Passage Lines Research Articles

166 Development of non-model organoids in animal research: A paradigm shift in in vitro models

Abstract For decades, two-dimensional (2D) cell cultures have been used as the gold standard in vitro models for basic research, drug discovery and preclinical screening applications. However, since 2009, the field has quickly shifted towards the use of three-dimensional (3D) stem cell-derived organoids. Organoids offer a variety of advantages compared with conventional 2D models, including being self-organized, retaining their cellular polarity, and being simplified versions of organs in vitro. Adult stem cells can self-renew, differentiate into multiple cell types, and are genomically stable over multiple passage lines, providing a variety of technical benefits and offering the closest recapitulation of the in vivo environment. Our laboratory has focused on expanding, characterizing, and utilizing animal-derived 3D organoid models, specifically focusing on canines; but also including other animals such as swine, turtles, snakes, and squirrels to study their unique adaptations. Practically, we can use 3D canine colonoids for the screening of anti-inflammatory candidates using our tumor necrosis factor (TNF)-α stimulation model. Additionally, we have grown and used swine lung organoids as a model for studying H1N1 viral uptake. Furthermore, we have grown and bio-banked tumor organoids/spheroids from a variety of canine cancers, including urothelial carcinoma (derived from urine and biopsies) and nasal carcinoma, among others, with future applications in precision veterinary medicine and comparative oncology. To accomplish this goal, we have carried out in vitro screening of a variety of chemotherapies and combination therapies at different concentrations across patients to predict in vivo responses and demonstrate the predictive ability of the organoid model. Finally, our laboratory has derived and characterized a variety of healthy organoid lines from canines including the pancreas, lung, liver, bladder, colon, and pituitary to act as healthy controls in mechanistic studies looking at disease pathogenesis and for uses in basic biology. In summary, in vitro organoid models from various animal species and tissues can be used in a variety of basic and applied research fields, including virology, toxicology, and oncology, with the potential to broaden our scientific understanding of disease and overall improve human and veterinary medicine.

Passaging Phyllosphere Microbial Communities Develop Suppression Towards Bacterial Speck Disease in Tomato

Microbial community-based disease management approaches have the potential to substitute or combine with currently employed strategies. Suppressive soils are great examples of microbial communities that suppress soilborne plant disease after severe outbreaks and are maintained over multiple years or crop cycles. Although there are many suppressive soil examples, to our knowledge, there are no descriptions of disease-suppressive phyllosphere communities. Therefore, we investigated whether a phyllosphere microbial community could be developed through a selective passaging method to suppress disease using the model pathosystem of Pseudomonas syringae pv. tomato ( Pto) and tomato. Field tomato phyllosphere microbial communities were recovered and spray transferred to greenhouse tomato plants, after which they were inoculated with Pto. Disease severity was visually estimated and the microbial communities were recovered to be independently applied to the next passage. Overall, greenhouse passaging resulted in an increase in disease severity for all passage lines from the initial passage, which peaked at passage 4 to 5, followed by a sharp decline until passage 9. The disease severity at passage 9 was also significantly lower than a non-passaged Pto-only comparison. Heat treatment of passage 9 communities resulted in elevated disease severity over several subsequent passages in the growth chamber, whereas the untreated community maintained low disease. Community-only passage lines (without Pto inclusion during passaging) did not show disease suppression after subsequent pathogen introduction. This work presents an experimental approach to develop phyllosphere microbial communities in the presence of a phytopathogen to enrich for a low disease phenotype and results in maintained disease suppression. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Mutations in PB2 and HA are crucial for the increased virulence and transmissibility of H1N1 swine influenza virus in mammalian models

Genetic analyses indicated that the pandemic H1N1/2009 influenza virus originated from a swine influenza virus (SIV). However, SIVs bearing the same constellation of genetic features as H1N1/2009 have not been isolated. Understanding the adaptation of SIVs with such genotypes in a new host may provide clues regarding the emergence of pandemic strains such as H1N1/2009. In this study, an artificial SIV with the H1N1/2009 genotype (rH1N1) was sequentially passaged in mice through two independent series, yielding multiple mouse-adapted mutants with high genetic diversity and increased virulence. These experiments were meant to mimic genetic bottlenecks during adaptation of wild viruses with rH1N1 genotypes in a new host. Molecular substitutions in the mouse-adapted variants mainly occurred in genes encoding surface proteins (hemagglutinin [HA] and neuraminidase [NA]) and polymerase proteins (polymerase basic 2 [PB2], polymerase basic 1 [PB1], polymerase acid [PA] proteins and nucleoprotein [NP]). The PB2D309N and HAL425M substitutions were detected at high frequencies in both passage lines and enhanced the replication and pathogenicity of rH1N1 in mice. Moreover, these substitutions also enabled direct transmission of rH1N1 in other mammals such as guinea pigs. PB2D309N showed enhanced polymerase activity and HAL425M showed increased stability compared with the wild-type proteins. Our findings indicate that if SIVs with H1N1/2009 genotypes emerge in pigs, they could undergo rapid adaptive changes during infection of a new host, especially in the PB2 and HA genes. These changes may facilitate the emergence of pandemic strains such as H1N1/2009.

Experimental evolution of the megaplasmid pMPPla107 in Pseudomonas stutzeri enables identification of genes contributing to sensitivity to an inhibitory agent.

Horizontally transferred elements, such as plasmids, can burden host cells with various metabolic and fitness costs and may lead to other potentially detrimental phenotypic effects. Acquisition of the Pseudomonas syringae megaplasmid pMPPla107 by various Pseudomonads causes sensitivity to a growth-inhibiting substance that is produced in cultures by Pseudomonads during growth under standard laboratory conditions. After approximately 500 generations of laboratory passage of Pseudomonas stutzeri populations containing pMPPla107, strains from two out of six independent passage lines displayed resistance to this inhibitory agent. Resistance was transferable and is, therefore, associated with mutations occurring on pMPPla107. Resequencing experiments demonstrated that resistance is likely due to a large deletion on the megaplasmid in one line, and to a nonsynonymous change in an uncharacterized megaplasmid locus in the other strain. We further used allele exchange experiments to confirm that resistance is due to this single amino acid change in a previously uncharacterized megaplasmid protein, which we name SkaA. These results provide further evidence that costs and phenotypic changes associated with horizontal gene transfer can be compensated through single mutational events and emphasize the power of experimental evolution and resequencing to better understand the genetic basis of evolved phenotypes. This article is part of the theme issue 'The secret lives of microbial mobile genetic elements'.

Potential neoplastic evolution of Vero cells: in vivo and in vitro characterization.

Vero cell lines are extensively employed in viral vaccine manufacturing. Similarly to all established cells, mutations can occur during Vero cells in vitro amplification which can result in adverse features compromising their biological safety. To evaluate the potential neoplastic evolution of these cells, in vitro transformation test, gene expression analysis and karyotyping were compared among low- (127 and 139 passages) and high-passage (passage 194) cell lines, as well as transformed colonies (TCs). In vivo tumorigenicity was also tested to confirm preliminary in vitro data obtained for low passage lines and TCs. Moreover, Vero cells cultivated in foetal bovine serum-free medium and derived from TCs were analysed to investigate the influence of cultivation methods on tumorigenic evolution. Low-passage Vero developed TCs in soft agar, without showing any tumorigenic evolution when inoculated in the animal model. Karyotyping showed a hypo-diploid modal chromosome number and rearrangements with no difference among Vero cell line passages and TCs. These abnormalities were reported also in serum-free cultivated Vero. Gene expression revealed that high-passage Vero cells had several under-expressed and a few over-expressed genes compared to low-passage ones. Gene ontology revealed no significant enrichment of pathways related to oncogenic risk. These findings suggest that in vitro high passage, and not culture conditions, induces Vero transformation correlated to karyotype and gene expression alterations. These data, together with previous investigations reporting tumour induction in high-passage Vero cells, suggest the use of low-passage Vero cells or cell lines other than Vero to increase the safety of vaccine manufacturing.

Mutation tendency of mutator Plasmodium berghei with proofreading-deficient DNA polymerase δ.

In this study, we investigated the mutation tendency of a mutator rodent malaria parasite, Plasmodium berghei, with proofreading-deficient DNA polymerase δ. Wild-type and mutator parasites were maintained in mice for over 24 weeks, and the genome-wide accumulated mutations were determined by high-throughput sequencing. The mutator P. berghei had a significant preference for C/G to A/T substitutions; thus, its genome had a trend towards a higher AT content. The mutation rate was influenced by the sequence context, and mutations were markedly elevated at TCT. Some genes mutated repeatedly in replicate passage lines. In particular, knockout mutations of the AP2-G gene were frequent, which conferred strong growth advantages on parasites during the blood stage but at the cost of losing the ability to form gametocytes. This is the first report to demonstrate a biased mutation tendency in malaria parasites, and its results help to promote our basic understanding of Plasmodium genetics.

THE SADNESS OF EPARCHIUS AVITUS (SIDONIUS,CARM. 7.519-21)

In his panegyric of Avitus, his father-in-law, the poet Sidonius gives a vivid and often detailed picture of the career of the future emperor from his boyhood until he gained the supreme power in the West in the year 455, which he owed to his ability and accomplishments in warfare, diplomacy and administration. He also enjoyed strong support from both Goths and Gauls, and his repeated success in managing the volatility and the aspirations of the Goths is a major theme. This short note seeks to contribute to the understanding of his emergence as Augustus by proposing a new interpretation of a pivotal passage (lines 519–24), quoted below. The context can be briefly given. Sidonius has painted a graphic picture of the reaction among the Goths to the news of the Vandal capture of Rome in the year 455 (lines 441–57). In the ensuing assembly of Gothic elders Avitus (who happens to be with them at Toulouse at this moment) gives a speech that vigorously praises peace (lines 458–86), and this is favourably received. There follows a speech from the Gothic king, Theoderic II, in which he puts his own strong case for an agreement between Goths and Gauls, and seeks to persuade Avitus to fill the vacuum in Rome and Italy caused by the recent death of the emperor Petronius Maximus and take the name of Augustus (lines 489–518).

Abstract 2300: Human CD133-specific chimeric antigen receptor (CAR) modified T cells target patient-derived glioblastoma brain tumors

Abstract Glioblastoma (GBM), an aggressive primary brain tumor in adults, is feared for its near uniformly fatal prognosis and is characterized by a diverse cellular phenotype and genetic heterogeneity. Despite the use of aggressive multi-modal treatment including surgical resection, radiotherapy and chemotherapy, the outcome of patients with GBM has failed to improve significantly. We developed patient-derived brain tumor initiating cell (BTIC) early passage lines that describe the extent of intertumoral heterogeneity, presenting a powerful preclinical model of GBM. Numerous studies have implicated CD133+ BTICs as drivers of chemo- and radio-resistance in GBM. CD133 expression correlates with disease progression, recurrence, and poor overall survival of GBM patients. Here, we describe the preclinical evaluation of chimeric antigen receptor (CAR) T-cell strategy that specifically targets CD133+ GBM cells. From the previously generated CD133-specific humanized monoclonal antibody, we derived the single chain variable fragment (scFv) and cloned it into an antigen-specific second-generation CAR. Anti-CD133 scFv with a myc tag was cloned in frame with a human CD8 leader sequence, CD8a transmembrane domain, CD28, and hCD3ζ signaling tail in the lentiviral construct pCCL-ΔNGFR vector in two different orientations: Light chain-linker-Heavy chain (CD133 CAR-LH) and Heavy chain-linker-Light chain (CD133 CAR-HL). Following lentiviral preparation, the T cells isolated from PBMCs were transduced with CD133 CAR-LH and CD133 CAR-VH constructs. After successful T cell engineering, the expression of ΔNGFR and myc tag was analyzed using flow cytometry to confirm the efficiency of transduction and surface expression of anti-CD133 respectively. While expression of ΔNGFR was observed in all CAR T cells (including controls), we found expression of the myc tag in both variations of CARs, CD133 CAR-HL and CD133 CAR-LH. Furthermore, we used Presto Blue-based killing assays to test the ability of CD133 CARs to selectively bind and kill CD133+ GBM BTICs. Our data shows that CD133-specific CAR T cells not only recognized, but killed the CD133+ GBM cells selectively, validating this adoptive T-cell therapeutic strategy. CAR-expressing T cells were activated in presence of CD133high GBM cells showed increase surface expression of activation markers CD69 and CD25. Both, CD4+ and CD8+ CD133-specific CAR-T cells showed upregulation in surface expression levels of activation markers. This rigorously obtained data offers compelling evidence that CAR-T induced cytotoxicity against treatment-resistant and evasive CD133+ GBM BTICs could provide a very potent, specific and novel therapeutic strategy for GBM patients. Citation Format: Parvez Vora, Chitra Venugopal, Sujeivan Mahendram, Chirayu Chokshi, Maleeha Qazi, Minomi Subapanditha, Mohini Singh, David Bakhshinyan, Ksenia Bezverbnaya, Jarrett Adams, Nicole McFarlane, Sachdev Sidhu, Jason Moffat, Jonathan Bramson, Sheila Singh. Human CD133-specific chimeric antigen receptor (CAR) modified T cells target patient-derived glioblastoma brain tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2300.

Abstract 1481: Preclinical validation of a novel CD133/CD3 bispecific T-cell engager (BiTE) antibody to target patient-derived glioblastoma cells

Abstract Glioblastoma (GBM), an aggressive primary brain tumor in adults, is feared for its near uniformly fatal prognosis and is characterized by a diverse cellular phenotype and genetic heterogeneity. Despite the use of aggressive multi-modal treatment including surgical resection, radiotherapy and chemotherapy, the outcome of patients with GBM has failed to improve significantly. We developed patient-derived brain tumor initiating cell (BTIC) early passage lines that describe the extent of intertumoral heterogeneity, presenting a powerful preclinical model of GBM. Numerous studies have implicated CD133+ BTICs as drivers of chemo- and radio-resistance in GBM. CD133 expression correlates with disease progression, recurrence, and poor overall survival of GBM patients. Here, we describe the preclinical evaluation of a recombinant RW03xCD3 bispecific T-cell engager (BiTE) antibody that redirects human polyclonal T cells to CD133+ GBM cells, inducing very potent anti-tumor response. Using CellectSeq, a novel methodology that combines use of phage-displayed synthetic antibody libraries and high-throughput DNA sequencing technology, we developed the CD133-specific monoclonal antibody ‘RW03’. We constructed CD133-specific BiTEs or RW03xCD3 that consist of two arms; one arm recognizes the tumor antigen (CD133) while the second is specific to CD3 antigen. The BiTEs were constructed in four different conformations and dual binding specificity was confirmed using flow cytometry. Using CD133high and CD133low primary GBM lines, we validated the binding of BiTEs to CD133+ cells. Further analysis showed binding of BiTEs to human T cells known to express CD3 within a population of healthy donor peripheral blood mononuclear cells. In order to test the ability of BiTEs to functionally elicit CD133-specific cytotoxic responses in vitro, we performed Presto blue-based killing assays. We observed CD133-specific BiTEs redirect T cells to kill CD133-expressing GBM cells in a coculture of T cells and GBM cells. The killing was more efficient in CD133high GBMs compared to CD133low GBMs, validating its specificity to target CD133+ BTICs. Incubating T cells with BiTEs and GBMs resulted in increased surface expression of T-cell activation markers CD69 and CD25 in both, CD4+ and CD8+ T cells populations. Treatment with BiTEs yielded extended survival in mice and significant reductions in brain tumor burden. This rigorously obtained data offers compelling evidence that BiTE-mediated cytotoxicity against treatment-resistant and evasive CD133+ GBM BTICs could provide a very potent, specific and novel therapeutic strategy for GBM patients. Citation Format: Parvez Vora, Chitra Venugopal, Jarrett Adams, James Pan, Chirayu Chokshi, Maleeha Qazi, Minomi Subapanditha, Mohini Singh, David Bakhshinyan, Ksenia Bezverbnaya, Nicole McFarlane, Jonathan Bramson, Sachdev Sidhu, Jason Moffat, Sheila Singh. Preclinical validation of a novel CD133/CD3 bispecific T-cell engager (BiTE) antibody to target patient-derived glioblastoma cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1481.

Temozolomide resistance in glioblastoma cells occurs partly through epidermal growth factor receptor-mediated induction of connexin 43

Glioblastoma Multiforme (GBM) is an aggressive adult primary brain tumor with poor prognosis. GBM patients develop resistance to the frontline chemotherapy, temozolomide (TMZ). As the connexins (Cx) have been shown to have a complex role in GBM, we investigated the role of Cx43 in TMZ resistance. Cx43 was increased in the TMZ-resistant low passage and cell lines. This correlated with the data in The Cancer Genome Atlas. Cx43 knockdown, reporter gene assays, chromatin immunoprecipitation assay, real-time PCR and western blots verified a role for Cx43 in TMZ resistance. This occurred by TMZ-resistant GBM cells being able to activate epidermal growth factor receptor (EGFR). In turn, EGFR activated the JNK-ERK1/2-AP-1 axis to induce Cx43. The increased Cx43 was functional as indicated by gap junctional intercellular communication among the resistant GBM cells. Cell therapy could be a potential method to deliver drugs, such as anti-EGF to tumor cells. Similar strategies could be used to reverse the expression of Cx43 to sensitize GBM cells to TMZ. The studies showed the potential for targeting EGF in immune therapy. These agents can be used in conjunction with stem cell therapy to treat GBM.

Genomic characterization and high-throughput therapeutic screening of malignant mesothelioma to reveal novel tumor dependencies.

7581 Background: Clinical trials of targeted therapeutics in mesothelioma have demonstrated limited efficacy. It has been suggested that combinations of targeted agents may be more effective in this disease, but given the unknown status of tractable oncogenic mutations it has been difficult to ascertain which key signalling nodes drive these tumours that may lend themselves to therapeutic intervention. We therefore aimed to characterise a representative panel of mesothelioma cell lines at the genomic level, and then determine critical “nodes” utilising a high throughput screen of targeted agents. Methods: 19 mesothelioma cell lines and 6 mesothelioma primary tumour early passage lines underwent Illumina whole exome sequencing and copy number analysis. In parallel a high throughput screen was performed utilising a panel of targeted therapeutics enabling each mesothelioma to be screened across 48 compounds. Efficacy was confirmed in 3D spheroid culture. Results: Exome sequencing of the mesothelioma panel revealed mutations in tumour suppressor genes previously described in mesothelioma including NF2, as well as previously unreported mutations in this disease affecting histone modifying genes MLL2 and SETD2. Notably an absence of mutated “driver” oncogenes was observed. High throughput screening demonstrated limited activity for most small molecule inhibitors as single agents. However, despite an absence of mutations in PIK3CAor related genes, PI3K/mTORC inhibition had the broadest single agent efficacy. Conclusions: We demonstrate that mutations in cell lines are similar to those found in patient-derived tumours implying fidelity at the genomic level. Mesotheliomas harbour multiple mutations in tumour suppressors, but lack commonly tractable oncogenic mutations which may explain poor efficacy seen in clinical trials to date. We further demonstrate that PI3K may represent a critical node therapeutically that may be useful in combinatorial approaches.

Comparison of responses of human melanoma cell lines to MEK and BRAF inhibitors

The NRAS and BRAF genes are frequently mutated in melanoma, suggesting that the NRAS-BRAF-MEK-ERK signaling pathway is an important target for therapy. Two classes of drugs, one targeting activated BRAF and one targeting MEK, are currently undergoing clinical evaluation. We have analysed the NRAS and BRAF mutational status of a series of 44 early passage lines developed from New Zealand patients with metastatic melanoma. 41% of the lines analysed had BRAF mutations, 23% had NRAS mutations, and 36% had neither. We then determined IC50 values (drug concentrations for 50% growth inhibition) for CI-1040, a commonly used inhibitor of MEK kinase; trametinib, a clinical agent targeting MEK kinase; and vemurafenib, an inhibitor of mutant BRAF kinase. Cell lines with activating BRAF mutations were significantly more sensitive to vemurafenib than lines with NRAS mutations or lines lacking either mutation (p < 0.001). IC50 values for CI-1040 and trametinib were strongly correlated (r = 0.98) with trametinib showing ~100-fold greater potency. Cell lines sensitive to vemurafenib were also sensitive to CI-1040 and trametinib, but there was no relationship between IC50 values and NRAS mutation status. A small number of lines lacking a BRAF mutation were sensitive to CI-1040 but resistant to vemurafenib. We used western blotting to investigate the effect on ERK phosphorylation of CI-1040 in four lines, of vemurafenib in two lines and of trametinib in two lines. The results support the view that MEK inhibitors might be combined with BRAF inhibitors in the treatment of melanomas with activated BRAF. The high sensitivity to trametinib of some lines with wildtype BRAF status also suggests that MEK inhibitors could have a therapeutic effect against some melanomas as single agents.

The Prologue of Iphigenia Aulidensis Reconsidered

AbstractThe authenticity of the prologue ofIphigenia Aulidensis, as it is preserved in the MSS, has been questioned by many scholars. Apart from its metrical form and style (which are discussed only briefly here), the students of the play point out two crucial problems concerning the internal consistency of the plot. First, lines 124-32 become superfluous when they are confronted with lines 106-7. Secondly, there is an inconsistency between the passages implying that Calchas’ oracle and the plan of luring Iphigenia to Aulis was known only to Calchas, Odysseus, Menelaus and Agamemnon and those according to which the whole Greek army was aware of Agamemnon’s ruse. The present discussion aims to show that the simplest way to remove both inconsistencies is to exclude as interpolated lines 105-10, without the necessity either of deleting the subsequent passage (lines 111-14), suspected by many scholars, or of questioning the authenticity of the prologue as a whole. Additionally, it is suggested that the interpolator was probably thinking of E.IT15-25 and 521-49.

High-temperature-mediated spontaneous mutations in the coat protein of cucumber mosaic virus in Nicotiana tabacum

Since temperature effects on cucumber mosaic virus (CMV) have not been extensively studied, we examined the effects of a moderate increase in temperature on the virulence and the genetic diversity of CMV in Nicotiana tabacum (cv. Samsun NN). Two passage lines were initiated by inoculation of tobacco plants with a chlorosis strain of CMV (designated FFM-CMV) derived from full-length infectious CMV clones. Symptom changes in the tobacco plants were monitored during five successive passages at 25 and 33°C. At the fifth passage, tobacco plants inoculated with FFM-CMV at 33°C showed typical chlorosis symptoms at 7 days post-inoculation (dpi), and the plants further developed mosaic symptoms on the upper leaves at 20 dpi. In contrast, tobacco plants inoculated with FFM-CMV at 25°C did not show changes in symptoms on the upper leaves within 20 days. Sequence analysis of the coat protein (CP) genes of FFM-CMV revealed that a variety of spontaneous sequence changes were generated from the 14th and the 18th leaves of 33°C lineage tobacco plants, but not from those of 25°C lineage tobacco plants. The major CP mutation from the upper leaves of 33°C lineage tobacco plants was an L129P mutation, indicating a change in symptoms correlating to the population composition of the mutant viruses. Although relatively low mutation rates were observed from 33°C-lineage tobacco plants, some CP mutations may have been selected for systemic movement of CMV at the higher temperature, providing evidence for the importance of temperature in the process of genetic diversification of CMV populations.

Altered patterns of differentiation in karyotypically abnormal human embryonic stem cells

Upon prolonged culture, human embryonic stem (hES) cells undergo adaptation, exhibiting decreased population doubling times and increased cloning efficiencies, often associated with karyotypic changes. To test whether culture adaptation influences the patterns of differentiation of hES cells, we compared the expression of genes indicative of distinct embryonic lineages in the embryoid bodies produced from two early passage, karyotypically normal hES cell lines, and two late passage, karyotypically abnormal hES cell lines. One of the abnormal lines was a subline of one of the normal early passage lines. The embryoid bodies from each of the lines showed evidence of extensive differentiation. However, there were differences in the expression of several genes, indicating that the culture adapted hES cells show altered patterns of differentiation compared to karyotypically normal hES cells. The loss of induction of alphafetoprotein in the culture-adapted cells was especially marked, suggesting that they had a reduced capacity to produce extra-embryonic endoderm. These changes may contribute to the growth advantages of genetically variant cells, not only by reflecting an increased tendency to self renewal rather than to differentiate, but also by reducing spontaneous differentiation to derivatives that themselves may produce factors that could induce further differentiation of undifferentiated stem cells.

The Impact of Extended Voice Use on the Acoustic Characteristics of Phonation After Training and Performance of Actors From the La MaMa Experimental Theater Club

To test the hypothesis that extensive use of La MaMa vocal technique may result in symptoms of vocal abuse, an evaluation of the acoustic and perceptual characteristics of voice for eight performers from the Great Jones Repertory Company of the La MaMa Experimental Theater was conducted. This vocal technique includes wide ranges of frequency from 46 to 2003 Hz and vocal intensity that is sustained at 90-108 dB sound pressure level with a mouth-to-microphone distance of 30 cm for 3-4 hours per performance. The actors rehearsed for 4 hours per day, 5 days per week for 14 weeks before the series of performances. Thirty-nine performances were presented in 6 weeks. Three pretraining, three posttraining, and two postperformance series data collection sessions were carried out for each performer. Speech samples were gathered using the CSL 4500 and analyzed using Real-Time Pitch program and Multidimensional Voice Program. Acoustic analysis was performed on 48 tokens of sustained vowel phonation for each subject. Statistical analysis was performed using the Friedman test of related samples. Perceptual analysis included professional listeners rating voice quality in pretraining, posttraining, and postperformance samples of the Rainbow Passage and sample lines from the plays. The majority of professional listeners (11/12) judged that this technique would result in symptoms of vocal abuse; however, acoustic data revealed statistically stable or improved measurements for all subjects in most dependent acoustic variables when compared with both posttraining and postperformance trials. These findings add support to the notion that a technique that may be perceived as vocally abusive, generating 90-100 dB sound pressure level and sustained over 6 weeks of performances, actually resulted in improved vocal strength and flexibility.

Marvell, Nathaniel Whiting, and Cowley

ONE of the intriguing features of Andrew Marvell’s poetic composition process is the manner in which he appropriated words, phrases, images, and themes from other writers and transmuted them in poems that are in some sense distinctively ‘Marvellian’. What is equally intriguing is that notwithstanding his intimate knowledge of the major classical (particularly Latin) poets and his wide reading in the works of his precursors and contemporaries, many of the sources on which he appears to have drawn are ones that are rarely read today, poems that lie outside the core of the canon (sometimes for good reason). Marvell’s use of Nathaniel Whiting appears to provide an important instance.1 In 1637, when both Marvell and Abraham Cowley were at Cambridge, Whiting (1611/12–1682), who had studied at Queens’ College and graduated MA in 1635, published Le hore di recreatione: or, The Pleasant Historie of Albino and Bellama (hereinafter Albino and Bellama or simply AB), together with ‘Il Insonio Insonadada’, a defence of poetry in the form of a satire of some its characteristic detractors. Albino and Bellama is a lengthy, disjointed, and uneven but spirited combination of comedy, picaresque romance, satire (particularly of uncouth nobles, religious houses, and unlettered rustics), parody, Boccaccian bawdy, and some extravagant but beautiful and effective lyric passages. To the best of my knowledge, Pierre Legouis is the sole scholar to have commented on Marvell’s use of Whiting. Legouis focused exclusively on the affinities between AB and the nunnery passage (lines 81–272) of Marvell’s ‘Upon Appleton House’, remarking that ‘By comparison with [Whiting’s] burlesque Marvell’s treatment of monastic life sounds gentle.’2 Marvell, however, appears to have drawn on AB in a number of poems, and his appropriations cast light on both his poetic production process and the interpretation of the poems.

Adaptation of plum pox virus to a herbaceous host (Pisum sativum) following serial passages.

Plum pox virus (PPV) populations from peaches are able to adapt consistently to herbaceous hosts, characterized by a reduction in time to symptom development, increases in inoculation efficiency and increased titres. PPV adaptation was studied by using pea (Pisum sativum) as an alternative host. Two isolates of PPV from peaches were inoculated and passaged in peas ten times using either aphid or mechanical inoculation, generating four independent passage lines. Mechanical-transmission efficiency from peach to pea improved from 3 % at passage 1 to 100 % by serial passage 4 on peas. Inoculation using aphid vectors required six to ten serial passages in pea to reach a peak of 50-60 % transmission efficiency. Sequence analyses of all four PPV population lines inoculated sequentially to pea identified a specific mutation occurring consistently in the NIb gene when compared with the same PPV isolates passaged in parallel in peach. The mutation allowed PPV to replicate up to 20 times faster in the new host. Pea-adapted strains of PPV at every passage were also tested for their ability to infect the original host, peach. Regardless of the number of previous passages, all pea-adapted PPV strains consistently infected peach at low levels using aphid inoculation.

Short-Term Viral Evolution in Response to Passaging I. Consequences for Population Size

The Red Queen hypothesis for the advantage of sex predicts that pathogens will evolve by increasing fitness with frequent encounters with specific host genotypes. In this study, BMV population size, measured as an indicator of fitness, was investigated during repeated passages through the same, or different host genotypes of the crop host, Hordeum vulgare (barley). Overall, mean BMV concentration within individual hosts was significantly higher in genetically homogeneous compared to heterogeneous host passage lines. In addition, BMV populations, passaged through a specific host variety, showed higher growth in that host variety compared to BMV passaged through varying varieties. These results supports the Red Queen hypothesis. However, the decrease in viral populations during passages contradicts the Red Queen. Nevertheless, the results found here show that even under simplified conditions, pathogens do not evolve in simple, predictable ways. Constraints on pathogen evolution may lead to counterintuitive results.

Restoration of telomeres in human papillomavirus-immortalized human anogenital epithelial cells.

Loss of telomeres has been hypothesized to be important in cellular senescence and may play a role in carcinogenesis. In this study, we have measured telomere length in association with the immortalization and transformation of human cervical and foreskin epithelial cells by the human papillomavirus type 16 or 18 E6 and E7 open reading frames. By using a telomeric TTAGGG repeat probe, it was shown that the telomeres of precrisis normal and E6-, E7-, and E6/E7-expressing cells gradually shortened with passaging (30 to 100 bp per population doubling). Cells that expressed both E6 and E7 went through a crisis period and gave rise to immortalized lines. In contrast to precrisis cells, E6/E7-immortalized cells generally showed an increase in telomere length as they were passaged in culture, with some later passage lines having telomeres that were similar to or longer than the earliest-passage precrisis cells examined. No consistent association could be made between telomere length and tumorigenicity of cells in nude mice. However, of the three cell lines that grew in vivo, two had long telomeres, thus arguing against the hypothesis that cancer cells favor shortened telomeres. Our results indicate that arrest of telomere shortening may be important in human papillomavirus-associated immortalization and that restoration of telomere length may be advantageous to cells with regard to their ability to proliferate.