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BackgroundAlthough the naturally occurring reovirus causes only mild symptoms in humans, it shows considerable potential as an oncolytic agent because of its innate ability to target cancer cells. In immunocompromised hosts, however, wild-type reovirus can target healthy tissues, including heart, liver, pancreas and neural structures.MethodsWe characterized an attenuated form of reovirus (AV) derived from a persistently infected cell line through sequence analysis, as well as western blot and in vitro transcription and translation techniques. To examine its pathogenesis and oncolytic potential, AV reovirus was tested on healthy embryonic stem cells, various non-transformed and transformed cell lines, and in severe combined immunodeficiency (SCID) mice with tumour xenografts.ResultsSequence analysis of AV reovirus revealed a premature STOP codon in its sigma 1 attachment protein. Western blot and in vitro translation confirmed the presence of a truncated σ1. In comparison to wild-type reovirus, AV reovirus did not kill healthy stem cells or induce black tail formation in SCID mice. However, it did retain its ability to target cancer cells and reduce tumour size.ConclusionDespite containing a truncated attachment protein, AV reovirus still preferentially targets cancer cells, and compared with wild-type reovirus it shows reduced toxicity when administered to immunodeficient hosts, suggesting the potential use of AV reovirus in combination cancer therapy.

This descriptive study examines the prevalence of obesity-related cardiometabolic (CM) risk factors using CM laboratory metrics, in 3 to 19 year olds presenting to a rural American Academy of Pediatrics stage 3 multidisciplinary weight management clinic based on gender, age ranges, and obesity classes. From 2009 to 2016, 382 children (body mass index ≥85th percentile) enrolled. Multiple logistic regression determined the effects of age, gender, or obesity class on CM risk factors. Odds of elevated insulin were more significant in 15 to 19 year olds than in 3 to 5 year olds, or in 6 to 11 year olds. Obesity class III had higher odds than class II, class I, and overweight in having elevated insulin; twice likely than class II for having low high-density lipoprotein; and twice as likely than class I for high triglycerides. Adolescents and obesity class III categories have significant CM risk but the burden in younger and less severe obesity cohorts cannot be underestimated.

The change in transfer coefficient () with increasing heart rate during exercise was studied in 25 normal subjects and in 21 patients with pulmonary sarcoidosis. The slope of the response against heart rate was found to be 0·0053 mmol min kPa l per beat in the normal group but in many of the patients was two standard deviations or more below this normal slope, even when their routine function tests were normal. This response of to exercise is a more sensitive index of changed function than more routine function tests in pulmonary sarcoidosis. [ABSTRACT FROM PUBLISHER]

Reovirus, a naturally occurring oncolytic virus, initiates the lysis of tumor cells while simultaneously releasing tumor antigens or proapoptotic cytokines in the tumor microenvironment to augment anticancer immunity. However, reovirus has developed a strategy to evade antiviral immunity via its inhibitory effect on interferon production, which negatively affects the induction of antitumor immune responses. The mammalian adaptor protein Stimulator of Interferon Genes (STING) was identified as a key regulator that orchestrates immune responses by sensing cytosolic DNA derived from pathogens or tumors, resulting in the production of type I interferon. Recent studies reported the role of STING in innate immune responses to RNA viruses leading to the restriction of RNA virus replication. In the current study, we found that reovirus had a reciprocal reaction with a STING agonist regarding type I interferon responses in vitro; however, we found that the combination of reovirus and STING agonist enhanced anti-tumor immunity by enhancing cytotoxic T cell trafficking into tumors, leading to significant tumor regression and survival benefit in a syngeneic colorectal cancer model. Our data indicate the combination of reovirus and a STING agonist to enhance inflammation in the tumor microenvironment might be a strategy to improve oncolytic reovirus immunotherapy., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Aim: The aim of the study was to explore undergraduate nursing and midwifery students' perspectives of participating in a student-led collaborative online international learning (COIL) program., Background: Research on COIL programs is limited. This program, developed across three global universities, was implemented to provide an internationalization-at-home experience for students during the COVID-19 pandemic., Method: An exploratory descriptive qualitative design was employed using nursing students' reflections and interviews., Results: Data analysis revealed four key themes: student-led learning experiences, personal gains, influence on professional practice, and becoming a global citizen. Students' experiences offer insights into positive aspects of the program and highlight challenges to overcome., Conclusion: The student-led COIL experience increased nursing students' understanding of the dynamics of culture and nursing practices across different countries. Students' personal growth and professional gains will potentially prepare them to work in multicultural environments and develop global citizenship attributes., Competing Interests: The authors have declared no conflict of interest., (Copyright © 2023 National League for Nursing.)

Patients with stage IV gastric cancer suffer from dismal outcomes, a challenge especially in many Asian populations and for which new therapeutic options are needed. To explore this issue, we used oncolytic reovirus in combination with currently used chemotherapeutic drugs (irinotecan, paclitaxel, and docetaxel) for the treatment of gastric and other gastrointestinal cancer cells in vitro and in a mouse model. Cell viability in vitro was quantified by WST-1 assays in human cancer cell lines treated with reovirus and/or chemotherapeutic agents. The expression of reovirus protein and caspase activity was determined by flow cytometry. For in vivo studies, athymic mice received intratumoral injections of reovirus in combination with irinotecan or paclitaxel, after which tumor size was monitored. In contrast to expectations, we found that reoviral oncolysis was only poorly correlated with Ras pathway activation. Even so, the combination of reovirus with chemotherapeutic agents showed synergistic cytopathic effects in vitro, plus enhanced reovirus replication and apoptosis. In vivo experiments showed that reovirus alone can reduce tumor size and that the combination of reovirus with chemotherapeutic agents enhances this effect. Thus, we find that oncolytic reovirus therapy is effective against gastric cancer. Moreover, the combination of reovirus and chemotherapeutic agents synergistically enhanced cytotoxicity in human gastric cancer cell lines in vitro and in vivo. Our data support the use of reovirus in combination with chemotherapy in further clinical trials, and highlight the need for better biomarkers for reoviral oncolytic responsiveness.

Oncolytic viruses (OVs) are an emerging cancer therapeutic that are intended to act by selectively targeting and lysing cancerous cells and by stimulating anti-tumour immune responses, while leaving normal cells mainly unaffected. Reovirus is a well-studied OV that is undergoing advanced clinical trials and has received FDA approval in selected circumstances. However, the mechanisms governing reoviral selectivity are not well characterised despite many years of effort, including those in our accompanying paper where we characterize pathways that do not consistently modulate reoviral cytolysis. We have earlier shown that reovirus is capable of infecting and lysing both certain types of cancer cells and also cancer stem cells, and here we demonstrate its ability to also infect and kill healthy pluripotent stem cells (PSCs). This led us to hypothesize that pathways responsible for stemness may constitute a novel route for the modulation of reoviral tropism. We find that reovirus is capable of killing both murine and human embryonic and induced pluripotent stem cells. Differentiation of PSCs alters the cells' reoviral-permissive state to a resistant one. In a breast cancer cell line that was resistant to reoviral oncolysis, induction of pluripotency programming rendered the cells permissive to cytolysis. Bioinformatic analysis indicates that expression of the Yamanaka pluripotency factors may be associated with regulating reoviral selectivity. Mechanistic insights from these studies will be useful for the advancement of reoviral oncolytic therapy.

Background: Escherichia coli are mostly commensals but also contain pathogenic lineages. It is largely unclear whether the commensal E. coli as the potential origins of pathogenic lineages may consist of monophyletic or polyphyletic populations, elucidation of which is expected to lead to novel insights into the associations of E. coli diversity with human health and diseases., Methods: Using genomic sequencing and pulsed field gel electrophoresis (PFGE) techniques, we analyzed E. coli from the intestinal microbiota of three groups of healthy individuals, including preschool children, university students, and seniors of a longevity village, as well as colorectal cancer (CRC) patients, to probe the commensal E. coli populations for their diversity., Results: We delineated the 2280 fresh E. coli isolates from 185 subjects into distinct genome types (genotypes) by PFGE. The genomic diversity of the sampled E. coli populations was so high that a given subject may have multiple genotypes of E. coli, with the general diversity within a host going up from preschool children through university students to seniors. Compared to the healthy subjects, the CRC patients had the lowest diversity level among their E. coli isolates. Notably, E. coli isolates from CRC patients could suppress the growth of E. coli bacteria isolated from healthy controls under nutrient-limited culture conditions., Conclusions: The coexistence of multiple E. coli lineages in a host may help create and maintain a microbial environment that is beneficial to the host. As such, the low diversity of E. coli bacteria may be associated with unhealthy microenvironment in the intestine and hence facilitate the pathogenesis of diseases such as CRC.

Ovarian Clear Cell Carcinoma (OCCC) displays distinctive clinical and molecular characteristics and confers the worst prognosis among all ovarian carcinoma histotypes when diagnosed at advanced stage, because of the lack of effective therapy. IGF2BP3 is an RNA binding protein that modulates gene expression by post-transcriptional action. In this study, we investigated the roles of IGF2BP3 in the progression of OCCC. We used 328 OCCCs from the AOVT (the Alberta Ovarian Tumor Type study) and the COEUR (the Canadian Ovarian Experimental Unified Resource) cohorts to elucidate the associations between IGF2BP3 expression and clinicopathological parameters, with positive IGF2BP3 expression defined as diffuse block staining, being more frequently observed at stage III ( P = 0.0056) and significantly associated with unfavorable overall survival (HR = 1.59, 95% CI 1.09-2.33) in multivariate analysis. IGF2BP3 mRNA gene expression was markedly increased in OCCC cell lines compared to normal tissues such as ovarian surface epithelium. We chose two IGF2BP3-overexpressing cell lines ES2 and OVMANA for in vitro and in vivo knockdown experiments. The proliferation and viability of both cell lines were significantly inhibited by two IGF2BP3 siRNAs and similar suppression was observed in cell migration and invasion by Wound Healing and Transwell assays. The percentage of apoptotic cancer cells was enhanced by both IGF2BP3 siRNAs. In vivo experiments showed significantly reduced sizes of tumors when treated with IGF2BP3 siRNA compared to controls. Furthermore, cancer metastasis-indicators MMP2 and MMP9 proteins were down-regulated. In conclusion, our study shows that IGF2BP3 expression is a promising biomarker for prognostication of women diagnosed with OCCC with multiple effects on key cell functions, supporting its role as an important cellular regulator with potential oncogenic activity, and as a potential target for future intervention strategies., (Copyright © 2020 Liu, Zeng, Afsharpad, Lin, Wang, Yang, Liu, Kelemen, Xu, Ma, Xiang, Mastriani, Wang, Wang, Liu, Johnston and Köbel.)

Background: Salmonella bongori infect mainly cold-blooded hosts, but infections by S. bongori in warm-blooded hosts have been reported. We hypothesized that S. bongori might have diverged into distinct phylogenetic lineages, with some being able to infect warm-blooded hosts., Results: To inspect the divergence status of S. bongori, we first completely sequenced the parakeet isolate RKS3044 and compared it with other sequenced S. bongori strains. We found that RKS3044 contained a novel T6SS encoded in a pathogenicity island-like structure, in addition to a T6SS encoded in SPI-22, which is common to all S. bongori strains so far reported. This novel T6SS resembled the SPI-19 T6SS of the warm-blooded host infecting Salmonella Subgroup I lineages. Genomic sequence comparisons revealed different genomic sequence amelioration events among the S. bongori strains, including a unique CTAG tetranucleotide degeneration pattern in RKS3044, suggesting non-overlapping gene pools between RKS3044 and other S. bongori lineages/strains leading to their independent accumulation of genomic variations. We further proved the existence of a clear-cut genetic boundary between RKS3044 and the other S. bongori lineages/strains analyzed in this study., Conclusions: The warm-blooded host-infecting S. bongori strain RKS3044 has diverged with distinct genomic features from other S. bongori strains, including a novel T6SS encoded in a previously not reported pathogenicity island-like structure and a unique genomic sequence degeneration pattern. These findings alert cautions about the emergence of new pathogens originating from non-pathogenic ancestors by acquiring specific pathogenic traits.

Oncolytic viruses show intriguing potential as cancer therapeutic agents. These viruses are capable of selectively targeting and killing cancerous cells while leaving healthy cells largely unaffected. The use of oncolytic viruses for cancer treatments in selected circumstances has recently been approved by the Food and Drug Administration (FDA) of the US and work is progressing on engineering viral vectors for enhanced selectivity, efficacy and safety. However, a better fundamental understanding of tumour and viral biology is essential for the continued advancement of the oncolytic field. This knowledge will not only help to engineer more potent and effective viruses but may also contribute to the identification of biomarkers that can determine which patients will benefit most from this treatment. A mechanistic understanding of the overlapping activity of viral and standard chemotherapeutics will enable the development of better combinational approaches to improve patient outcomes. In this review, we will examine each of the factors that contribute to productive viral infections in cancerous cells versus healthy cells. Special attention will be paid to reovirus as it is a well-studied virus and the only wild-type virus to have received orphan drug designation by the FDA. Although considerable insight into reoviral biology exists, there remain numerous deficiencies in our understanding of the factors regulating its successful oncolytic infection. Here we will discuss what is known to regulate infection as well as speculate about potential new mechanisms that may enhance successful replication. A joint appreciation of both tumour and viral biology will drive innovation for the next generation of reoviral mediated oncolytic therapy.

Imatinib, a multitargeted receptor tyrosine kinase inhibitor, is used as the standard initial therapy against inoperable gastrointestinal stromal tumor (GIST). However, GIST can acquire resistance to imatinib within several years of therapy. The development of oncolytic reovirus as an anticancer agent has expanded to many clinical trials for various tumors. Here, we investigated whether reovirus has antitumor activity against GIST cells in the setting of imatinib sensitivity in vitro and in vivo . Cell proliferation and apoptosis assays were performed using a human GIST cell line, GIST-T1, and imatinib-resistant GIST (GIST-IR) cells that we established. The molecular pathways responsible for cell damage by reovirus were explored using PCR-arrays and Western blots. Reovirus significantly induced apoptotic cell death in GIST-T1 and GIST-IR cells in vitro , despite differences in the activation of receptor tyrosine kinase pathways between GIST-T1 and GIST-IR. Molecular assays indicated the possibility that reovirus induces apoptotic cell death via Fas signaling. Furthermore, in vivo mouse tumor xenograft models demonstrated a significant anti-tumor effect of reovirus on both GIST-T1 and GIST-IR cells. Our results demonstrate the therapeutic potential of reovirus against GIST., Competing Interests: CONFLICTS OF INTEREST All authors state that they have no conflicts of interest.

Ovarian cancer is the third most common cancer in the female reproductive organs and epithelial ovarian cancer has the highest lethality of all gynecological cancers. Pomegranate fruit juice (PFJ) has been shown to inhibit the growth of several types of cancer other than ovarian cancer. In this study, we exposed the ovarian cancer cell line A2780 to PFJ and two of its components (ellagic acid and luteolin). MTT and wound healing assays demonstrated that all three treatments suppressed the proliferation and migration of the ovarian cancer cells. In addition, western blotting and ELISA assays showed that the expression levels of MMP2 and MMP9 gradually decreased after treatment with increasing concentrations of ellagic acid and luteolin. To confirm our findings in the in vitro experiments, we used another ovarian cancer cell line, ES-2, in nude mice experiments. All three treatments inhibited tumor growth without obvious side-effects. Furthermore, compared with the control group, the expression levels of MMP2 and MMP9 were depressed. Ellagic acid induced a greater effect than luteolin, suggesting that ellagic acid might be a promising candidate for further preclinical testing for treatment of human ovarian cancer.

Background: Previously, it has been found that the cancer upregulated gene 2 (CUG2) and the epidermal growth factor receptor (EGFR) both contribute to drug resistance of cancer cells. Here, we explored whether CUG2 may exert its anticancer drug resistance by increasing the expression of EGFR., Methods: EGFR expression was assessed using Western blotting, immunofluorescence and capacitance assays in A549 lung cancer and immortalized bronchial BEAS-2B cells, respectively, stably transfected with a CUG2 expression vector (A549-CUG2; BEAS-CUG2) or an empty control vector (A549-Vec; BEAS-Vec). After siRNA-mediated EGFR, Stat1 and HDAC4 silencing, antioxidant and multidrug resistance protein and mRNA levels were assessed using Western blotting and RT-PCR. In addition, the respective cells were treated with doxorubicin after which apoptosis and reactive oxygen species (ROS) levels were measured. Stat1 acetylation was assessed by immunoprecipitation., Results: We found that exogenous CUG2 overexpression induced EGFR upregulation in A549 and BEAS-2B cells, whereas EGFR silencing sensitized these cells to doxorubicin-induced apoptosis. In addition, we found that exogenous CUG2 overexpression reduced the formation of ROS during doxorubicin treatment by enhancing the expression of antioxidant and multidrug resistant proteins such as MnSOD, Foxo1, Foxo4, MRP2 and BCRP, whereas EGFR silencing congruently increased the levels of ROS by decreasing the expression of these proteins. We also found that EGFR silencing and its concomitant Akt, ERK, JNK and p38 MAPK inhibition resulted in a decreased Stat1 phosphorylation and, thus, a decreased activation. Since also acetylation can affect Stat1 activation via a phospho-acetyl switch, HDAC inhibition may sensitize cells to doxorubicin-induced apoptosis. Interestingly, we found that exogenous CUG2 overexpression upregulated HDAC4, but not HDAC2 or HDAC3. Conversely, we found that HDAC4 silencing sensitized the cells to doxorubicin resistance by decreasing Stat1 phosphorylation and EGFR expression, thus indicating an interplay between HDAC4, Stat1 and EGFR., Conclusion: Taken together, we conclude that CUG2-induced EGFR upregulation confers doxorubicin resistance to lung (cancer) cells through Stat1-HDAC4 signaling.

When bacteria diverge, they need to adapt to the new environments, such as new hosts or different tissues of the same host, by accumulating beneficial genomic variations, but a general scenario is unknown due to the lack of appropriate methods. Here we profiled the ACTAGT sequence and its degenerated forms (i.e., hexa-nucleotide sequences with one of the six nucleotides different from ACTAGT) in Salmonella to estimate the nucleotide amelioration processes of bacterial genomes. ACTAGT was mostly located in coding sequences but was also found in several intergenic regions, with its degenerated forms widely scattered throughout the bacterial genomes. We speculated that the distribution of ACTAGT and its degenerated forms might be lineage-specific as a consequence of different selection pressures imposed on ACTAGT at different genomic locations (in genes or intergenic regions) among different Salmonella lineages. To validate this speculation, we modelled the secondary structures of the ACTAGT-containing sequences conserved across Salmonella and many other enteric bacteria. Compared to ACTAGT at conserved regions, the degenerated forms were distributed throughout the bacterial genomes, with the degeneration patterns being highly similar among bacteria of the same phylogenetic lineage but radically different across different lineages. This finding demonstrates biased amelioration under distinct selection pressures among the bacteria and provides insights into genomic evolution during bacterial divergence.

Background: Ovarian cancer is one of the three leading gynecological malignancies, characterized by insidious growth, highly frequent metastasis, and quick development of drug resistance. As a result, this disease has low 5-year survival rates. Estrogen receptor inhibitors were commonly used for the treatment, but only 7% to 18% of patients respond to anti-estrogen therapies. Therefore, more effective therapies to inhibit estrogen-related tumors are urgently needed. Recently, phytoestrogens, such as lignans with estrogen-like biological activities, have attracted attention for their potential effects in the prevention or treatment of estrogen-related diseases. Enterodiol (END) and enterolactone (ENL) are mammalian lignans, which can reduce the risk of various cancers. However, the effects of END and ENL on ovarian cancer are not adequately documented., Methods: We used in vitro assays on the ES-2 cell line to evaluate the inhibiting effects of END and ENL on ovarian cancer cell proliferation, invasion and migration ability and in vivo xenograft experiments on nude mice to validate the anticancer effects of END and ENL., Results: The in vitro assays demonstrated that high-dose END and ENL could obviously inhibit ovarian malignant properties, including cancerous proliferation, invasion, and metastasis. Compared to END, ENL behaved in a better time-dose dependent manner on the cancer cells. The in vivo experiments showed that END (1 mg/kg), ENL (1 mg/kg) and ENL (0.1 mg/kg) suppressed tumor markedly, and there were statistically significant differences between the experimental and control groups in tumor weight and volume. Compared to END, which have serious side effects to the animals at high concentration such as 1 mg/kg, ENL had higher anticancer activities and less side effects in the animals than END at the same concentrations, so it would be a better candidate for drug development., Conclusion: END and ENL both have potent inhibitory effects on ovarian cancer but ENL possesses a more effective anti-cancer capability and less side effects than END. Findings in this work provide novel insights into ovarian cancer therapeutics with phytoestrogens and encourage their clinical applications.

Reoviruses, like many eukaryotic viruses, contain an inverted 7-methylguanosine (m7G) cap linked to the 5' nucleotide of mRNA. The traditional functions of capping are to promote mRNA stability, protein translation, and concealment from cellular proteins that recognize foreign RNA. To address the role of mRNA capping during reovirus replication, we assessed the benefits of adding the African swine fever virus NP868R capping enzyme during reovirus rescue. C3P3, a fusion protein containing T7 RNA polymerase and NP868R, was found to increase protein expression 5- to 10-fold compared to T7 RNA polymerase alone while enhancing reovirus rescue from the current reverse genetics system by 100-fold. Surprisingly, RNA stability was not increased by C3P3, suggesting a direct effect on protein translation. A time course analysis revealed that C3P3 increased protein synthesis within the first 2 days of a reverse genetics transfection. This analysis also revealed that C3P3 enhanced processing of outer capsid μ1 protein to μ1C, a previously described hallmark of reovirus assembly. Finally, to determine the rate of infectious-RNA incorporation into new virions, we developed a new recombinant reovirus S1 gene that expressed the fluorescent protein UnaG. Following transfection of cells with UnaG and infection with wild-type virus, passage of UnaG through progeny was significantly enhanced by C3P3. These data suggest that capping provides nontraditional functions to reovirus, such as promoting assembly and infectious-RNA incorporation. IMPORTANCE Our findings expand our understanding of how viruses utilize capping, suggesting that capping provides nontraditional functions to reovirus, such as promoting assembly and infectious-RNA incorporation, in addition to enhancing protein translation. Beyond providing mechanistic insight into reovirus replication, our findings also show that reovirus reverse genetics rescue is enhanced 100-fold by the NP868R capping enzyme. Since reovirus shows promise as a cancer therapy, efficient reovirus reverse genetics rescue will accelerate production of recombinant reoviruses as candidates to enhance therapeutic potency. NP868R-assisted reovirus rescue will also expedite production of recombinant reovirus for mechanistic insights into reovirus protein function and structure., (Copyright © 2017 American Society for Microbiology.)

Enzastaurin is an orally administered drug that was intended for the treatment of solid and haematological cancers. It was initially developed as an isozyme specific inhibitor of protein kinase Cβ (PKCβ), which is involved in both the AKT and MAPK signalling pathways that are active in many cancers. Enzastaurin had shown encouraging preclinical results for the prevention of angiogenesis, inhibition of proliferation and induction of apoptosis as well as showing limited cytotoxicity within phase I clinical trials. However, during its assessment in phase II and III clinical trials the efficacy of enzastaurin was poor both in combination with other drugs and as a single agent. In this review, we will discuss the development of enzastaurin from drug design to clinical testing, exploring target identification, validation and preclinical assessment. Finally, we will consider the clinical evaluation of enzastaurin as an example of the challenges associated with drug development. In particular, we discuss the poor translation of drug efficacy from preclinical animal models, inappropriate end point analysis, limited standards in phase I clinical trials, insufficient use of biomarker analysis and also patient stratification, all of which contributed to the failure to achieve approval of enzastaurin as an anticancer therapeutic., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Highly conserved short sequences help identify functional genomic regions and facilitate genomic annotation. We used Salmonella as the model to search the genome for evolutionarily conserved regions and focused on the tetranucleotide sequence CTAG for its potentially important functions. In Salmonella, CTAG is highly conserved across the lineages and large numbers of CTAG-containing short sequences fall in intergenic regions, strongly indicating their biological importance. Computer modeling demonstrated stable stem-loop structures in some of the CTAG-containing intergenic regions, and substitution of a nucleotide of the CTAG sequence would radically rearrange the free energy and disrupt the structure. The postulated degeneration of CTAG takes distinct patterns among Salmonella lineages and provides novel information about genomic divergence and evolution of these bacterial pathogens. Comparison of the vertically and horizontally transmitted genomic segments showed different CTAG distribution landscapes, with the genome amelioration process to remove CTAG taking place inward from both terminals of the horizontally acquired segment.

Viruses that specifically replicate in tumor over normal cells offer promising cancer therapies. Oncolytic viruses (OV) not only kill the tumor cells directly; they also promote anti-tumor immunotherapeutic responses. Other major advantages of OVs are that they dose-escalate in tumors and can be genetically engineered to enhance potency and specificity. Unmodified wild type reovirus is a propitious OV currently in phase I-III clinical trials. This review summarizes modifications to reovirus that may improve potency and/or specificity during oncolysis. Classical genetics approaches have revealed reovirus variants with improved adaptation towards tumors or with enhanced ability to establish specific steps of virus replication and cell killing among transformed cells. The recent emergence of a reverse genetics system for reovirus has provided novel strategies to fine-tune reovirus proteins or introduce exogenous genes that could promote oncolytic activity. Over the next decade, these findings are likely to generate better-optimized second-generation reovirus vectors and improve the efficacy of oncolytic reotherapy.

Salmonella arizonae (also called Salmonella subgroup IIIa) is a Gram-negative, non-spore-forming, motile, rod-shaped, facultatively anaerobic bacterium. S. arizonae strain RKS2983 was isolated from a human in California, USA. S. arizonae lies somewhere between Salmonella subgroups I (human pathogens) and V (also called S. bongori; usually non-pathogenic to humans) and so is an ideal model organism for studies of bacterial evolution from non-human pathogen to human pathogens. We hence sequenced the genome of RKS2983 for clues of genomic events that might have led to the divergence and speciation of Salmonella into distinct lineages with diverse host ranges and pathogenic features. The 4,574,836 bp complete genome contains 4,203 protein-coding genes, 82 tRNA genes and 7 rRNA operons. This genome contains several characteristics not reported to date in Salmonella subgroup I or V and may provide information about the genetic divergence of Salmonella pathogens.

Background: The pre-eminent goal of biobanks is to accelerate scientific discovery and support improvements in healthcare through the supply of high quality biospecimens to enable excellent science. Despite the need for retrospective future-proofed cancer repositories, they are presented with significant fiscal challenges. While it was once thought that biobanks could recover most, if not all, operational costs through distribution fees, biobanks have been consistently unable to fully realize this dream., Methods: Using data from three mature Canadian cancer biobanks, common attributes and assumptions related to cost recovery were evaluated. The values were entered into a simple financial model to determine the cost recovery potential for biobanks., Results: Over a 5-year period analyzed, aliquots from almost 40% (8990) of 23055 cases collected have been distributed in whole or in part to researchers. The financial modeling demonstrates that, based on values derived from the real life experiences of three major Canadian biobanks, full cost recovery through distribution is not feasible. A more realistic, experience based, expectation of cost recovery from distribution fees is in the range of 5%-25%, and this range is lower if only academic research is supported as opposed to also supporting industry researchers., Conclusions: Biobanks are expensive and, to mitigate costs, are frequently challenged to operate under "self-sustainable" financial models. However, the only possible route to self-sustainability through distribution fees in today's market would require an almost exclusive targeting of commercial researchers and, even then, evidence suggests this is an impossible goal to attain. Support for biobanks should recognize that they exist to further development of personalized treatments and diagnostics essential for precision medicine. For biobanks to continue to achieve this goal, pro bono publicum, funders need to be aware of the full funding requirements of biobanks and create appropriate funding streams.

Background: The bacterial genus Salmonella contains thousands of serotypes that infect humans or other hosts, causing mild gastroenteritis to potentially fatal systemic infections in humans. Pathogenically distinct Salmonella serotypes have been classified as individual species or as serological variants of merely one or two species, causing considerable confusion in both research and clinical settings. This situation reflects a long unanswered question regarding whether the Salmonella serotypes exist as discrete genetic clusters (natural species) of organisms or as phenotypic (e.g. pathogenic) variants of a single (or two) natural species with a continuous spectrum of genetic divergence among them. Our recent work, based on genomic sequence divergence analysis, has demonstrated that genetic boundaries exist among Salmonella serotypes, circumscribing them into clear-cut genetic clusters of bacteria., Methodologies/principal Findings: To further test the genetic boundary concept for delineating Salmonella into clearly defined natural lineages (e.g., species), we sampled a small subset of conserved genomic DNA sequences, i.e., the endonuclease cleavage sites that contain the highly conserved CTAG sequence such as TCTAGA for XbaI. We found that the CTAG-containing cleavage sequence profiles could be used to resolve the genetic boundaries as reliably and efficiently as whole genome sequence comparisons but with enormously reduced requirements for time and resources., Conclusions: Profiling of CTAG sequence subsets reflects genetic boundaries among Salmonella lineages and can delineate these bacteria into discrete natural clusters.

Background: Acquisition of exogenous genetic material is a key event in bacterial speciation. It seems reasonable to assume that recombination of the incoming DNA into genome would be more efficient with higher levels of relatedness between the DNA donor and recipient. If so, bacterial speciation would be a smooth process, leading to a continuous spectrum of genomic divergence of bacteria, which, however, is not the case as shown by recent findings. The goal of this study was todetermine if DNA transfer efficiency is correlated with the levels of sequence identity., Results: To compare the relative efficiency of exogenous DNA acquisition among closely related bacteria, we carried out phage-mediated transduction and plasmid-mediated transformation in representative Salmonella strains with different levels of relatedness. We found that the efficiency was remarkably variable even among genetically almost identical bacteria. Although there was a general tendency that more closely related DNA donor-recipient pairs had higher transduction efficiency, transformation efficiency exhibited over a thousand times difference among the closely related Salmonella strains., Conclusion: DNA acquisition efficiency is greatly variable among bacteria that have as high as over 99% identical genetic background, suggesting that bacterial speciation involves highly complex processes affected not only by whether beneficial exogenous DNA may exist in the environment but also the "readiness" of the bacteria to accept it.

Each year funding agencies and academic institutions spend millions of dollars and euros on biobanking. All funding providers assume that after initial investments biobanks should be able to operate sustainably. However the topic of sustainability is challenging for the discipline of biobanking for several major reasons: the diversity in the biobanking landscape, the different purposes of biobanks, the fact that biobanks are dissimilar to other research infrastructures and the absence of universally understood or applicable value metrics for funders and other stakeholders. In this article our aim is to delineate a framework to allow more effective discussion and action around approaches for improving biobank sustainability. The term sustainability is often used to mean fiscally self-sustaining, but this restricted definition is not sufficient for biobanking. Instead we propose that biobank sustainability should be considered within a framework of three dimensions - financial, operational, and social. In each dimension, areas of focus or elements are identified that may allow different types of biobanks to distinguish and evaluate the relevance, likelihood, and impact of each element, as well as the risks to the biobank of failure to address them. Examples of practical solutions, tools and strategies to address biobank sustainability are also discussed.

The deadly human typhoid agent was initially classified as a species called Salmonella typhi but later reclassified as a serovar of Salmonella enterica together with other pathogenically diverse serovars. The dynamic changes of Salmonella taxonomy reflect the need to clarify the phylogenetic status of the Salmonella serovars: are they discrete lineages or variants of a genetic lineage? To answer this question, we compared S. typhi and other Salmonella serotypes. We found that the S. typhi and Salmonella typhimurium strains had over 90% and ca. 80%, respectively, of their genes identical; however, between S. typhi and S. typhimurium, this percentage dropped to 6%, suggesting the existence of genetic boundaries between them. We conclude that S. typhi and the other compared Salmonella serovars have developed into distinct lineages circumscribed by the genetic boundary. This concept and methods may be used to delineate other Salmonella serotypes, many of which are polyphyletic, needing differentiation., (© 2013 Published by Elsevier Inc.)

Background: Previous studies have shown successful antitumor effects of systemically delivered double-deleted vaccinia virus (vvDD) against a number of adult tumor models, including glioma, colon and ovarian cancers. The purpose of this study was to investigate the oncolytic potential of vvDD against a panel of cell lines representative of pediatric solid tumors that are currently difficult to cure., Methods: Cell lines derived from central nervous system atypical teratoid rhabdoid tumor (AT/RT) (BT12, BT16 and KCCF1), sarcoma (143B, HOS, RD and RH30), and neuroblastoma (SKNAS, SKNBE2, IMR-5 and IMR-32) were examined for vvDD mediated cytotoxicity defined by virus expansion followed by loss of tumor cell viability. The normal human fibroblast cell line HS68 was used as a control. Next, relevant orthotopic, subcutaneous and lung metastasis xenograft models were treated with intravenous doses of live vvDD or killed virus controls (DV). Tumor growth inhibition and viral replication were quantified and survival outcomes of these animals were assessed., Results: vvDD was able to infect and kill nine of eleven of the pediatric tumor cells (81.8%) in vitro. In xenograft models, intravenous administration of a single dose of vvDD significantly inhibited the growth of tumors and prolonged the survival of intracranial and metastatic tumors., Conclusions: Oncolytic vvDD administered i.v. shows activity in preclinical models of pediatric malignancies that are resistant to many currently available treatments. Our data support further evaluation of vvDD virotherapy for refractory pediatric solid tumors., (Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Background: Bacteria are currently classified into arbitrary species, but whether they actually exist as discrete natural species was unclear. To reveal genomic features that may unambiguously group bacteria into discrete genetic clusters, we carried out systematic genomic comparisons among representative bacteria., Results: We found that bacteria of Salmonella formed tight phylogenetic clusters separated by various genetic distances: whereas over 90% of the approximately four thousand shared genes had completely identical sequences among strains of the same lineage, the percentages dropped sharply to below 50% across the lineages, demonstrating the existence of clear-cut genetic boundaries by a steep turning point in nucleotide sequence divergence. Recombination assays supported the genetic boundary hypothesis, suggesting that genetic barriers had been formed between bacteria of even very closely related lineages. We found similar situations in bacteria of Yersinia and Staphylococcus., Conclusions: Bacteria are genetically isolated into discrete clusters equivalent to natural species.

To investigate the role of gene localization and genome organization in cell-cell signalling and regulation, we mapped the distribution pattern of gene families that comprise core components of intercellular communication networks. Our study is centered on the distinct evolutionarily conserved metazoan signalling pathways that employ proteins in the receptor tyrosine kinase, WNT, hedgehog, NOTCH, Janus kinase/STAT, transforming growth factor beta, and nuclear hormone receptor protein families. Aberrant activity of these signalling pathways is closely associated with the promotion and maintenance of human cancers. The cataloguing and mapping of genes encoding these signalling proteins and comparisons across species has led us to propose that the genome can be subdivided into six genome-wide primary linkage groups (PLGs). PLGs are composed of assemblages of gene families that are often mutually exclusive, raising the possibility of unique functional identities for each group. Examination of the localization patterns of genes with distinct functions in signal transduction demonstrates dichotomous segregation patterns. For example, gene families of cell-surface receptors localize to genomic compartments that are distinct from the locations of their cognate ligand gene families. Additionally, genes encoding negative-acting components of signalling pathways (inhibitors and antagonists) are topologically separated from their positive regulators and other signal transducer genes. We, therefore, propose the existence of conserved genomic territories that encode key proteins required for the proper activity of metazoan signaling and regulatory systems. Disruption in this pattern of topologic genomic organization may contribute to aberrant regulation in hereditary or acquired diseases such as cancer. We further propose that long-range looping genomic regulatory interactions may provide a mechanism favouring the remarkable retention of these conserved gene clusters during chordate evolution.

We have recently found a novel oncogene, named cancer upregulated gene 2 (CUG2), which activates Ras and mitogen-activated protein kinases (MAPKs), including ERK, JNK and p38 MAPK. Because activation of these signaling pathways has previously been shown to enhance cancer cell susceptibility to oncolysis by certain viruses, we examined whether vesicular stomatitis virus (VSV) could function as a potential therapeutic agent by efficiently inducing cytolysis in cells transformed by CUG2. Unexpectedly, NIH3T3 cells stably expressing CUG2 (NIH-CUG2) were resistant to VSV because of the activation of signal transducers and activators of transcription 1 (STAT1). The result was supported by evidence showing that suppression of STAT1 with short interference RNA (siRNA) renders cells susceptible to VSV. Furthermore, 2'-5' oligoadenylate synthetase-like (OASL) 2 was the most affected by STAT1 expression level among anti-viral proteins and furthermore suppression of OASL2 mRNA level caused NIH-CUG2 cells to succumb to VSV as seen in NIH-CUG2 cells treated with STAT1 siRNA. In addition, Colon26L5 carcinoma cells stably expressing CUG2 (Colon26L5-CUG2) exhibited resistance to VSV, whereas Colon26L5 stably expressing a control vector yielded to VSV infection. Moreover, Colon26L5-CUG2 cells stably suppressing STAT1 succumbed to VSV infection, resulting in apoptosis. Taken together, we propose that VSV treatment combined with the selective regulation of genes such as STAT1 and OASL2 will improve therapeutic outcomes for CUG2-overexpressing tumors.

Background: Salmonella serovars Enteritidis and Gallinarum are closely related, but their host ranges are very different: the former is host-promiscuous and the latter can infect poultry only. Comparison of their genomic sequences reveals that Gallinarum has undergone much more extensive degradation than Enteritidis. This phenomenon has also been observed in other host restricted Salmonella serovars, such as Typhi and Paratyphi A. The serovar Gallinarum can be further split into two biovars: Gallinarum and Pullorum, which take poultry as their common host but cause distinct diseases, with the former eliciting typhoid and the latter being a dysentery agent. Genomic comparison of the two pathogens, with a focus on pseudogenes, would provide insights into the evolutionary processes that might have facilitated the formation of host-restricted Salmonella pathogens., Methodologies/principal Findings: We sequenced the complete genome of Pullorum strains and made comparison with Gallinarum and other Salmonella lineages. The gene contents of Gallinarum and Pullorum were highly similar, but their pseudogene compositions differed considerably. About one fourth of pseudogenes had the same inactivation mutations in Gallinarum and Pullorum but these genes remained intact in Enteritidis, suggesting that the ancestral Gallinarum may have already been restricted to poultry. On the other hand, the remaining pseudogenes were either in the same genes but with different inactivation sites or unique to Gallinarum or Pullorum, reflecting unnecessary functions in infecting poultry., Conclusions: Our results support the hypothesis that the divergence between Gallinarum and Pullorum was initiated and facilitated by host restriction. Formation of pseudogenes instead of gene deletion is the major form of genomic degradation. Given the short divergence history of Gallinarum and Pullorum, the effect of host restriction on genomic degradation is huge and rapid, and such effect seems to be continuing to work. The pseudogenes may reflect the unnecessary functions for Salmonella within the poultry host.

Salmonella enterica serovar Pullorum is a chicken-adapted pathogen, causing pullorum disease. Its strict host adaptation has been suspected to result in gene decay. To validate this hypothesis and identify the decayed genes, we sequenced the complete genome of S. Pullorum RKS5078. We found 263 pseudogenes in this strain and conducted functional analyses of the decayed genes.

The hepatitis B virus X (HBX) protein, a regulatory protein of the hepatitis B virus (HBV), has been shown to generate reactive oxygen species (ROS) in human liver cell lines; however, the mechanism by which cells protect themselves under this oxidative stress is poorly understood. Here, we show that HBX induces the up-regulation of Forkhead box class O 4 (Foxo4) not only in Chang cells stably expressing HBX (Chang-HBX) but also in primary hepatic tissues from HBX-transgenic mice. HBX also increased ROS, but reduction of the abundance of ROS using N-acetylcystein (NAC) diminished the levels of Foxo4. Elevated Foxo4 was also detected in nuclei of Chang-HBX cells but not in Chang cells stably expressing the vector (Chang-Vec), suggesting that HBX activates the transcriptional activity of Foxo4. When we examined whether HBX bypasses JNK signaling that targets Foxo4, we found that the activity of JNK but not of ERK is required for the up-regulation of Foxo4 even in the presence of HBX. Furthermore, the reduction of Foxo4 levels using siRNA or a JNK inhibitor rendered Chang-HBX cells sensitive to apoptosis under oxidative stress, suggesting that up-regulation of Foxo4 mediated by HBX enhances resistances to oxidative stress-induced cell death. Accordingly, we propose that Foxo4 may be a useful target for suppression in the treatment of HBV-associated hepatocellular carcinoma cells.

It is not yet understood how the enhanced expression of pancreatic adenocarcinoma up-regulated factor (PAUF; a novel oncogene identified in our recent studies), contributes to the oncogenesis of pancreatic cells. We herein report that PAUF up-regulates the expression and transcriptional activity of β-catenin while the suppression of PAUF by shRNA down-regulates β-catenin. The induction of b-catenin by PAUF is mediated by the activities of Akt and GSK-3β, but inhibition of downstream ERK does not reduce β-catenin expression. To test whether PAUF emulates either the Wnt3a-mediated or the protein kinase A-mediated signaling pathway for the stabilization of β-catenin, we examined the phosphorylation status of β-catenin in the presence of PAUF compared with that of β-catenin during treatment with Wnt3a or dibutyryl cAMP, a cell permeable cyclic AMP analogue. PAUF expression induces phosphorylation at Ser-33/37/Thr-41 and Ser-675 of β-catenin but no phosphorylation at Ser-45, indicating that a unique phosphorylation pattern of b-catenin is caused by PAUF. Finally, the expression of PAUF up-regulates both cyclin-D1 and c-Jun, target genes of β-catenin, leading to a rapid proliferation of pancreatic cells; conversely decreased PAUF expression (by shRNA) results in the reduced proliferation of pancreatic cells. Treatment with hexachlorophene (an inhibitor of β-catenin) reduces the proliferation of pancreatic cells despite the presence of PAUF. Taken together, we propose that PAUF can up-regulate and stabilize β-catenin via a novel pattern of phosphorylation, thereby contributing to the rapid proliferation of pancreatic cancer cells.

Background: Salmonella paratyphi C is one of the few human-adapted pathogens along with S. typhi, S. paratyphi A and S. paratyphi B that cause typhoid, but it is not clear whether these bacteria cause the disease by the same or different pathogenic mechanisms. Notably, these typhoid agents have distinct sets of large genomic insertions, which may encode different pathogenicity factors. Previously we identified a novel prophage, SPC-P1, in S. paratyphi C RKS4594 and wondered whether it might be involved in pathogenicity of the bacteria., Results: We analyzed the sequence of SPC-P1 and found that it is an inducible phage with an overall G+C content of 47.24%, similar to that of most Salmonella phages such as P22 and ST64T but significantly lower than the 52.16% average of the RKS4594 chromosome. Electron microscopy showed short-tailed phage particles very similar to the lambdoid phage CUS-3. To evaluate its roles in pathogenicity, we lysogenized S. paratyphi C strain CN13/87, which did not have this prophage, and infected mice with the lysogenized CN13/87. Compared to the phage-free wild type CN13/87, the lysogenized CN13/87 exhibited significantly increased virulence and caused multi-organ damages in mice at considerably lower infection doses., Conclusions: SPC-P1 contributes pathogenicity to S. paratyphi C in animal infection models, so it is possible that this prophage is involved in typhoid pathogenesis in humans. Genetic and functional analyses of SPC-P1 may facilitate the study of pathogenic evolution of the extant typhoid agents, providing particular help in elucidating the pathogenic determinants of the typhoid agents.

Background: All life forms need both high genetic stability to survive as species and a degree of mutability to evolve for adaptation, but little is known about how the organisms balance the two seemingly conflicting aspects of life: genetic stability and mutability. The DNA mismatch repair (MMR) system is essential for maintaining genetic stability and defects in MMR lead to high mutability. Evolution is driven by genetic novelty, such as point mutation and lateral gene transfer, both of which require genetic mutability. However, normally a functional MMR system would strongly inhibit such genomic changes. Our previous work indicated that MMR gene allele conversion between functional and non-functional states through copy number changes of small tandem repeats could occur spontaneously via slipped-strand mis-pairing during DNA replication and therefore may play a role of genetic switches to modulate the bacterial mutability at the population level. The open question was: when the conversion from functional to defective MMR is prohibited, will bacteria still be able to evolve by accepting laterally transferred DNA or accumulating mutations?, Results: To prohibit allele conversion, we "locked" the MMR genes through nucleotide replacements. We then scored changes in bacterial mutability and found that Salmonella strains with MMR locked at the functional state had significantly decreased mutability. To determine the generalizability of this kind of mutability 'switching' among a wider range of bacteria, we examined the distribution of tandem repeats within MMR genes in over 100 bacterial species and found that multiple genetic switches might exist in these bacteria and may spontaneously modulate bacterial mutability during evolution., Conclusions: MMR allele conversion through repeats-mediated slipped-strand mis-pairing may function as a spontaneous mechanism to switch between high genetic stability and mutability during bacterial evolution.

Replication-competent oncolytic viruses hold great potential for the clinical treatment of many cancers. Importantly, many oncolytic virus candidates, such as reovirus and myxoma virus, preferentially infect cancer cells bearing abnormal cellular signaling pathways. Reovirus and myxoma virus are highly responsive to activated Ras and Akt signaling pathways, respectively, for their specificity for viral oncolysis. However, considering the complexity of cancer cell populations, it is possible that other tumor-specific signaling pathways may also contribute to viral discrimination between normal versus cancer cells. Because carcinogenesis is a multistep process involving the accumulation of both oncogene activations and the inactivation of tumor suppressor genes, we speculated that not only oncogenes but also tumor suppressor genes may have an important role in determining the tropism of these viruses for cancer cells. It has been previously shown that many cellular tumor suppressor genes, such as p53, ATM and Rb, are important for maintaining genomic stability; dysfunction of these tumor suppressors may disrupt intact cellular antiviral activity due to the accumulation of genomic instability or due to interference with apoptotic signaling. Therefore, we speculated that cells with dysfunctional tumor suppressors may display enhanced susceptibility to challenge with these oncolytic viruses, as previously seen with adenovirus. We report here that both reovirus and myxoma virus preferentially infect cancer cells bearing dysfunctional or deleted p53, ATM and Rb tumor suppressor genes compared to cells retaining normal counterparts of these genes. Thus, oncolysis by these viruses may be influenced by both oncogenic activation and tumor suppressor status.

Many oncolytic viruses are currently being tested as potential cancer therapeutic agents. To be effective, these viruses must replicate and propagate efficiently through the tumor mass. However, it is possible that the hypoxia that characterizes many tumors may be an obstacle to viral therapy because of its inhibition of viral replication and propagation. We, therefore, decided to test how oncolytic reovirus and its target cells respond to hypoxia. We found that reovirus infection suppresses hypoxia inducible factor (HIF)-1alpha protein levels (but not transcript abundance) in colon cancer HCT116 cells under CoCl(2) or hypoxia. Reovirus infection was able to reduce HIF-1alpha levels in both von Hippel Lindau (VHL)-/- renal carcinoma A498 and p53-/- HCT116 cells, indicating that the decrease of HIF-1alpha mediated by reovirus requires neither VHL nor p53 proteins. However, treatment with the inhibitor MG132 restored HIF-1alpha levels, suggesting that reovirus-induced HIF-1alpha decrease needs proteosomal activity. A498 VHL-/- cells with constitutive expression of HIF-1alpha were relatively resistant to reovirus-induced apoptosis when compared with A498 VHL+/+ cells. However, we found that the use of YC-1 to target HIF-1alpha promoted reovirus-induced apoptosis in A498 VHL-/- cells. Accordingly, we propose that reovirus may be used together with YC-1 as a potential therapeutic agent against chemoresistant or radioresistant tumors that are hypoxic and show increased levels of HIF-1alpha.

As we have recently found a novel oncogene, the cancer-upregulated gene 2 (CUG2), which was elevated in a variety of tumor tissues such as the ovary, liver, lung and pancreas, we examined whether reovirus could efficiently induce cytolysis in cancer cells expressing CUG2 and thus be used as a potential cancer therapeutic agent. In this study, we describe experiments in which we use reovirus to treat NIH3T3 cells stably expressing either CUG2 (NIH-CUG2) or vector only (NIH-Vec). NIH-CUG2 cells readily support reoviral proliferation and undergo apoptosis, whereas NIH-Vec cells are highly resistant to reoviral infection and virus-induced apoptosis. This notable result may be explained by the observation that CUG2 expression inhibits PKR activation, leading to reoviral proliferation in nonpermissive NIH3T3 cells. Furthermore, reovirus infection results in almost complete regression of tumorgenic NIH-CUG2 cells in transplanted nude mice. As we found that CUG2 enhances activation of MAPK (ERK, JNK and p38), Src kinase and Ras, we examined whether CUG2 confers reoviral replication independent of the Ras or p38 MAPK signaling pathway. From these experiments we found that either inhibition of p38 MAPK or Ras blocks reoviral proliferation even in the presence of CUG2 but inhibition of ERK, JNK and Src kinase does not, indicating that activation of p38 MAPK and Ras has critical roles in reoviral replication in CUG2-expressing tumor cells. Accordingly, we propose that reovirus can be useful in the treatment of transformed cells expressing CUG2, which is commonly detected in various tumor tissues.

Reovirus is a nonattenuated double-stranded RNA virus that exploits aberrant signaling pathways allowing selective cytotoxicity against multiple cancer histologies. The use of reovirus as a potential treatment modality for prostate cancer has not previously been described, and in this study evidence of in vitro and in vivo activity against prostate cancer was seen both in preclinical models and in six patients. The human prostate carcinoma cell lines PC-3, LN-CaP, and DU-145 exposed to replication-competent reovirus showed evidence of infection as illustrated by viral protein synthesis, cytopathic effect, and release of viral progeny. This oncolytic effect was found to be manifested through apoptosis, as DNA fragmentation, Apo 2.7 expression, Annexin V binding, and poly(ADP-ribose) polymerase cleavage were observed in live reovirus-infected cells, but not in uninfected or dead virus-treated cells. In vivo, hind flank severe combined immunodeficient/nonobese diabetic murine xenograft showed reduction in tumor size when treated with even a single intratumoral injection of reovirus. Finally, intralesional reovirus injections into a cohort of six patients with clinically organ-confined prostate cancer resulted in minimal side effects and evidence of antitumor activity. Histologic analysis after prostatectomy found a significant CD8 T-cell infiltration within the reovirus-injected areas as well as evidence of increased caspase-3 activity. These findings suggest that reovirus therapy may provide a promising novel treatment for prostate cancer and also imply a possible role for viral immune targeting of tumor.

Although reovirus has been used in tests as a potential cancer therapeutic agent against a variety of cancer cells, its application to hepatocellular carcinoma cells, in which the hepatitis B virus (HBV) X (HBX) protein of HBV plays a primary role, has not yet been explored. Here, we describe experiments in which we use reovirus to treat Chang liver carcinoma cells expressing either a vector only (Chang-vec) or a vector encoding HBX protein (Chang-HBX). Although Chang-vec cells readily support reoviral proliferation and undergo apoptosis, Chang-HBX cells are highly resistant to reoviral infection and virus-induced apoptosis, even though HBX protein induces activation of Ras and inactivation of PKR, which are normally thought to enhance reoviral oncolysis. The resistance of Chang-HBX cells to reovirus may instead be explained by HBX-induced downregulation of death receptor 5 and activation of Stat1. Phosphorylated Stat1 activates interferon (IFN)-stimulated regulatory element (ISRE)- and IFN-gamma-activated sequence (GAS)-mediated transcription, leading to the production of IFN-beta, whereas the reduced expression of Stat1 with its siRNA results in a decrease in IFN-beta production, by which Chang-HBX cells eventually succumb to reovirus infection. This result further indicates that HBX induces the establishment of an antiviral state through Stat1 activation. Thus, it appears that active Ras does not override the antiviral effect mediated by the activation of Stat1. Accordingly, we report that HBX, an oncoprotein of HBV, can prevent reoviral oncolysis of hepatocellular carcinoma. This suggests there may be limits to the practical application of reovirus in the treatment of human cancers already expressing other oncoviral proteins.

Background: Although over 1400 Salmonella serovars cause usually self-limited gastroenteritis in humans, a few, e.g., Salmonella typhi and S. paratyphi C, cause typhoid, a potentially fatal systemic infection. It is not known whether the typhoid agents have evolved from a common ancestor (by divergent processes) or acquired similar pathogenic traits independently (by convergent processes). Comparison of different typhoid agents with non-typhoidal Salmonella lineages will provide excellent models for studies on how similar pathogens might have evolved., Methodologies/principal Findings: We sequenced a strain of S. paratyphi C, RKS4594, and compared it with previously sequenced Salmonella strains. RKS4594 contains a chromosome of 4,833,080 bp and a plasmid of 55,414 bp. We predicted 4,640 intact coding sequences (4,578 in the chromosome and 62 in the plasmid) and 152 pseudogenes (149 in the chromosome and 3 in the plasmid). RKS4594 shares as many as 4346 of the 4,640 genes with a strain of S. choleraesuis, which is primarily a swine pathogen, but only 4008 genes with another human-adapted typhoid agent, S. typhi. Comparison of 3691 genes shared by all six sequenced Salmonella strains placed S. paratyphi C and S. choleraesuis together at one end, and S. typhi at the opposite end, of the phylogenetic tree, demonstrating separate ancestries of the human-adapted typhoid agents. S. paratyphi C seemed to have suffered enormous selection pressures during its adaptation to man as suggested by the differential nucleotide substitutions and different sets of pseudogenes, between S. paratyphi C and S. choleraesuis., Conclusions: S. paratyphi C does not share a common ancestor with other human-adapted typhoid agents, supporting the convergent evolution model of the typhoid agents. S. paratyphi C has diverged from a common ancestor with S. choleraesuis by accumulating genomic novelty during adaptation to man.

Oral neoplasm constitutes a predominant class of cancer that is encountered in South India. This is in large part due to the elevated risk of oncogenesis as a result of the habit of chewing of quids containing betel leaves, areca nut and smokeless tobacco. An array of molecular events are induced during the transformation of the buccal epithelium, among them the over-expression of oncogene products plays a key role. The c-Myc protein, a regulator of a number of key cellular signalling pathways, plays a pivotal role in a number of malignancies. The present study was undertaken to evaluate expression of the c-Myc protein in tumours of the oral cavity from the South Indian population, predominantly oral squamous cell carcinoma (OSCC). The c-Myc protein was over-expressed in 80% of the cases studied. Taking into account the pivotal role demonstrated for c-Myc in tumourigenesis, our observations suggest a key role for Myc oncoprotein in the genesis of OSCC as well as its potential as a therapeutic target in this population.

Previously, we reported the phenomenon of genome diversification in Salmonella typhimurium LT7, i.e., individual strains derived from LT7 kept changing the genome structure by inversions, translocations, duplications, and mutations. To elucidate the genetic basis, we sequenced selected genes of the mismatch repair (MMR) system for correlations between MMR defects and genome diversification. We chose S. typhimurium LT7 mutants 8111F2 and 9052D1 for mut gene sequence analyses and found that both mutants had a deletion of one of three tandem 6-bp repeats, GCTGGC GCTGGC GCTGGC, within mutL, which was designated 6 bpDeltamutL. mutS and mutH genes were unchanged in the mutants analyzed. Some sublines of 8111F2 and 9052D1 spontaneously stopped the genome diversification process at certain stages during single-colony restreaking passages, and in these strains the 6 bpDeltamutL genotype also became wild-type mutL. We conclude that conversion between mutL and 6 bpDeltamutL occurs spontaneously and that transient defects of mutL facilitate genome diversification without leading to the accumulation of multiple detrimental genetic changes. Spontaneous conversion between mutL and 6 bpDeltamutL may be an important mechanism used by bacteria to regulate genetic stability in adaptation to changing environments.

Reovirus type 3 Dearing has demonstrated oncolytic efficacy in vitro and in vivo against a variety of cancer cell lines, tumor xenografts and syngeneic cancer models. In this study, we investigated the effectiveness of reovirus against aberrant crypt foci (ACF) and colon cancer induced by the carcinogen azoxymethane (AOM) in an immunocompetent rat model. Sprague-Dawley rats received 15 mg/kg AOM intraperitoneally once per week for 4 weeks and reovirus was administered rectally once a week for 5 weeks starting 20 weeks after the last dose of AOM. Two weeks after completion of reovirus therapy, animals were examined for tumor burden in the colon and other tissues. Reovirus-treated animals showed a decrease in total ACF numbers (P=0.014), in large ACFs (P=0.0069) and in tumor number (P=0.03) compared to vehicle-treated animals. Fewer obstructing tumors in the colon (P=0.07) and duodenum (P=0.03) and reduced hepatic metastases were also noted. In addition, a tumor cell line derived from hepatic metastases was found to be susceptible to reovirus in vitro. Our results show that repeated rectal reovirus administration had some efficacy in the treatment and prevention of AOM-induced ACFs, colon cancers and metastases.

Background: Salmonella paratyphi C, like S. typhi, is adapted to humans and causes typhoid fever. Previously we reported different genome structures between two strains of S. paratyphi C, which suggests that S. paratyphi C might have a plastic genome (large DNA segments being organized in different orders or orientations on the genome). As many but not all host-adapted Salmonella pathogens have large genomic insertions as well as the supposedly resultant genomic rearrangements, bacterial genome plasticity presents an extraordinary evolutionary phenomenon. Events contributing to genomic plasticity, especially large insertions, may be associated with the formation of particular Salmonella pathogens., Results: We constructed a high resolution genome map in S. paratyphi C strain RKS4594 and located four insertions totaling 176 kb (including the 90 kb SPI7) and seven deletions totaling 165 kb relative to S. typhimurium LT2. Two rearrangements were revealed, including an inversion of 1602 kb covering the ter region and the translocation of the 43 kb I-CeuI F fragment. The 23 wild type strains analyzed in this study exhibited diverse genome structures, mostly as a result of recombination between rrn genes. In at least two cases, the rearrangements involved recombination between genomic sites other than the rrn genes, possibly homologous genes in prophages. Two strains had a 20 kb deletion between rrlA and rrlB, which is a highly conservative region and no deletion has been reported in this region in any other Salmonella lineages., Conclusion: S. paratyphi C has diverse genome structures among different isolates, possibly as a result of large genomic insertions, e.g., SPI7. Although the Salmonella typhoid agents may not be more closely related among them than each of them to other Salmonella lineages, they may have evolved in similar ways, i.e., acquiring typhoid-associated genes followed by genome structure rearrangements. Comparison of multiple Salmonella typhoid agents at both single sequenced genome and population levels will facilitate the studies on the evolutionary process of typhoid pathogenesis, especially the identification of typhoid-associated genes.

Reovirus shows considerable potential as an oncolytic agent for Ras-activated tumors and is currently in clinical trials. Here we ask whether such tumor cell lines can acquire resistance to reoviral oncolysis. We challenged human HT1080 fibrosarcoma cells that carry a Ras mutation by prolonged exposure to reovirus, thereby yielding highly virus-resistant HTR1 cells. These cells are persistently infected with reovirus, exhibit high Ras activity and retain the original Ras gene mutation, showing that resistance to reovirus can be displayed in cells with active Ras. The HTR1 cells also exhibit reduced cellular cathepsin B activity, which normally contributes to viral entry and activation. Persistently infected HTR1 cells were not tumorigenic in vivo, whereas immunologically cured virus-free HTR1 cells were highly tumorigenic. Thus, acquisition of resistance to reovirus may constrain therapeutic strategies. To determine whether reoviral resistance is associated with a general reduction in apoptotic potential, we challenged the HTR1 cells with apoptotic inducers and E1B-defective adenovirus, resulting in significant apoptosis and cell death following both approaches. Therefore, even if resistance to reoviral oncolysis should arise in tumor cells in vivo, other therapeutic strategies may nevertheless remain effective.

Background: An ideal virus for the treatment of cancer should have effective delivery into multiple sites within the tumor, evade immune responses, produce rapid viral replication, spread within the tumor, and infect multiple tumors. Vesicular stomatitis virus (VSV) has been shown to be an effective oncolytic virus in a variety of tumor models, and mutations in the matrix (M) protein enhance VSV's effectiveness in animal models., Methods: We evaluated the susceptibility of 14 glioma cell lines to infection and killing by mutant strain VSV(deltaM51), which contains a single-amino acid deletion in the M protein. We also examined the activity and safety of this strain against the U87 and U118 experimental models of human malignant glioma in nude mice and analyzed the distribution of the virus in the brains of U87 tumor-bearing mice using fluorescence labeling. Finally, we examined the effect of VSV(deltaM51) on 15 primary human gliomas cultured from surgical specimens. All statistical tests were two-sided., Results: All 14 glioma cell lines were susceptible to VSV(deltaM51) infection and killing. Intratumoral administration of VSV(deltaM51) produced marked regression of malignant gliomas in nude mice. When administered systemically, live VSV(deltaM51) virus, as compared with dead virus, statistically significantly prolonged survival of mice with unilateral U87 tumors (median survival: 113 versus 46 days, P = .0001) and bilateral U87 tumors (median survival: 73 versus 46 days, P = .0025). VSV(deltaM51) infected multifocal gliomas, invasive glioma cells that migrated beyond the main glioma, and all 15 primary human gliomas. There was no evidence of toxicity., Conclusions: Systemically delivered VSV(deltaM51) was an effective and safe oncolytic agent against laboratory models of multifocal and invasive malignant gliomas, the most challenging clinical manifestations of this disease.

The use of oncolytic viruses has received considerable attention in recent years and many viruses have proved to be effective against a variety of cancer models and a few are currently being used in clinical trials. However, the possible emergence and outcome of virus-resistant tumour cells has not been addressed. We previously reported the effective use of reovirus against lymphoid malignancies, including the Burkitt's lymphoma cell line Raji. Here we isolated in vitro persistently infected (PI) Raji cells, and cells 'cured' of persistent reovirus infection ('cured' cells). Both PI and cured Raji cells resisted reovirus infection and cell killing in vitro. In vivo, the PI cells were non-tumorigenic in SCID mice, but cured cells regained the parental cells' ability to form tumours. Tumour xenografts from the cured cells, however, were highly susceptible to reovirus oncolysis in vivo. This susceptibility was due to the proteolytic environment within tumours that facilitates reovirus infection and cell killing. Our results show that persistent infection by reovirus impedes tumour development and that although PI cells cleared of reovirus are tumorigenic, they are killed upon rechallenge with reovirus. Both the PI and cured states are therefore not likely to be significant barriers to reovirus oncolytic therapy.