Smoking and Radiation-induced Skin Injury: Analysis of a Multiracial, Multiethnic Prospective Clinical Trial.

INTRODUCTION: Smoking during breast radiotherapy (RT) may be associated with radiation-induced skin injury (RISI). We aimed to determine if a urinary biomarker of tobacco smoke exposure is associated with increased rates of RISI during and after breast RT. PATIENTS AND METHODS: Women with Stage 0-IIIA breast cancer treated with breast-conserving surgery or mastectomy followed by RT to the breast or chest wall with or without regional nodal irradiation were prospectively enrolled on a multicenter study assessing acute/late RISI. 980 patients with urinary cotinine (UCot) measurements (baseline and end-RT) were categorized into three groups. Acute and late RISI was assessed using the ONS Acute Skin Reaction scale and the LENT-SOMA Criteria. RESULTS: Late Grade 2+ and Grade 3+ RISI occurred in 18.2% and 1.9% of patients, respectively-primarily fibrosis, pain, edema, and hyperpigmentation. Grade 2+ late RISI was associated with UCot group (P= 006). Multivariable analysis identified UCot-based light smoker/secondhand smoke exposure (HR 1.79, P= .10) and smoking (HR 1.60, p = .06) as non-significantly associated with an increased risk of late RISI. Hypofractionated breast RT was associated with decreased risk of late RISI (HR 0.51, P=.03). UCot was not associated with acute RISI, multivariable analysis identified race, obesity, RT site/fractionation, and bra size to be associated with acute RISI. CONCLUSIONS: Tobacco exposure during breast RT may be associated with an increased risk of late RISI without an effect on acute toxicity. Smoking cessation should be encouraged prior to radiotherapy to minimize these and other ill effects of smoking.

Association Between Inflammatory Biomarker C-Reactive Protein and Radiotherapy-Induced Early Adverse Skin Reactions in a Multiracial/Ethnic Breast Cancer Population.

Purpose This study examined an inflammatory biomarker, high-sensitivity C-reactive protein (hsCRP), in radiotherapy (RT)-induced early adverse skin reactions or toxicities in breast cancer. Patients and Methods Between 2011 and 2013, 1,000 patients with breast cancer who underwent RT were evaluated prospectively for skin toxicities through the National Cancer Institute-funded Wake Forest University Community Clinical Oncology Program Research Base. Pre- and post-RT plasma hsCRP levels and Oncology Nursing Society skin toxicity criteria (0 to 6) were used to assess RT-induced skin toxicities. Multivariable logistic regression analyses were applied to ascertain the associations between hsCRP and RT-induced skin toxicities after adjusting for potential confounders. Results The study comprised 623 white, 280 African American, 64 Asian/Pacific Islander, and 33 other race patients; 24% of the patients were Hispanic, and 47% were obese. Approximately 42% and 15% of patients developed RT-induced grade 3+ and 4+ skin toxicities, respectively. The hsCRP levels differed significantly by race and body mass index but not by ethnicity. In multivariable analysis, grade 4+ skin toxicity was significantly associated with obesity (odds ratio [OR], 2.17; 95% CI, 1.41 to 3.34], post-RT hsCRP ≥ 4.11 mg/L (OR, 1.61; 95% CI, 1.07 to 2.44), and both factors combined (OR, 3.65; 95% CI, 2.18 to 6.14). Above-median post-RT hsCRP (OR, 1.93; 95% CI, 1.03 to 3.63), and change in hsCRP (OR, 2.80; 95% CI, 1.42 to 5.54) were significantly associated with grade 4+ skin toxicity in nonobese patients. Conclusion This large prospective study is the first to our knowledge of hsCRP as an inflammatory biomarker in RT-induced skin toxicities in breast cancer. We demonstrate that nonobese patients with elevated RT-related change in hsCRP levels have a significantly increased risk of grade 4+ skin toxicity. The outcomes may help to predict RT responses and guide decision making.

A Decellularized Porcine Xenograft-Derived Bone Scaffold for Clinical Use as a Bone Graft Substitute: A Critical Evaluation of Processing and Structure.

BACKGROUND: Bone grafts are used in approximately one half of all musculoskeletal surgeries. Autograft bone is the historic gold standard but is limited in supply and its harvest imparts significant morbidity to the patient. Alternative sources of bone graft include allografts, synthetics and, less commonly, xenografts which are taken from animal species. Xenografts are available in unlimited supply from healthy animal donors with controlled biology, avoiding the risk of human disease transmission, and may satisfy current demand for bone graft products. METHODS: In the current study, cancellous bone was harvested from porcine femurs and subjected to a novel decellularization protocol to derive a bone scaffold. RESULTS: The scaffold was devoid of donor cellular material on histology and DNA sampling (p < 0.01). Microarchitectural properties important for osteoconductive potential were preserved after decellularization as shown by high resolution imaging modalities. Proteomics data demonstrated similar profiles when comparing the porcine bone scaffold against commercially available human demineralized bone matrix approved for clinical use. CONCLUSION: We are unaware of any porcine-derived bone graft products currently used in orthopaedic surgery practice. Results from the current study suggest that porcine-derived bone scaffolds warrant further consideration to serve as a potential bone graft substitute.

Self-reported adherence and biomarker levels of CoQ10 and alpha-tocopherol.

PURPOSE: Women with breast cancer were randomized to receive coenzyme Q10 (CoQ10) plus Vitamin E or placebo in a clinical trial. The objective of this evaluation is to examine the association between participant self-reported adherence to the study supplements and changes in plasma biomarker levels. PATIENTS AND METHODS: Correlation coefficients quantified the association between changes in alpha-tocopherol and CoQ10 levels and the association between self-reported adherence and changes in biomarkers. Participants were categorized by self-reported adherence; Kruskal- Wallis tests compared changes in alpha-tocopherol and CoQ10 levels between self-reported adherence groups. RESULTS: Women (N=155) provided baseline and post-treatment biomarkers; 147 completed at least one diary. While changes in alpha-tocopherol and CoQ10 levels were moderately correlated, correlations ranged from 0.40 to 0.48, association between self-reported adherence and plasma alpha-tocopherol or CoQ10 levels was weak; correlations ranged from 0.10 to 0.29 at weeks 8, 16, and 24. Some participants with high self-reported adherence actually had decreases in their biomarker levels. CONCLUSION: These findings support that self-reported adherence is likely to be overestimated. Biological and other measures of adherence that can better identify true adherence to study pills provided in clinical trials are greatly needed as they may assist in improving the interpretation of findings of future clinical trials.

The Development of a Xenograft-Derived Scaffold for Tendon and Ligament Reconstruction Using a Decellularization and Oxidation Protocol.

PURPOSE: To evaluate the biological, immunological, and biomechanical properties of a scaffold derived by architectural modification of a fresh-frozen porcine patella tendon using a decellularization protocol that combines physical, chemical, and enzymatic modalities. METHODS: Porcine patellar tendons were processed using a decellularization and oxidation protocol that combines physical, chemical, and enzymatic modalities. Scaffolds (n = 88) were compared with native tendons (n = 70) using histologic, structural (scanning electron microscopy, porosimetry, and tensile testing), biochemical (mass spectrometry, peracetic acid reduction, DNA quantification, alpha-galactosidase [α-gal] content), as well as in vitro immunologic (cytocompatibility, cytokine induction) and in vivo immunologic nonhuman primate analyses. RESULTS: A decrease in cellularity based on histology and a significant decrease in DNA content were observed in the scaffolds compared with the native tendon (P < .001). Porosity and pore size were increased significantly (P < .001). Scaffolds were cytocompatible in vitro. There was no difference between native tendons and scaffolds when comparing ultimate tensile load, stiffness, and elastic modulus. The α-gal xenoantigen level was significantly lower in the decellularized scaffold group compared with fresh-frozen, nondecellularized tissue (P < .001). The in vivo immunological response to implanted scaffolds measured by tumor necrosis factor-α and interleukin-6 levels was significantly (P < .001) reduced compared with untreated controls in vitro. These results were confirmed by an attenuated response to scaffolds in vivo after implantation in a nonhuman primate model. CONCLUSIONS: Porcine tendon was processed via a method of decellularization and oxidation to produce a scaffold that possessed significantly less inflammatory potential than a native tendon, was biocompatible in vitro, of increased porosity, and with significantly reduced amounts of α-gal epitope while retaining tensile properties. CLINICAL RELEVANCE: Porcine-derived scaffolds may provide a readily available source of material for musculoskeletal reconstruction and repair while eliminating concerns regarding disease transmission and the morbidity of autologous harvest.

Optically Triggered Immune Response through Photocaged Oligonucleotides.

Bacterial and viral CpG oligonculeotides are unmethylated cytosine-phosphate-guanosine dinucleotide sequences and trigger an innate immune response through activation of the toll-like receptor 9 (TLR9). We have developed synthetic photocaged CpGs via site-specific incorporation of nitropiperonyloxymethyl (NPOM)-caged thymidine residues. These oligonucleotides enable the optical control of TLR9 function and thereby provide light-activation of an immune response. We provide a proof-of-concept model by applying a reporter assay in live cells and by quantification of endogenous production of interleukin 6.

Induction of robust type-I CD8+ T-cell responses in WHO grade 2 low-grade glioma patients receiving peptide-based vaccines in combination with poly-ICLC.

PURPOSE: WHO grade 2 low-grade gliomas (LGG) with high risk factors for recurrence are mostly lethal despite current treatments. We conducted a phase I study to evaluate the safety and immunogenicity of subcutaneous vaccinations with synthetic peptides for glioma-associated antigen (GAA) epitopes in HLA-A2(+) adults with high-risk LGGs in the following three cohorts: (i) patients without prior progression, chemotherapy, or radiotherapy (RT); (ii) patients without prior progression or chemotherapy but with prior RT; and (iii) recurrent patients. EXPERIMENTAL DESIGN: GAAs were IL13Rα2, EphA2, WT1, and Survivin. Synthetic peptides were emulsified in Montanide-ISA-51 and given every 3 weeks for eight courses with intramuscular injections of poly-ICLC, followed by q12 week booster vaccines. RESULTS: Cohorts 1, 2, and 3 enrolled 12, 1, and 10 patients, respectively. No regimen-limiting toxicity was encountered except for one case with grade 3 fever, fatigue, and mood disturbance (cohort 1). ELISPOT assays demonstrated robust IFNγ responses against at least three of the four GAA epitopes in 10 and 4 cases of cohorts 1 and 3, respectively. Cohort 1 patients demonstrated significantly higher IFNγ responses than cohort 3 patients. Median progression-free survival (PFS) periods since the first vaccine are 17 months in cohort 1 (range, 10-47+) and 12 months in cohort 3 (range, 3-41+). The only patient with large astrocytoma in cohort 2 has been progression-free for more than 67 months since diagnosis. CONCLUSION: The current regimen is well tolerated and induces robust GAA-specific responses in WHO grade 2 glioma patients. These results warrant further evaluations of this approach. Clin Cancer Res; 21(2); 286-94. ©2014 AACR.

Site-specific DNA-doxorubicin conjugates display enhanced cytotoxicity to breast cancer cells.

Doxorubicin (Dox) is widely used for breast cancer treatment but causes serious side effects including cardiotoxicity that may adversely impact patient lifespan even if treatment is successful. Herein, we describe selective conjugation of Dox to a single site in a DNA hairpin resulting in a highly stable complex that enables Dox to be used more effectively. Selective conjugation of Dox to G15 in the hairpin loop was verified using site-specific labeling with [2-(15)N]-2'-deoxyguanosine in conjunction with [(1)H-(15)N] 2D NMR, while 1:1 stoichiometry for the conjugate was validated by ESI-QTOF mass spectrometry and UV spectroscopy. Molecular modeling indicated covalently bound Dox also intercalated into the stem of the hairpin and stability studies demonstrated the resulting Dox-conjugated hairpin (DCH) complex had a half-life >30 h, considerably longer than alternative covalent and noncovalent complexes. Secondary conjugation of DCH with folic acid (FA) resulted in increased internalization into breast cancer cells. The dual conjugate, DCH-FA, can be used for safer and more effective chemotherapy with Dox and this conjugation strategy can be expanded to include additional anticancer drugs.

Optochemical control of RNA interference in mammalian cells.

Short interfering RNAs (siRNAs) and microRNAs (miRNAs) have been widely used in mammalian tissue culture and model organisms to selectively silence genes of interest. One limitation of this technology is the lack of precise external control over the gene-silencing event. The use of photocleavable protecting groups installed on nucleobases is a promising strategy to circumvent this limitation, providing high spatial and temporal control over siRNA or miRNA activation. Here, we have designed, synthesized and site-specifically incorporated new photocaged guanosine and uridine RNA phosphoramidites into short RNA duplexes. We demonstrated the applicability of these photocaged siRNAs in the light-regulation of the expression of an exogenous green fluorescent protein reporter gene and an endogenous target gene, the mitosis motor protein, Eg5. Two different approaches were investigated with the caged RNA molecules: the light-regulation of catalytic RNA cleavage by RISC and the light-regulation of seed region recognition. The ability to regulate both functions with light enables the application of this optochemical methodology to a wide range of small regulatory RNA molecules.

Cellular delivery and photochemical activation of antisense agents through a nucleobase caging strategy.

Antisense oligonucleotides are powerful tools to regulate gene expression in cells and model organisms. However, a transfection or microinjection is typically needed for efficient delivery of the antisense agent. We report the conjugation of multiple HIV TAT peptides to a hairpin-protected antisense agent through a light-cleavable nucleobase caging group. This conjugation allows for the facile delivery of the antisense agent without a transfection reagent, and photochemical activation offers precise control over gene expression. The developed approach is highly modular, as demonstrated by the conjugation of folic acid to the caged antisense agent. This enabled targeted cell delivery through cell-surface folate receptors followed by photochemical triggering of antisense activity. Importantly, the presented strategy delivers native oligonucleotides after light-activation, devoid of any delivery functionalities or modifications that could otherwise impair their antisense activity.

A randomized, double-blind, placebo-controlled study of oral coenzyme Q10 to relieve self-reported treatment-related fatigue in newly diagnosed patients with breast cancer.

BACKGROUND: Coenzyme Q10 (CoQ10) is a common antioxidant supplement with known cardioprotective effects and potential anticancer benefits. OBJECTIVES: We performed a randomized, double-blind, placebo-controlled study of oral CoQ10 in female breast cancer patients with the primary objective of determining CoQ10's effects on self-reported fatigue, depression, and quality of life (QOL). Methods Eligible women with newly diagnosed breast cancer and planned adjuvant chemotherapy were randomized to oral supplements of 300 mg CoQ10 or placebo, each combined with 300 IU vitamin E, divided into 3 daily doses. Treatment was continued for 24 weeks. Blood tests, QOL measures, and levels of plasma CoQ10 and vitamin E were obtained at baseline and at 8, 16, and 24 weeks. Mixed-effects models were used to assess treatment differences in outcomes over time. RESULTS: Between September 2004 and March 2009, 236 women were enrolled. Treatment arms were well balanced with respect to age (range, 28-85 years), pathologic stage (stage 0, 91%; stage 1, 8%; stage II, 1%), ethnicity (white, 87%; black, 11%; Hispanic, 2%), and planned therapy. Baseline CoQ10 levels in the CoQ10 and placebo arms were 0.70 and 0.73 microg/mL, respectively; the 24-week CoQ10 levels were 1.83 and 0.79 microg/mL, respectively. There were no significant differences between the CoQ10 and placebo arms at 24 weeks for scores on the Profile of Mood States-Fatigue questionnaire (least squares means, 7.08 vs 8.24, P = .257), the Functional Assessment of Chronic Illness Therapy-Fatigue tool (37.6 vs 37.6, P = .965), the Functional Assessment of Cancer Therapy-Breast Cancer instrument (111.9 vs 110.4, P = .577), or the Center for Epidemiologic Studies-Depression scale (11.6 vs 12.3, P = .632). CONCLUSIONS: Supplementation with conventional doses of CoQ10 led to sustained increases in plasma CoQ10 levels but did not result in improved self-reported fatigue or QOL after 24 weeks of treatment.

EphrinA1 is released in three forms from cancer cells by matrix metalloproteases.

EphrinA1 is a glycosylphosphatidylinositol (GPI)-linked ligand for the EphA2 receptor, which is overexpressed in glioblastoma (GBM), among other cancers. Activation of the receptor by ephrinA1 leads to a suppression of oncogenic properties of GBM cells. We documented that a monomeric functional form of ephrinA1 is released from cancer cells and thus explored the mechanism of ephrinA1 release and the primary protein sequence. We demonstrate here that multiple metalloproteases (MMPs) are able to cleave ephrinA1, most notably MMP-1, -2, -9, and -13. The proteolytic cleavage that releases ephrinA1 occurs at three positions near the C terminus, producing three forms ending in valine-175, histidine-177, or serine-178. Moreover, deletion of amino acids 174 to 181 or 175 to 181 yields ephrinA1 that is still GPI linked but not released by proteolysis, underlining the necessity of amino acids 175 to 181 for release from the membrane. Furthermore, recombinant ephrinA1 ending at residue 175 retains activity toward the EphA2 receptor. These findings suggest a mechanism of release and provide evidence for the existence of several forms of monomeric ephrinA1. Moreover, ephrinA1 should be truncated at a minimum at amino acid 175 in fusions or conjugates with other molecules in order to prevent likely proteolysis within physiological and pathobiological environments.

Regulation of transcription through light-activation and light-deactivation of triplex-forming oligonucleotides in mammalian cells.

Triplex-forming oligonucleotides (TFOs) are efficient tools to regulate gene expression through the inhibition of transcription. Here, nucleobase-caging technology was applied to the temporal regulation of transcription through light-activated TFOs. Through site-specific incorporation of caged thymidine nucleotides, the TFO:DNA triplex formation is blocked, rendering the TFO inactive. However, after a brief UV irradiation, the caging groups are removed, activating the TFO and leading to the inhibition of transcription. Furthermore, the synthesis and site-specific incorporation of caged deoxycytidine nucleotides within TFO inhibitor sequences was developed, allowing for the light-deactivation of TFO function and thus photochemical activation of gene expression. After UV-induced removal of the caging groups, the TFO forms a DNA dumbbell structure, rendering it inactive, releasing it from the DNA, and activating transcription. These are the first examples of light-regulated TFOs and their application in the photochemical activation and deactivation of gene expression. In addition, hairpin loop structures were found to significantly increase the efficacy of phosphodiester DNA-based TFOs in tissue culture.

Photochemical control of DNA decoy function enables precise regulation of nuclear factor κB activity.

DNA decoys have been developed for the inhibition of transcriptional regulation of gene expression. However, the present methodology lacks the spatial and temporal control of gene expression that is commonly found in nature. Here, we report the application of photoremovable protecting groups on nucleobases of nuclear factor κB (NF-κB) DNA decoys to regulate NF-κB-driven transcription of secreted alkaline phosphatase using light as an external control element. The NF-κB family of proteins is comprised of important eukaryotic transcription factors that regulate a wide range of cellular processes and are involved in immune response, development, cellular growth, and cell death. Several diseases, including cancer, arthritis, chronic inflammation, asthma, neurodegenerative diseases, and heart disease, have been linked to constitutively active NF-κB. Through the direct incorporation of caging groups into an NF-κB decoy, we were able to disrupt DNA:DNA hybridization and inhibit the binding of the transcription factor to the DNA decoy until UV irradiation removed the caging groups and restored the activity of the oligonucleotide. Excellent light-switching behavior of transcriptional regulation was observed. This is the first example of a caged DNA decoy for the photochemical regulation of gene expression in mammalian cells and represents an important addition to the toolbox of light-controlled gene regulatory agents.

Evidence for a novel serum factor distinct from zinc alpha-2 glycoprotein that promotes body fat loss early in the development of cachexia.

We provide evidence that a factor other than the previously identified lipid mobilizing factor, zinc alpha-2 glycoprotein, promotes lipolysis in the MCA-induced sarcoma-bearing cachexia model. Cachexia is characterized by progressive loss of adipose tissue and skeletal muscle without a concurrent increase in food intake to restore lost tissue stores. We compared tumor-bearing ad lib fed (TB) animals to nontumor bearing ad lib fed (NTB) animals or nontumor-bearing pair-fed (PF) animals at various time points throughout development of tumor derived cachexia. Prior to cachexia, the TB animals lost more than 10 +/- 0.7% of their body fat before losing protein mass and decreasing their food intake. Fat loss occurred because adipocyte size, not number, was reduced. Increased turnover of palmitate and significantly higher serum triglyceride levels prior to cachexia were further indicators of an early loss of lipid from the adipocytes. Yet, circulating levels of norepinephrine, epinephrine, TNF-alpha, and zinc alpha-2 glycoprotein were not increased prior to the loss of fat mass. We provide evidence for a serum factor(s), other than zinc alpha-2 glycoprotein, that stimulates release of glycerol from 3T3-L1 adipocytes and promotes the loss of stored adipose lipid prior to the loss of lean body mass in this model.

Activation and deactivation of DNAzyme and antisense function with light for the photochemical regulation of gene expression in mammalian cells.

The photochemical regulation of biological systems represents a very precise means of achieving high-resolution control over gene expression in both a spatial and a temporal fashion. DNAzymes are enzymatically active deoxyoligonucleotides that enable the site-specific cleavage of RNA and have been used in a variety of in vitro applications. We have previously reported the photochemical activation of DNAzymes and antisense agents through the preparation of a caged DNA phosphoramidite and its site-specific incorporation into oligonucleotides. The presence of the caging group disrupts either DNA:RNA hybridization or catalytic activity until removed via a brief irradiation with UV light. Here, we are expanding this concept by investigating the photochemical deactivation of DNAzymes and antisense agents. Moreover, we report the application of light-activated and light-deactivated antisense agents to the regulation of gene function in mammalian cells. This represents the first example of gene silencing antisense agents that can be turned on and turned off in mammalian tissue culture.

Photochemical regulation of restriction endonuclease activity.

Removal by the light: The photochemical regulation of restriction endonucleases, which are important enzymes in molecular biology, has been investigated. Photolabile protecting groups have been installed on DNA substrates and have been demonstrated to inhibit restriction endonuclease activity until removed by UV light irradiation. Interestingly, these groups do not appear to dramatically affect initial binding of the enzyme to the DNA substrate, but rather prevent recognition of the specific cleavage site.

Noncatalytic interactions between glutathione S-transferases and nitroalkene fatty acids modulate nitroalkene-mediated activation of peroxisomal proliferator-activated receptor gamma.

The naturally occurring nitroalkenes, nitrolinoleic (NO(2)-LA) and nitrooleic (NO(2)-OA) acids, are among the most potent endogenous ligand activators of PPARgamma-dependent transcription. In order to understand mechanisms that regulate cellular response to these nitroalkenes, we previously demonstrated that glutathione conjugation of NO(2)-LA and MRP1-mediated efflux of the conjugates were associated with significant attenuation of PPARgamma activation by this nitroalkene [(2006) Biochemistry 45, 7889-7896]. Here we show that NO(2)-OA activation of PPARgamma is similarly affected by nonenzymatic conjugation and MRP1-mediated efflux. Moreover, the roles of glutathione S-transferases (GSTs) in the glutathione conjugation and bioactivities of NO(2)-LA and NO(2)-OA were investigated. While none of the GST isozymes tested (GSTA1-1, A4-4, M1a-1a, and P1a-1a) enhanced the rate of glutathione conjugation, expression of GSTA1-1, M1a-1a, or P1a-1a in MCF7 cells significantly reduced the magnitude of PPARgamma-dependent reporter gene transcription in response to NO(2)-LA and NO(2)-OA treatment, with GSTP1a-1a expression mediating the most potent inhibition of PPARgamma. Although these GSTs failed to catalyze nitroalkene conjugation with glutathione, the nitroalkenes were found to associate avidly with all four GST isozymes as indicated by their ability to inhibit GST activity with K(i)'s in the nanomolar range. Treatment of purified GSTP1a-1a with excess NO(2)-LA and NO(2)-OA resulted in the formation of covalent adducts between GSTP1a monomers and nitroalkenes, although separate experiments indicated that such covalent bond formation was not necessary for avid GST-nitroalkene interactions. These results suggest that GSTs can inhibit the activation of transcription by nitroalkenes via noncatalytic sequestration of these ligands, and their glutathione conjugates, away from their nuclear target, PPARgamma.

Restriction enzyme-free mutagenesis via the light regulation of DNA polymerization.

The effects of photocaged nucleosides on the DNA polymerization reaction was investigated, finding that most polymerases are unable to recognize and read through the presence of a single caging group on the DNA template. Based on this discovery, a new method of introducing mutations into plasmid DNA via a light-mediated mutagenesis protocol was developed. This methodology is advantageous over several common approaches in that it requires the use of only two polymerase chain reaction primers, and does not require any restriction sites or use of restriction enzymes. Additionally, this approach enables not only site-directed mutations, but also the insertion of DNA strands of any length into plasmids and the deletion of entire genes from plasmids.