Presence Of Selective Pressure Research Articles

A multispecies framework for modeling adaptive immunity and immunotherapy in cancer.

Predator-prey theory is commonly used to describe tumor growth in the presence of selective pressure from the adaptive immune system. These interactions are mediated by the tumor immunopeptidome (what the tumor "shows" the body) and the T-cell receptor (TCR) repertoire (how well the body "sees" cancer cells). The tumor immunopeptidome comprises neoantigens which can be gained and lost throughout tumorigenesis and treatment. Heterogeneity in the immunopeptidome is predictive of poor response to immunotherapy in some tumor types, suggesting that the TCR repertoire is unable to support a fully polyclonal response against every neoantigen. Importantly, while tumor and T-cell populations are known to compete with each other for intratumoral resources, whether between-lineage competition among peripheral T cells influences the TCR repertoire is unknown and difficult to interrogate experimentally. Computational models may offer a way to investigate these phenomena and deepen our understanding of the tumor-immune axis. Here, we construct a predator-prey-like model and calibrate it to preclinical and clinical data to describe tumor growth and immunopeptidome diversification. Simultaneously, we model the expansion of antigen-specific T-cell lineages and their consumption of both lineage-specific antigenic resources and lineage-agnostic, shared resources. This predator-prey-like framework accurately described clinically observed immunopeptidomes; recapitulated response-associated effects of immunotherapy, including immunoediting; and allowed exploration of treatment of tumors with varying growth and mutation rates.

Abstract PR003: OXPHOS impairing mitochondrial DNA mutations suppress melanoma growth and metastatic progression

Abstract Cancers acquire somatic mitochondrial DNA (mtDNA) mutations undetected in surrounding tissues. Cancer-specific mtDNA mutations primarily constitute variants of unknown significance whose oncogenic consequences are poorly understood. Analysis of large-scale sequencing datasets suggests that human cancers generally select against severe mtDNA mutations; however, the presence of selective pressure on the mtDNA genome has yet to be experimentally demonstrated. Here we optimized a FACS-based protocol to reconstitute the highly metastatic human melanoma cell line A375 with exogenous mtDNA, thereby generating nuclear-isogenic cytoplasmic hybrid (cybrid) lines. Homoplasmic cybrids were generated exclusively harboring either exogenous wildtype mtDNA, mtDNA with partial loss of OXHPHOS function (SNPs in mt.ND1 (of Complex I) or mt.ATP6 (of Complex V)), or mtDNA with complete loss of OXPHOS function (derived from a human melanoma harboring a truncating frameshift mutation in mt.CO1 (of Complex IV)). Compared to wildtype cybrids, subcutaneous xenografts of ND1 and ATP6 cybrids into NOD–SCID Il2rg−/− (NSG) mice had elevated primary tumor ROS levels and similar primary tumor growth rates. Xenografts of our most dysfunctional OXPHOS cybrid, CO1, formed tumors albeit with lowered growth rates. Uniquely, the ND1 and CO1 xenografts respectively exhibited diminished metastatic burden and no metastatic burden. Our results suggest that OXPHOS impairing mtDNA mutations are tolerable during primary tumor growth but reduce the severity and incidence of metastasis. Lastly, we generated heteroplasmic A375 cybrids, harboring both wild-type and mutant alleles, and developed a ddPCR method to characterize allelic frequency with single-cell precision. Extended in vitro passage of the heteroplasmic cybrids failed to significantly shift the heteroplasmic mtDNA allelic frequency. However, following subcutaneous xenografting of the heteroplasmic cybrids, primary tumors significantly shifted their variant allele frequency to favor the wildtype allele. In summary, these results indicate that a functional mtDNA haplotype is not absolutely required for tumorigenesis but contributes to metastatic efficiency. In addition, we experimentally demonstrated the existence of selective pressure in favor of functional mtDNA during tumorigenesis. Citation Format: Spencer Shelton, Sara House, Vijayashree Ramesh, Zhengkang Chen, Claire Llamas, Alpaslan Tasdogan, Ralph DeBerardinis, Sean Morrison, Prashant Mishra. OXPHOS impairing mitochondrial DNA mutations suppress melanoma growth and metastatic progression [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr PR003.

Structural and molecular rationale for the diversification of resistance mediated by the Antibiotic_NAT family

The environmental microbiome harbors a vast repertoire of antibiotic resistance genes (ARGs) which can serve as evolutionary predecessors for ARGs found in pathogenic bacteria, or can be directly mobilized to pathogens in the presence of selection pressures. Thus, ARGs from benign environmental bacteria are an important resource for understanding clinically relevant resistance. Here, we conduct a comprehensive functional analysis of the Antibiotic_NAT family of aminoglycoside acetyltransferases. We determined a pan-family antibiogram of 21 Antibiotic_NAT enzymes, including 8 derived from clinical isolates and 13 from environmental metagenomic samples. We find that environment-derived representatives confer high-level, broad-spectrum resistance, including against the atypical aminoglycoside apramycin, and that a metagenome-derived gene likely is ancestral to an aac(3) gene found in clinical isolates. Through crystallographic analysis, we rationalize the molecular basis for diversification of substrate specificity across the family. This work provides critical data on the molecular mechanism underpinning resistance to established and emergent aminoglycoside antibiotics and broadens our understanding of ARGs in the environment.

PSXIV-23 Polymorphism of the LCORL gene in the reindeer

Abstract Reindeer are one of the main representatives of the ecosystem of the Arctic zone and the only possible object of economic activity of the indigenous peoples of the polar regions of Russia. Reindeer meat is a valuable source of complete protein in the diet of the Evenks, Nenets, Yakuts and other peoples of the Far North. Four breeds of reindeer have been bred in Russia - Nenets, Evenk, Chukchi and Even. Evenk and Nenets are the most numerous. They live in different climatic zones and differ significantly in phenotypic characteristics (size and body weight). Populations of wild reindeer are also widespread. The problem of increasing the meat productivity of the main breeds of domesticated reindeer can be solved by searching for casual polymorphisms in candidate genes responsible for the growth and development of muscle tissue. Thus, polymorphism of the LCORL gene (mRNA ligand-dependent nuclear receptor of the corepressor type, which is a transcription factor) is associated with body weight and size in many mammals. We sequenced exon 7 of the LCORL gene and found seven SNPs, six of which are nonsynonymous and lead to a change in the amino acid composition of the protein. There was a significant difference in the occurrence of wild and mutant alleles in the three analyzed populations (wild reindeer, Nenets breed, Evenk breed), which indicates the presence of selection pressure on the LСORL gene region, and also indirectly confirms the significance of the polymorphism of this locus for phenotypic variability in height and size of the reindeer. Studies of associations of LCORL gene polymorphisms on live weight and growth in other animal species suggest that some of the found SNPs may be true causal quantitative trait loci (QTL) in domestic reindeer breeds. Project No. 0045-2021-0010.

Assessment of Genetic Stability During Serial In Vitro Passage and In Vivo Carriage.

In this study, our objective was to evaluate the genetic stability of foodborne bacterial pathogens during serial passage in vitro and persistent in vivo carriage. Six strains of Listeria, Campylobacter, Escherichia, Salmonella, and Vibrio were serially passaged 20 times. Three colonies were picked for whole-genome sequencing (WGS) from passes P0, P5, P10, P15, and P20. In addition, isolates of Salmonella and Escherichia from three patients with persistent infections were sequenced. Genetic stability was evaluated in terms of variations detected in high-quality single-nucleotide polymorphism (hqSNP), core genome multilocus sequence typing (cgMLST), seven-gene MLST, and determinants encoding serotype, antimicrobial resistance (AMR), and virulence. During serial passage, increasing diversity was observed in Listeria, Salmonella, and Vibrio as measured by hqSNPs (from median of 0 SNPs to median of 3-5 SNPs, depending on the organism) and to a lesser extent with cgMLST (from median of 0 alleles to median of 0-5 alleles), while Escherichia and Campylobacter genomes showed minimal variation. The serotype, AMR, and virulence markers remained stable in all organisms. Isolates from persistent infections lasting up to 10 weeks remained genetically stable. However, isolates from a persistent Salmonella enterica ser. Montevideo infection spanning 9 years showed early heterogeneity leading to the emergence of one predominant genotype that continued to evolve over the years, including gains and losses of AMR markers. While the hqSNP and cgMLST variation observed during the serial passage was minimal, culture passages should be limited to as few times as possible before WGS. Our WGS data show that in vivo carriage lasting for a few weeks did not appear to alter the genotype. Longer persistent infections spanning for years, particularly in the presence of selective pressure, may cause changes in the genotype making it challenging to differentiate persistent infections from reinfections.

Antibiotic selective pressure in microcosms: Pollution influences the persistence of multidrug resistant Shigella flexneri 2a YSH6000 strain in polluted river water samples

Abstract Some urban rivers reach dangerous concentrations of residual antibiotics imposing a certain level of selective pressure on microorganisms to develop various antibiotic resistance mechanisms. In the current work, we have measured the persistence and growth of a multidrug resistant strain of Shigella flexneri 2a YSH6000 under a mock release of lethal concentration of oxytetracycline in microcosms of River Thames water. The water was sampled from upstream (lower levels of pollution) and downstream (higher levels of pollution) of London city centre. In our in-vitro microcosms, in the presence of 160 μ g/mL of oxytetracycline, growth of S. flexneri in the downstream sector was up to 2 log(cfu/mL) higher relative to the upstream sector. This difference in growth is a sum of undefined interactions of different chemicals with the antibiotic. We extrapolated the contribution of two abundant pollutants in downstream sector: iron and phenanthrene. In the presence of selection pressure, iron at a concentration of 6.49 mg/L was found to foster the growth of resistant bacteria while phenanthrene at concentration of 160 μ g/L reduced the growth of the resistant strain. In addition, label free proteomics analysis showed that there are 64 proteins that were differentially expressed by the bacteria exposed to the upstream section versus the downstream sector. In the presence of oxytetracycline, at concentration of 160 μ g/mL, the differences reduced to only a few proteins, demonstrating that environmental stress impacts protein synthesis. Such mock studies contribute to our knowledge of chemicals that reduce growth of resistant strains and aids in the identification of selective biomarkers.

Salmonellosis: Serotypes, prevalence and multi-drug resistant profiles of Salmonella enterica in selected poultry farms, Kwara State, North Central Nigeria.

Salmonellosis is a major threat facing the poultry industry globally. This study was conducted to investigate the level of Salmonella contaminations and determine the resistance pattern of isolates obtained from selected poultry farms in Kwara State, a transition state between southern and northern regions of Nigeria. A total of 900 samples were collected between January and August 2017, from the poultry environment, apparently including healthy and dead birds. Salmonella was isolated and identified using standard bacteriological methods. All presumptive Salmonella isolates were serotyped and tested for antimicrobial susceptibility using 11 different antimicrobials. A total of 58 (6.4%) Salmonella isolates were obtained, and the isolation rate was only statistically significant (p < 0.05) in live birds. The isolates comprised of 13 serovars. The three predominant serovars, Salmonella enterica ser. 6.7:d:- (29.0%), Salmonella Agama (28.0%) and Salmonella Typhimurium (16.0%), were isolated from all three sample types. Rare serovars like Salmonella Albany, Salmonella Colindale, Salmonella Istanbul, Salmonella Larochelle, Salmonella Nigeria and Salmonella Orion were also isolated in this study. A high frequency of resistance was generally observed with all the isolates exhibiting a total of (100%) resistance to ampicillin, cefotaxime and ceftazidime. This study documents the first predominant isolation of S. enterica ser. 6.7:d:- and S. Agama from chickens. It also documents the high frequency of fluoroquinolone and cephalosporins resistance of the isolates indicating the presence of selective pressure in the environment. Controls and targeted interventions against Salmonella and the frequent occurrence of antimicrobial resistance in chickens should be initiated to prevent the spread of this organism.

Identifying anti-EGFR (EGFRi) response subgroups using evidence of ctDNA selective pressure.

3587 Background: Metastatic colorectal cancers (mCRC) that respond to EGFRi display a robust circulating tumor DNA (ctDNA) signature that reflects selective pressure and clonal evolution. Conversely, non-responding tumors do not exhibit this signature. On this basis, we developed a novel method that defines EGFRi sensitivity with improved biological confidence with fewer patients (pts), and does not rely on clinical trial outcomes where responses may be confounded by concurrent chemotherapy. We used this method to further elucidate the association of several features that have been previously reported to be associated with EGFRi resistance, namely tumor sidedness, BRAF, PIK3CA, or ERBB2 ( HER2) MTs, and the absence of APC/ TP53MT. Methods: We analyzed 112 pts with baseline tissue based RASWT mCRC who had progressed following EGFRi, and with plasma samples available for ctDNA sequencing using a blood based NGS assay. Using our previously validated EGFRi exposure signature, we identified pts with evidence of selective pressure. Results: Post EGFRi ctDNA found 37% and 33% of pts with left sided and transverse tumors displayed evidence of selective pressure, respectively. 0 pts with right sided tumors displayed evidence of selective pressure; p= 0.01. Similarly, BRAFV600EMT displayed no evidence of selective pressure vs 30% of WT pts; in contrast, selective pressure was evident in pts with PIK3CAMT, ERBB2MT and pts with absence of APC/TP53MT (42% vs 28%, 67% vs 28%, 24% vs 43%, respectively for MT vs WT, p= NS for all). BRAF, PIK3CA, ERBB2, and APC/TP53 MT were present in 4/117, 12/108, 3/118 and 30/91 pts, respectively. ctDNA shedding was similar for all subgroups, as was time from previous EGFRi, indicating that these factors were not confounders. Conclusions: Consistent with prior large randomized studies, no pts with right sided tumors or BRAFMT had evidence of biologic benefit as assessed by presence of selective pressure. In contrast a number of pts with transverse tumors, ERBB2MT, PIK3CAMT or absence of APC/TP53MT had evidence of EGFR selective pressure, confirming that these are not absolute predictors of EGFR resistance and suggesting a subset of these pts were deriving benefit from EGFR inhibition. This biology based approach has the potential to more efficiently evaluate biomarkers of targeted therapy in the future without reliance on large randomized datasets.

Transient AID expression for in situ mutagenesis with improved cellular fitness

Activation induced cytidine deaminase (AID) in germinal center B cells introduces somatic DNA mutations in transcribed immunoglobulin genes to increase antibody diversity. Ectopic expression of AID coupled with selection has been successfully employed to develop proteins with desirable properties. However, this process is laborious and time consuming because many rounds of selection are typically required to isolate the target proteins. AID expression can also adversely affect cell viability due to off target mutagenesis. Here we compared stable and transient expression of AID mutants with different catalytic activities to determine conditions for maximum accumulation of mutations with minimal toxicity. We find that transient (3–5 days) expression of an AID upmutant in the presence of selection pressure could induce a high rate of mutagenesis in reporter genes without affecting cells growth and expansion. Our findings may help improve protein evolution by ectopic expression of AID and other enzymes that can induce DNA mutations.

Effect of anthropogenic pollution on the fitness of tetracycline sensitive Shigella flexneri in Thames river water

Urban rivers may be source of antibiotics contamination that could support spread of antibiotic resistant bacteria (ARB) to the population. It is important to understand to what extent the presence of pollutants in urban rivers influences fitness of ARB. In an exercise to estimate this contribution, microcosms were generated from Thames river (London, UK) from different locations: upstream and downstream the city center. The concentration of the polycyclic aromatic hydrocarbons (PAHs) benzo(a)pyrene, pyrene and phenantrene was found to be 128, 171 and 128 times higher in downstream sector when compared to upstream sector, respectively. Filtered microcosms for each sector were enriched with tetracycline at lethal (10μg/mL) and sub-lethal (10ng/mL) concentrations and the fitness of an isogenic pair of Shigella flexneri 2a YSH6000 (tetR) and S. flexneri 2a 1363 (tetS) was then measured. In the presence of selective pressure in upstream microcosms, the resistant strain outcompeted the sensitive one, as expected. In contrast, sensitive S. flexneri tetS was found to significantly compete with resistant S. flexneri tetR at lethal concentrations of tetracycline in downstream microcosms, where levels of PAHs were the highest. Further experiments showed that PAHs rendered the resistant S. flexneri tetR ∼20% more sensitive to tetracycline. Sensitive S. flexneri tetS strain was able to persist at lethal concentration of tetracycline in downstream microcosms, at higher concentrations of PAHs. Our findings suggest that in a polluted river sensitive S. flexneri cells may still thrive in presence of selective pressure. Fitness tests provide an additional tool to measure bioavailability.

Transformation of the cucurbit powdery mildew pathogen Podosphaera xanthii by Agrobacterium tumefaciens.

The obligate biotrophic fungal pathogen Podosphaera xanthii is the main causal agent of powdery mildew in cucurbit crops all over the world. A major limitation of molecular studies of powdery mildew fungi (Erysiphales) is their genetic intractability. In this work, we describe a robust method based on the promiscuous transformation ability of Agrobacterium tumefaciens for reliable transformation of P.xanthii. The A.tumefaciens-mediated transformation (ATMT) system yielded transformants of P.xanthii with diverse transferred DNA (T-DNA) constructs. Analysis of the resultant transformants showed the random integration of T-DNA into the P.xanthii genome. The integrations were maintained in successive generations in the presence of selection pressure. Transformation was found to be transient, because in the absence of selection agent, the introduced genetic markers were lost due to excision of T-DNA from the genome. The ATMT system represents a potent tool for genetic manipulation of P.xanthii and will likely be useful for studying other biotrophic fungi. We hope that this method will contribute to the development of detailed molecular studies of the intimate interaction established between powdery mildew fungi and their host plants.

FISH-Based Analysis of Clonally Derived CHO Cell Populations Reveals High Probability for Transgene Integration in a Terminal Region of Chromosome 1 (1q13)

A basic goal in the development of recombinant proteins is the generation of cell lines that express the desired protein stably over many generations. Here, we constructed engineered Chinese hamster ovary cell lines (CHO-S) with a pCHO-hVR1 vector that carried an extracellular domain of a VEGF receptor (VR) fusion gene. Forty-five clones with high hVR1 expression were selected for karyotype analysis. Using fluorescence in situ hybridization (FISH) and G-banding, we found that pCHO-hVR1 was integrated into three chromosomes, including chromosomes 1, Z3 and Z4. Four clones were selected to evaluate their productivity under non-fed, non-optimized shake flask conditions. The results showed that clones 1 and 2 with integration sites on chromosome 1 revealed high levels of hVR1 products (shake flask of approximately 800 mg/L), whereas clones 3 and 4 with integration sites on chromosomes Z3 or Z4 had lower levels of hVR1 products. Furthermore, clones 1 and 2 maintained their productivity stabilities over a continuous period of 80 generations, and clones 3 and 4 showed significant declines in their productivities in the presence of selection pressure. Finally, pCHO-hVR1 localized to the same region at chromosome 1q13, the telomere region of normal chromosome 1. In this study, these results demonstrate that the integration of exogenous hVR1 gene on chromosome 1, band q13, may create a high protein-producing CHO-S cell line, suggesting that chromosome 1q13 may contain a useful target site for the high expression of exogenous protein. This study shows that the integration into the target site of chromosome 1q13 may avoid the problems of random integration that cause gene silencing or also overcome position effects, facilitating exogenous gene expression in CHO-S cells.

Prevalence of α+-Thalassemia in the Scheduled Tribe and Scheduled Caste Populations of Damoh District in Madhya Pradesh, Central India

This study was carried out to ascertain the allelic frequency of α+-thalassemia (α+-thal) in Scheduled caste and scheduled tribe populations of the Damoh district of Madhya Pradesh, India. Random blood samples of Scheduled tribe (267) and Scheduled caste (168), considering the family as a sampling unit, were analyzed for the presence of the –α3.7 (rightward) (NG_000006.1: g.34164_37967del3804) and –α4.2 (leftward) (AF221717) deletions. α+-Thal was significantly higher in the Scheduled tribals (77.9%) as compared to the scheduled caste population (9.0%). About 58.0% scheduled tribals carried at least one chromosome with the –α3.7 deletion and 20.0% scheduled tribals carried the –α4.2 deletion. Frequency for the –α3.7 allele was 0.487 in the scheduled tribal populations in comparison to 0.021 in scheduled castes. Allelic frequency for –α4.2 was 0.103 and 0.024, respectively, in the above communities. No Hardy-Weinberg equilibrium for α-thal gene (p < 0.05) was detected in the tribal population, indicating the presence of selection pressures in favor of α-thal mutation and adaptation.

Ecophysiological forecasting for environmental change adaptation

Ecophysiological forecasting for environmental change adaptation

Intrablastocyst injection with human CD34+/CD133+ cells increase survival of immunocompetent fumarylacetoacetate hydrolase knockout mice

Mice harbouring a humanized liver represent a powerful tool for translating preclinical studies of drug metabolism and pharmacokinetics into humans, as well as the exploitation of basic studies on liver pathophysiology including hepatitis C virus (HCV) infection. Human adult stem cells injected into immunocompetent mice at preimmune stages of development, generate chimeric animals harbouring a liver with relatively discrete foci of human hepatocyte-like cells. In this study, we have evaluated whether similar protocol of xenotransplantation in the presence of selective pressure might lead to a higher human-into-mouse liver repopulation, leading to a relevant improvement of liver function. Human CD34(+)/CD133(+) cells were microinjected into blastocysts from genetically-modified mice committed to develop a lethal hepatopathy, due to the absence of the enzyme fumarylacetoacetate hydrolase (FAH). Following xenotransplantation, mouse survival was followed over time and histochemical evidence of liver chimerism was assessed. The survival expectancy of seven out of 21 intrablastocyst xenotransplanted FAH knockout (Fah-/-) mice was significantly higher as compared with non-xenotransplanted mice. Several nodules of human hepatocyte-like cells were revealed by immunohistochemistry in the liver of rescued mice. Our data positively support the hypothesis that preimmune xenotransplantation of human stem cells into immunocompetent mice harbouring a lethal hepatic disease might lead to a functionally relevant human-mouse liver chimerism and marks a significant advancement towards the establishment of a novel translational preclinical model for liver diseases.

Occurrence and Characterization ofSalmonellaHiduddify from Chickens and Poultry Meat in Nigeria

The objective of this study was to determine the prevalence of Salmonella serovars and the antimicrobial susceptibility in chickens and poultry meat products in rural areas in Nigeria. The study was an observational cross-sectional investigation in which the target population included exotic and local chickens in Maiduguri main markets, chickens from farms, and free-range local chickens. A total of 865 samples were collected from feces, kidney, lungs, cecum, intestine, liver, heart, gizzard, and cloacal swabs from 525 different chickens. Salmonella was isolated from 130 of the samples. A stratified random sampling technique was used to select 41 isolates out of the 130 strains for serotyping, pulsed-field gel electrophoresis (PFGE), and antimicrobial susceptibility testing. Thirty-nine out of the 41 samples serotyped yielded Salmonella Hiduddify; two yielded a rough Salmonella serovar. The 39 Salmonella Hiduddify isolates and the two rough isolates were highly similar by PFGE typing, indicating that all of the isolates were of the same serovar. A low frequency of resistance was found among the isolates, except for resistance to ciprofloxacin for which 23 (56%) of the isolates tested exhibited resistance. This study documents for the first time the isolation of Salmonella Hiduddify in chickens and shows that this serovar is widespread in rural areas in Nigeria. It also documents a high frequency of fluoroquinone resistance in the isolates indicating the presence of selective pressure in the environment. Further studies should be conducted to reveal if the serovar is present in eggs and causes salmonellosis among the general population.

Production of 1,3-propanediol from Glycerol by Recombinant E. coli Using Incompatible Plasmids System

1,3-Propanediol (1,3-PD) has numerous applications in polymers, cosmetics, foods, lubricants, and medicines as a bifunctional organic compound. The genes for the production of 1,3-PD in Klebsiella pneumoniae, dhaB, which encodes glycerol dehydratase, and dhaT, which encodes 1,3-PD oxidoreductase, and gdrAB, which encodes glycerol dehydratase reactivating factor, are naturally under the control of different promoters and are transcribed in different directions. These genes were coexpressed in E. coli using two incompatible plasmids (pET28a and pET22b) in the presence of selective pressure. The recombinant E. coli coexpressed the glycerol dehydratase, 1,3-propanediol oxidoreductase and reactivating factor for the glycerol dehydratase at high levels. In a fed-batch fermentation of glycerol and glucose, the recombinant E. coli containing these two incompatible plasmids consumed 14.3 g/l glycerol and produced 8.6 g/l 1,3-propanediol. In the substitution case of yqhD (encoding alcohol dehydrogenase from E. coli) for dhaT, the final 1,3-propanediol concentration of the recombinant E. coli could reach 13.2 g/l.

Limitations to the Development of Humanized Antibody Producing Chinese Hamster Ovary Cells Using Glutamine Synthetase-Mediated Gene Amplification

Recombinant Chinese hamster ovary (CHO) cells expressing a humanized antibody were obtained by transfection of an antibody expression vector (pKC-GS-HC-huS) into CHO-K1 cells and subsequent glutamine synthetase (GS)-mediated gene amplification in media containing different concentrations of methionine sulfoximine (MSX). Concentrations consisted of 25, 200, 500, and 1000 microM of MSX. The highest producer (HP) subclones were isolated from each MSX level by the limiting dilution method and were characterized with respect to antibody production. No positive relationship was observed between specific antibody productivity (q(Ab)) and MSX concentration. Furthermore, it was found that the antibody production stability of these subclones was very poor even in the presence of selection pressure. During long-term cultures in the presence of the corresponding concentrations of MSX, q(Ab) of all HP subclones significantly decreased for the first six passages and thereafter stabilized. Southern and slot blot analyses showed that the loss of antibody gene copies was only partially responsible for the decreased q(Ab). Fluorescence in situ hybridization (FISH) analysis revealed some cytogenetic features indicative of antibody production instability. Unstable chromosomal structures including dicentrics, rings, and extremely long chromosomes were observed. Amplified sequences enclosed in nuclear projections were often observed. The telomeric repeat sequence, which may be involved in the stabilization of amplified arrays, was found to be absent at the ends of most marker chromosomes. Furthermore, FISH analysis revealed that the overall chromosome content was duplicated in some HP subclones. When metaphase of 12 high producing parental clones was examined, the frequency of occurrence of the polyploidy was 25%. Taken together, the data obtained here suggests that instability could be a concern in the development of CHO cells with GS-mediated gene amplification.

Increased Fidelity Reduces Poliovirus Fitness and Virulence under Selective Pressure in Mice

RNA viruses have high error rates, and the resulting quasispecies may aid survival of the virus population in the presence of selective pressure. Therefore, it has been theorized that RNA viruses require high error rates for survival, and that a virus with high fidelity would be less able to cope in complex environments. We previously isolated and characterized poliovirus with a mutation in the viral polymerase, 3D-G64S, which confers resistance to mutagenic nucleotide analogs via increased fidelity. The 3D-G64S virus was less pathogenic than wild-type virus in poliovirus-receptor transgenic mice, even though only slight growth defects were observed in tissue culture. To determine whether the high-fidelity phenotype of the 3D-G64S virus could decrease its fitness under a defined selective pressure, we compared growth of the 3D-G64S virus and 3D wild-type virus in the context of a revertible attenuating point mutation, 2C-F28S. Even with a 10-fold input advantage, the 3D-G64S virus was unable to compete with 3D wild-type virus in the context of the revertible attenuating mutation; however, in the context of a non-revertible version of the 2C-F28S attenuating mutation, 3D-G64S virus matched the replication of 3D wild-type virus. Therefore, the 3D-G64S high-fidelity phenotype reduced viral fitness under a defined selective pressure, making it likely that the reduced spread in murine tissue could be caused by the increased fidelity of the viral polymerase.

Field and soil microcosm studies on the survival and conjugation of a Pseudomonas putida strain bearing a recombinant plasmid, pADPTel.

Pseudomonas putida CR30RNS (pADPTel) is an antibiotic-resistant strain with a recombinant plasmid that confers resistance to tellurite and the ability to catabolize atrazine. The survival of this strain as well as its ability to transfer genes for atrazine degradation and tellurite resistance to indigenous soil bacteria were tested in both fallow soil and canola (Brassica napus) rhizosphere by the use of parallel field and laboratory releases. Culturable CR30RNS (pADPTel) were enumerated in field and microcosm soils at 7- to 14-day intervals over 49 d. Strain CR30RNS (pADPTel) survived for up to 7 weeks in microcosm soils at a density of 10(4) CFU/g soil, whereas in field soils the population declined to 10(3) CFU/g soil by the fourth week. In contrast, when CR30RNS (pADPTel) was introduced into the soil as a seed coating of canola (B. napus 'Karoo'), the bacterium established at higher cell densities in the rhizosphere (10(6)-10(5) CFU/g fresh root mass), with no subsequent decrease in numbers. The presence of selective pressure (i.e., atrazine) had no significant effect on the survival of CR30RNS (pADPTel) in either field or microcosm soils. One year postinoculation field sites were examined for the presence of CR30RNS (pADPTel) and no evidence of culturable parental cells was observed when samples were plated onto selective media. However, the atzC and telAB gene segments were amplified from the field soils at that time. Under laboratory conditions, indigenous soil bacteria were capable of receiving and expressing the engineered plasmid construct at frequencies ranging from 1 to 10(-3) transconjugants per donor. However, no plasmid transfer to indigenous soil bacteria was detected in the field or microcosm soils regardless of the presence of canola rhizosphere and (or) the application of atrazine. Our results show that the survival and population size of P. putida CR30RNS (pADPTel) might be sufficient for degradation of environmental pollutants but that the transfer frequency was too low to be detected under the conditions of this study.