous and particulate debris that did not resemble a rhabdovirus in morphology or size. SDS-PAGE analysis confirmed that the protein composition did not contain the typical five rhabdovirus structural proteins and LC-MS/MS analysis revealed primarily of exosomal marker proteins, the SfRV N protein, and truncated forms of SfRV N, P, and G proteins. The SfRV L gene fragment RNA sequence was recovered from the supernatant after ultracentrifugation of the 1.14 g/ml fraction treated with diethyl ether suggesting that the SfRV L gene fragment sequence is not associated with a diethyl ether resistant nucleocapsid. Interestingly, the 1.14 g/ml fraction was able to transfer baculovirus DNA into Sf9L5814 cells, consistent with the presence of functional exosomes. Our results demonstrate the absence of viral particles in ATCC CRL-1711 lot 5814 Sf9 cells in contrast to a previous study that suggested the presence of infectious rhabdoviral particles in Sf9 cells from a different lot. This study highlights how cell lines with different lineages may present different virosomes and therefore no general conclusions can be drawn across Sf9 cells from different laboratories.

Background Recombinant hemagglutinin (rHA) is the active component in Flublok®; a trivalent influenza vaccine produced using the baculovirus expression vector system (BEVS). HA is a membrane bound homotrimer in the influenza virus envelope, and the purified rHA protein assembles into higher order rosette structures in the final formulation of the vaccine. During purification and storage of the rHA, disulfide mediated cross-linking of the trimers within the rosette occurs and results in reduced potency. Potency is measured by the Single Radial Immuno-diffusion (SRID) assay to determine the amount of HA that has the correct antigenic form. Results The five cysteine residues in the transmembrane (TM) and cytoplasmic (CT) domains of the rHA protein from the H3 A/Perth/16/2009 human influenza strain have been substituted to alanine and/or serine residues to produce three different site directed variants (SDVs). These SDVs have been evaluated to determine the impact of the TM and CT cysteines on potency, cross-linking, and the biochemical and biophysical properties of the rHA. Modification of these cysteine residues prevents disulfide bond cross-linking in the TM and CT, and the resulting rHA maintains potency for at least 12 months at 25°C. The strategy of substituting TM and CT cysteines to prevent potency loss has been successfully applied to another H3 rHA protein (from the A/Texas/50/2012 influenza strain) further demonstrating the utility of the approach. Conclusion rHA potency can be maintained by preventing non-specific disulfide bonding and cross-linked multimer formation. Substitution of carboxy terminal cysteines is an alternative to using reducing agents, and permits room temperature storage of the vaccine.

The eukaryotic releasing factor eRF3 is a multifunctional protein that plays pivotal roles in translation termination as well as the initiation of mRNA decay. eRF3 also functions in the regulation of apoptosis; eRF3 is cleaved at Ala73 by an as yet unidentified protease into processed isoform of eRF3 (p-eRF3), which interacts with the inhibitors of apoptosis proteins (IAPs). The binding of p-eRF3 with IAPs leads to the release of active caspases from IAPs, which promotes apoptosis. Although full-length eRF3 is localized exclusively in the cytoplasm, p-eRF3 localizes in the nucleus as well as the cytoplasm. We here focused on the role of p-eRF3 in the nucleus. We identified leptomycin-sensitive nuclear export signal (NES) at amino acid residues 61–71 immediately upstream of the cleavage site Ala73. Thus, the proteolytic cleavage of eRF3 into p-eRF3 leads to release an amino-terminal fragment containing NES to allow the relocalization of eRF3 into the nucleus. Consistent with this, p-eRF3 more strongly interacted with the nuclear ARF tumor suppressor than full-length eRF3. These results suggest that while p-eRF3 interacts with IAPs to promote apoptosis in the cytoplasm, p-eRF3 also has some roles in regulating cell death in the nucleus.

A plasmid library of restriction DNA fragments of a Plutella xylostella granulovirus (PxGV) was constructed, which covered all 9 EcoRI and all 19 HindIII fragments, and 18 of 20 PstI fragments. Restriction endonuclease analysis and DNA-DNA hybridization analysis of the cloned viral DNA fragments in the library and the restricted viral DNA fragments were carried out for construction of a physical map of PxGV DNA for ApaI, BamHI, BglII, EcoRI, HindIII, KpnI, PstI, SacII, XbaI and XhoI. The size of PxGV genome was estimated to be 102.2 kb. Location of the left-most DNA fragment on the physical map that contained granulin gene was determined by hybridization using a DNA probe of granulin gene of Trichoplusia ni granulovirus. Four homologous regions interspersed on the genome were also determined by DNA-DNA hybridization analysis.

The genomic DNA of four Autographa californica multinucleocapsid nucleopolyhedrovirus (AcMNPV) variants isolated from Galleria mellonella, Spodoptera exigua, Spodoptera litura and Xestia c-nigrum was analyzed in comparison with the AcMNPV E2 strain. Restriction endonuclease analysis revealed a deletion and an insertion in collinear regions of the four variants. Polymerase chain reaction analysis indicated that, in the four variants, the deletion occurred in the region corresponding to AcMNPV C6 ORF86 (pnk/pnl). Also the insertion, with a length of approximately 1.1 kb, was commonly identified in the fragments corresponding to the PstI-J fragment (18.5 m.u.–21.2 m.u.) of AcMNPV E2. Sequencing analysis of the variant from S. litura showed that the insertion contains an additional open reading frame encoding 322 amino acids between homologues of AcMNPV ORF30 and ORF31 (the superoxide dismutase gene). This ORF has 82.8% amino acid identity to Bombyx mori NPV T3 ORF 22 (bro-a, one of the baculovirus repeated ORFs) and thus, it was named Splt-bro-a. Southern blot hybridization study indicated that the other three variants also contain Splt-bro-a homologue. In addition, the labeled Splt-bro-a gene weakly hybridized to the PstI-D fragment (99.0 m.u.–8.0 m.u.) of AcMNPV E2. This fragment contains AcMNPV ORF2, a member of bro family. The signal was also observed on the corresponding fragment of the four variants. This result suggested that two bro genes are present in the four variants, although AcMNPV E2 and C6 are known to contain a single bro gene.

A baculovirus virulence-associated protein, enhancin, was engineered for production in a baculovirus-insect cell expression system and used for transformation of tobacco plants. A recombinant Autographa californica nucleopolyhedrovirus (AcNPV) with the enhancin gene from Trichoplusia ni granulovirus, AcEnh26, was propagated in Sf9 cells. The infected cultured cells combined with either AcNPV occlusion bodies (OBs) or Spodoptera exigua NPV (SeNPV) OBs were fed to 3rd stadium larvae of S. exigua. Feeding larvae with AcEnh26-infected cells resulted in a 21-fold and 10-fold enhancement of infection by AcNPV and SeNPV, respectively, compared to controls. When a similar feeding assay was performed with engineered tobacco plants, a 10-fold enhancement of AcNPV infection was observed, but a marked enhancement of SeNPV infection was not observed. Thus, the engineering and expression of the baculovirus enhancin gene in homologous and heterologous organisms allowed us to analyze its effects on NPV infection of larvae which demonstrated its viral enhancement function.

We report a case of hyperammonemic encephalopathy related to 5-FU in an aged patient with recurrent colon cancer treated with FOLFIRI therapy. An 80-year-old man underwent right hemicolectomy for cecal cancer. After 10 months, surgical resection was performed for its local recurrence. He was then treated with FOLFIRI therapy, and during the fifth course, he presented with a sudden onset of congestive disturbances. Through radiographic examination and laboratory data, only hyperammonemia was found; he was therefore diagnosed with hyperammonemic encephalopathy. By starting branchedamino acid solutions for its treatment, his consciousness and serum ammonia were promptly improved. Hyperammonemic encephalopathy related 5-FU is caused by increasing ammonia production and its metabolic inhibition, and is worsened by renal dysfunction, dehydration, constipation, infections, or body weight loss. On account of the potential decrease of metabolic function of liver and kidney, an aged person tends to have hyperammonnemia more than a youth. Clinicians should be aware of the adverse events associated with hyperammonemia when then administer a large amount of 5-FU to elderly patients.

Baculoviruses are important insect pathogens that have been developed as protein expression vectors in insect cells and as transduction vectors for mammalian cells. They have large double-stranded DNA genomes containing approximately 156 tightly spaced genes, and they present significant challenges for transcriptome analysis. In this study, we report the first comprehensive analysis of AcMNPV transcription over the course of infection in Trichoplusia ni cells, by a combination of strand-specific RNA sequencing (RNA-Seq) and deep sequencing of 5′ capped transcription start sites and 3′ polyadenylation sites. We identified four clusters of genes associated with distinctive patterns of mRNA accumulation through the AcMNPV infection cycle. A total of 218 transcription start sites (TSS) and 120 polyadenylation sites (PAS) were mapped. Only 29 TSS were associated with a canonical TATA box, and 14 initiated within or near the previously identified CAGT initiator motif. The majority of viral transcripts (126) initiated within the baculovirus late promoter motif (T A AG), and late transcripts initiated precisely at the second position of the motif. Analysis of 3′ ends showed that 92 (77%) of the 3′ PAS were located within 30 nucleotides (nt) downstream of a consensus termination signal (AAUAAA or AUUAAA). A conserved U-rich region was found approximately 2 to 10 nt downstream of the PAS for 58 transcripts. Twelve splicing events and an unexpectedly large number of antisense RNAs were identified, revealing new details of possible regulatory mechanisms controlling AcMNPV gene expression. Combined, these data provide an emerging global picture of the organization and regulation of AcMNPV transcription through the infection cycle.

A restriction fragment library representing 89.3% of the genome of Trichoplusia ni granulosis virus (TnGV) was constructed. The library consisted of 13 of the 16 BamHI fragments, 18 of the 22 EcoRI fragments, and 6 of the 27 PstI fragments. By restriction endonuclease and Southern blot analysis of cloned or genomic viral DNA fragments, a complete physical map of TnGV was constructed for BamHI, EcoRI, PstI and XhoI. Three interspersed homologous regions (ihs1-ihs3) were identified from hybridization experiments and sequenced. Each TnGV ihs has an approximate size of 400 bp and shows homology to the other two. The orientation of ihs2 is inverted relative to ihs1 and ihs3. TnGV ihs regions do not have repetitive motifs or palindromic sequences, in contrast to homologous regions (hrs) of nuclear polyhedrosis viruses (NPVs). The genomic locations of TnGV ihs1-ihs3, represented in percentage map units, were very similar to those of ihs sequences previously reported in Bombyx mori NPV, suggesting that the ihs may be a novel type of cis-acting element common among baculoviruses. Additionally, an inverted repeat sequence, having overlapping, multiple inverted repeats of 400 bp, was identified to the left of ihs3 on the linearized genome map of TnGV.

家蚕核多角体病ウイルス(BmNPV) D1株をBUdRおよびNTG存在下でBmN4細胞へ感染させ,培養温度25°Cと33°Cでの出芽型ウイルス(extracellular virus)の産生量をTCID50値で表したとき,両温度区を比較して1桁以上の差が認められた温度感受性変異株(ts mutant)を6株分離した。そのうち,3株について,若干の性状を調査し,以下の結果を得た。1) 得られたts mutantを33°CでBmN4細胞に感染させた場合,野性株と同じプラークの形態を示した。2) 感染細胞中のポリペプチド合成をSDS-PAGEで分析したところ,ts mutantは野性株とは異なったバンドパターンを示したが,温度差による差異は認められなかった。3) ts mutant感染細胞よりウイルスDNAを抽出し,制限酵素HindIIIによる消化産物を野性株ゲノムのそれとアガロースゲル電気泳動法により比較検討したところ,HindIII認識部位に変異を生じていることが判明した。それらの位置を野性株ゲノムの制限酵素HindIII消化DNA断片に基づく物理地図を用いて調査したところ,TS1∼TS3の変異領域は20m.u.付近および75.0∼85.0m.u.であった。それらは,BmNPVのゲノム上に5か所分散して存在する相同性塩基配列(hr)を含む領域およびAcMNPVで分離された数種のts mutantの変異領域と近似していた。

A plaque-purified Bombyx mori nuclear polyhedrosis virus, BmNPV D1 isolate, was propagated for 30 generations by serial undiluted passages in BmN4 cells. During the passages, a few polyhedra phenotypes and defective viral genomes were generated. By contrast, the population of wild type polyhedra and genomes decreased. The main component of defective genomes contained most of the interspersed homologous sequences of the baculovirus genomes.

The viral surface protein hemagglutinin (HA) has been recognized as a key antigen in the host response to influenza virus in both natural infection and vaccination because neutralizing antibodies directed against HA can mitigate or prevent infection. The baculovirus-insect cell system can be used for the production of recombinant HA molecules and is suitable for influenza vaccine production where annual adjustment of the vaccine is required. This expression system is generally considered safe with minimal potential for growth of human pathogens. Extensive characterization of this novel cell substrate has been performed, none of which has revealed the presence of adventitious agents. Multiple clinical studies have demonstrated that the vaccine is safe, well-tolerated and immunogenic. The baculovirus-insect cell system could, therefore, be used for the expedited production of a safe and efficacious influenza vaccine. As a result, this technology should provide a fast track worldwide solution for newly emerging influenza strains or pandemic preparedness within a few years.

International audience; This letter to the editor brings to the attention of researchers an initiative to develop a baculovirus reference material repository. To be successful this initiative needs the support of a broad panel of researchers working with baculovirus vectors for recombinant protein production and gene delivery for either therapy or vaccination. First there is a need to reach a consensus on the nature of the reference material, the production protocols and the baculovirus characterization methods. It will also be important to define repository and distribution procedures so that the reference material is available to any researcher for calibrating experimental data and to compare experiments performed in the various laboratories. As more and more baculovirus-based products are licensed or in the final stages of development, the development of a repository of baculovirus reference material is timely. This letter describes the requirements for the reference material and for the project as a whole to be successful and calls for a partnership that would involve academic, industrial laboratories and governmental organizations to support this international initiative.

Background The insect cell line is a critical component in the production of recombinant proteins in the baculovirus expression system and new cell lines hold the promise of increasing both quantity and quality of protein production. Results Seventy cell lines were established by single-cell cloning from a primary culture of cells derived from eggs of the black witch moth (Ascalapha odorata; Lepidoptera, Noctuidae). Among 8 rapidly growing lines, cell line 38 (Ao38) was selected for further analysis, based on susceptibility to AcMNPV infection and production of secreted alkaline phosphatase (SEAP) from a baculovirus expression vector. In comparisons with low-passage High Five (BTI-Tn-5B1-4) cells, infected Ao38 cells produced β-galactosidase and SEAP at levels higher (153% and 150%, respectively) than those measured from High Five cells. Analysis of N-glycans of SEAP produced in Ao38 cells revealed two N-glycosylation sites and glycosylation patterns similar to those reported for High Five and Sf9 cells. Glycopeptide isoforms consisted of pauci- or oligomannose, with and without fucose on N-acetylglucosamine(s) linked to asparagine residues. Estimates of Ao38 cell volume suggest that Ao38 cells are approximately 2.5× larger than Sf9 cells but only approximately 74% of the size of High Five cells. Ao38 cells were highly susceptible to AcMNPV infection, similar to infectivity of Sf9 cells. Production of infectious AcMNPV budded virions from Ao38 cells peaked at approximately 4.5 × 107 IU/ml, exceeding that from High Five cells while lower than that from Sf9 cells. Ao38 cells grew rapidly in stationary culture with a population doubling time of 20.2 hr, and Ao38 cells were readily adapted to serum-free medium (Sf-900III) and to a suspension culture system. Analysis of Ao38 and a parental Ascalapha odorata cell line indicated that these lines were free of the alphanodavirus that was recently identified as an adventitious agent in High Five cell lines. Conclusions Ao38 cells represent a highly productive new insect cell line that will be useful for heterologous protein expression and other applications in biotechnology.

Purification of Solenopsis invicta virus 1 (SINV-1) from its host, S. invicta, and subsequent examination by electron microscopy revealed a homogeneous fraction of spherical particles with a diameter of 30-35 nm. Quantitative PCR with SINV-1-specific oligonucleotide primers verified that this fraction contained high copy numbers of the SINV-1 genome. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the SINV-1 purified fraction revealed three major and one minor protein bands. The protein bands were labeled VP1 (40.8+/-1.4 kDa), VP2 (35.7+/-2.8 kDa), VP3 (25.2+/-1.8 kDa), and VP4 (22.2+/-2.5 kDa) based on mass. N-terminal sequence was acquired successfully for VP1, VP2, and VP3, but not VP4, and delineated each capsid protein within the 3'-proximal open reading frame of SINV-1. Positional organization of the viral proteins within the SINV-1 structural polyprotein was consistent with dicistroviruses (when based on sequence similarity). Blastp analysis of SINV-1 VP1, VP2, and VP3 revealed significant identity with corresponding structural capsid proteins of positive-strand RNA viruses, particularly acute bee paralysis virus (ABPV), Kashmir bee virus (KBV) and Israeli acute paralysis virus (IAPV). Amino acid residues about the scissile bonds for VP1 and VP3 were consistent with dicistroviruses and insect-infecting picorna-like viruses. N-terminal sequencing of VP2 also established that translation initiation of the SINV-1 structural polyprotein was mediated by an internal ribosomal entry site and is AUG-independent.

Quantitative real-time PCR (QPCR) was used to quantify the genome of Solenopsis invicta virus-2 (SINV-2) from infected individual ants of S. invicta. Strand-specific cDNA synthesis oligonucleotide primers and RNase digestion after cDNA synthesis allowed quantification of plus (genomic) and minus (replicative) strands of the SINV-2 genome. Both strands were detected in adult workers and larval fire ants indicating that the virus was replicating within the ant. The differences between the genomic to replicative strand ranged from 199-fold in larvae to 479-fold in workers with an average ratio of 339:1.