Simplifying intensity-modulated radiotherapy plans with fewer beam angles for the treatment of oropharyngeal carcinoma.

The first aim of the present study was to investigate the feasibility of using fewer beam angles to improve delivery efficiency for the treatment of oropharyngeal cancer (OPC) with inverse-planned intensity-modulated radiation therapy (IP-IMRT). A secondary aim was to evaluate whether the simplified IP-IMRT plans could reduce the indirect radiation dose. The treatment plans for 5 consecutive OPC patients previously treated with a forward-planned IMRT (FP-IMRT) technique were selected as benchmarks for this study. The initial treatment goal for these patients was to deliver 70 Gy to > or = 95% of the planning gross tumor volume (PTV-70) and 59.4 Gy to > or = 95% of the planning clinical tumor volume (PTV-59.4) simultaneously. Each case was re-planned using IP-IMRT with multiple beam-angle arrangements, including four complex IP-IMRT plans using 7 or more beam angles, and one simple IMRT plan using 5 beam angles. The complex IP-IMRT plans and simple IP-IMRT plans were compared to each other and to the FPIMRT plans by analyzing the dose coverage of the target volumes, the plan homogeneity, the dose-volume histograms of critical structures, and the treatment delivery parameters including delivery time and the total number of monitor units (MUs). When comparing the plans, we found no significant difference between the complex IP-IMRT, simple IP-IMRT, and FP-IMRT plans for tumor target coverage (PTV-70: p = 0.56; PTV-59.4: p = 0.20). The plan homogeneity, measured by the mean percentage isodose, did not significantly differ between the IP-IMRT and FP-IMRT plans (p = 0.08), although we observed a trend toward greater inhomogeneity of dose in the simple IP-IMRT plans. All IP-IMRT plans either met or exceeded the quality of the FP-IMRT plans in terms of dose to adjacent critical structures, including the parotids, spinal cord, and brainstem. As compared with the complex IP-IMRT plans, the simple IP-IMRT plans significantly reduced the mean treatment time (maximum probability for four pairwise comparisons: p = 0.0003). In conclusion, our study demonstrates that, as compared with complex IP-IMRT, simple IP-IMRT can significantly improve treatment delivery efficiency while maintaining similar target coverage and sparing of critical structures. However, the improved efficiency does not significantly reduce the total number of MUs nor the indirect radiation dose

The fabM Gene Product of Streptococcus mutans Is Responsible for the Synthesis of Monounsaturated Fatty Acids and Is Necessary for Survival at Low pH

Previously, it has been demonstrated that the membrane fatty acid composition of Streptococcus mutans is affected by growth pH (E. M. Fozo and R. G. Quivey, Jr., Appl. Environ. Microbiol. 70:929-936, 2004; R. G. Quivey, Jr., R. Faustoferri, K. Monahan, and R. Marquis, FEMS Microbiol. Lett. 189:89-92, 2000). Specifically, the proportion of monounsaturated fatty acids increases when the organism is grown in acidic environments; if the shift to increased monounsaturated fatty acids is blocked by the addition of a fatty acid biosynthesis inhibitor, the organism is rendered more acid sensitive (E. M. Fozo and R. G. Quivey, Jr., Appl. Environ. Microbiol. 70:929-936, 2004). Recently, work with Streptococcus pneumoniae has identified a novel enzyme, FabM, responsible for the production of monounsaturated fatty acids (H. Marrakchi, K. H. Choi, and C. O. Rock, J. Biol. Chem. 277:44809-44816, 2002). Using the published S. pneumoniae sequence, a putative FabM was identified in the S. mutans strain UA159. We generated a fabM strain that does not produce unsaturated fatty acids as determined by gas chromatography of fatty acid methyl esters. The mutant strain was extremely sensitive to low pH in comparison to the wild type; however, the acid-sensitive phenotype was relieved by growth in the presence of long-chain monounsaturated fatty acids or through genetic complementation. The strain exhibited reduced glycolytic capability and altered glucose-PTS activity. In addition, the altered membrane composition was more impermeable to protons and did not maintain a normal ΔpH. The results suggest that altered membrane composition can significantly affect the acid survival capabilities, as well as several enzymatic activities, of S. mutans

Shifts in the Membrane Fatty Acid Profile of Streptococcus mutans Enhance Survival in Acidic Environments

Acid adaptation of Streptococcus mutans UA159 involves several different mechanisms, including the ability to alter its proportion of long-chain, monounsaturated membrane fatty acids (R. G. Quivey, Jr., R. Faustoferri, K. Monahan, and R. Marquis, FEMS Microbiol. Lett. 189:89-92, 2000). In the present study, we examined the mechanism and timing of changes in fatty acid ratios and the potential benefit that an increased proportion of long-chained fatty acids has for the organism during growth at low pH. Cells taken from steady-state cultures at intermediate pH values of 6.5, 6, and 5.5 showed incremental changes from the short-chained, saturated membrane fatty acid profile normally seen in pH 7 cultures to the long-chained, monounsaturated fatty acids more typically observed in acidic cultures (pH 5). Our observations showed that the bacterium was capable of effecting the majority of changes in approximately 20 min, far less than one generation time. However, reversion to the distribution of fatty acids seen in cells growing at a pH of 7 required a minimum of 10 generations. Fatty acid composition analysis of cells taken from cultures treated with chloramphenicol suggested that the changes in fatty acid distribution did not require de novo protein synthesis. Cells treated with the fatty acid biosynthesis inhibitor cerulenin were unable to alter their membrane fatty acid profiles and were unable to survive severe acidification. Results presented here indicate that membrane fatty acid redistribution is important for low pH survival and, as such, is a component of the S. mutans acid-adaptation arsenal

Role of Aminotransferases in Branched-Chain Amino Acid Metabolism: Acid Tolerance and Fatty Acid Synthesis in Streptococcus mutans

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Microbiology and Immunology, 2012.Streptococcus mutans relies on a variety of adaptive mechanisms to successfully colonize tooth surfaces in the human oral cavity and to become a dominant species in dental plaque. Organic acid production, arising from sugar metabolism, results in the accumulation of end‐products that damage tooth surfaces. Previous studies have shown that survival of S. mutans in under acidic conditions is predicated on an acid‐adaptive response. Branched‐chain amino acid (bcAA) metabolism is one of the pathways thought to be important for mitigating acidification. Synthesis of bcAAs allows S. mutans to reroute pyruvate, generate substrates for fatty acid synthesis, modulate carbon flow, and alter gene expression in response to its physiological needs. To elucidate the role of bcAA metabolism in the acid adaptive response of S. mutans, a strain carrying a mutation in the branched‐chain amino acid aminotransferase, ilvE, was characterized. Physiological and transcriptional studies demonstrated that ilvE is regulated by pH and by the global transcriptional regulator CodY. CodY acts a repressor of ilvE transcription, mediated through a physical interaction between the ilvE promoter and CodY protein, via a consensus‐binding domain. Regulation of ilvE is dependent on both CodY and physiological levels of branched‐chain amino acids, which act as signaling molecules to enhance binding affinity of CodY. The carbon catabolite regulator, CcpA, was also demonstrated to regulate ilvE in a positive manner, by activating ilvE transcription. The role of amino acid metabolism in branched‐chain fatty acid (bcFA) synthesis was also determined. Degradation of bcAAs provides the substrates for bcFA synthesis, since increased levels of ATase activity correlated with strains whose membrane fatty acid composition contained bcFAs. Although bcFAs were hypothesized to be a compensatory mechanism, detailed membrane fatty acid analysis demonstrated otherwise, since S. mutans was shown to be unable to incorporate bcFAs within its membrane. The results from this study demonstrated that branched‐chain amino acid metabolism plays an important role in providing the substrates necessary for the changes in gene expression required during acid adaptation and for the synthesis of bcFAs in S. mutans

DNA Base Excision Repair: a Network of Defense, Mutagenesis, and Tolerance in Streptococcus mutans

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Microbiology and Immunology, 2012.The oral pathogen Streptococcus mutans possesses inducible DNA repair defenses for protection against pH fluctuations and reactive oxygen metabolites, such as hydrogen peroxide (H2O2), which occur in the oral cavity. DNA base excision repair (BER) plays a critical role in maintaining genomic integrity by preventing the accumulation of genetic mutations associated with environmental insult. In the present study, we examined the biological consequences that result from compromising the DNA glycosylases (Fpg and MutY) and endonucleases (Smx and Smn), of the BER pathway, and their relative role in stress-adaptation and virulence of the organism. Enzymatic characterization of the BER system revealed its protective role in dealing with oxidized DNA damage; specifically for detecting and repairing oxidized bases and AP sites. In vitro analysis revealed a complex interaction of the BER enzymes in protecting the cell against mutagenic lesions and the plethora of biological responses to DNA damage. S. mutans strains lacking a functional Fpg, MutY, or Smn showed elevated spontaneous mutation frequencies and these mutator phenotypes correlated with the ability of the strains to survive killing by acid and oxidative agents. In addition, use of the G. mellonella virulence model showed that loss of BER resulted in elevated virulence, as compared to other strains tested. The data indicate that, for S. mutans, mutator phenotypes arising from the loss of BER enzymes may confer advantage to the organism’s virulence potential. Finally, we determined the effects of acidification on genomic stability in S. mutans. The results indicated that chronic exposure to acid results in increased genomic instability, abnormal mutability, and temporary resistance to oxidative stress environments due to functional loss of DNA repair defenses. In summary, the present work demonstrates that the BER system can contribute to the regulation of many aspects of the biological networks in S. mutans that include DNA repair, mutagenesis, biofilms, adaptation, survival, persistence, tolerance to DNA-damaging agents, and virulence. This study provides a more complete perspective of the contributions of DNA repair in S. mutans, facilitating our understanding of the role of these enzymes in adaptation and virulence

Genetic and Biochemical Characterization of the F-ATPase Operon from Streptococcus sanguis 10904

Oral streptococci utilize an F-ATPase to regulate cytoplasmic pH. Previous studies have shown that this enzyme is a principal determinant of aciduricity in the oral streptococcal species Streptococcus sanguis and Streptococcus mutans. Differences in the pH optima of the respective ATPases appears to be the main reason that S. mutans is more tolerant of low pH values than S. sanguis and hence pathogenic. We have recently reported the genetic arrangement for the S. mutans operon. For purposes of comparative structural biology we have also investigated the F-ATPase from S. sanguis. Here, we report the genetic characterization and expression in Escherichia coli of the S. sanguis ATPase operon. Sequence analysis showed a gene order of atpEBFHAGDC and that a large intergenic space existed upstream of the structural genes. Activity data demonstrate that ATPase activity is induced under acidic conditions in both S. sanguis and S. mutans; however, it is not induced to the same extent in the nonpathogenic S. sanguis. Expression studies with an atpD deletion strain of E. coli showed that S. sanguis-E. coli hybrid enzymes were able to degrade ATP but were not sufficiently functional to permit growth on succinate minimal media. Hybrid enzymes were found to be relatively insensitive to inhibition by dicyclohexylcarbodiimide, indicating loss of productive coupling between the membrane and catalytic subunits

Acid and Oxidative Stress Responses in Streptococcus mutans

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Microbiology and Immunology, 2016.S. mutans, the Gram-positive opportunistic oral pathogen most frequently associated with the development of dental caries, uses a robust acid-adaptive response to avoid becoming a victim of its own acidogenic metabolism. S. mutans also encounters oxidative stress, stemming from oxygen gas present in the oral cavity, as well as bacteriocidal amounts of hydrogen peroxide, produced by other oral streptococci competing for the same ecological niche. In this study, the responses of S. mutans to acid stress, oxidative stress, and both stresses experienced concurrently were characterized at the transcriptomic level using cDNA microarray analysis. These studies have provided insight into the adjustments S. mutans makes as a continuous culture begins growth at neutral pH, experiences glucose-shock and a rapid pH drop, and transitions into steady-state growth at low pH, similar to conditions in the oral cavity following intake of host dietary carbohydrates and maturation of dental plaque. Oxidative stress was provided through addition of oxygen gas to the continuous culture or through deletion of the oxygen-metabolizing enzyme, Nox. The acid and oxidative stress responses appear to be synergistic, as during these conditions, levels of numerous transcripts were elevated several fold higher than the additive result of both stresses experienced independently. Study of the S. mutans transcriptome during oxidative stress has also led to the discovery of a novel regulatory loop, involving nox and the transcriptional regulators SpxA and Rex. In addition, the loss of nox unexpectedly resulted in a greatly elevated ratio of free, intracellular NAD+/NADH. This shift was at least in part due to Rex-mediated up-regulation of lactate dehydrogenase. Importantly, the loss of nox rendered S. mutans defective in its ability to compete directly with two species of commensal Streptococci, suggesting a role for nox in the pathogenic potential of this organism. The importance of all three genes in the tre operon during growth on trehalose was also confirmed and initial work characterizing the regulon of the TreR regulator is presented

The Role of PlsX in Fatty Acid Synthesis and Acid Adaptation in Streptococcus mutans

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Microbiology & Immunology, 2016.Streptococcus mutans is one of the primary causative agents of dental caries in humans. S. mutans ferments dietary sugars in the mouth to produce organic acids. These acids lower local pH values resulting in demineralization of the tooth enamel, leading to caries. To survive acidic environments, S. mutans employs several adaptive mechanisms, including a shift from saturated to unsaturated fatty acids in membrane phospholipids. Evidence suggests that this shift requires de novo fatty acid and phospholipid synthesis; therefore, understanding these synthesis pathways is crucial for understanding how S. mutans adapts to low pH and causes caries. PlsX is an acyl- ACP:phosphate transacylase that links the fatty acid synthesis pathway to the phospholipid synthesis pathway, and is central to the movement of unsaturated fatty acids into the membrane. It has recently been discovered that plsX is not essential in S. mutans. This study explores how the loss of plsX affects the ability of S. mutans to alter its membrane fatty acid profile and survive at low pH. The plsX deletion mutant (ΔplsX) is not a fatty acid or phospholipid auxotroph, indicating that some alternative pathway is capable of carrying out the first step of phospholipid synthesis. Gas chromatography of fatty acid methyl esters (GC-FAME) indicates that deletion of plsX impacts the regulation of fatty acid synthesis, altering the length and saturation of fatty acids. Surprisingly, ΔplsX survives significantly longer than the parent strain, UA159, when subjected to an acid challenge of pH 2.5. This enhanced survival may be due to the increased F-ATPase activity observed at low pH. This enhanced F-ATPase activity may be due to the altered fatty acid profile, or may be part of a response to membrane stress. Supplementing ΔplsX with exogenous unsaturated fatty acids does not restore any wild-type phenotypes; however, incorporation of exogenous fatty acids is 2-fold greater in ΔplsX, compared to UA159. Exogenous oleic acid was observed to decrease survival in acid challenge for both ΔplsX and UA159. These results clearly indicate that the loss of plsX affects both the fatty acid synthesis pathway and the acid-adaptive response of S. mutans

NADH Oxidase and Stress Responses in Streptococcus mutans: A Phenotypic and Regulatory Characterization

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Microbiology and Immunology, 2012.Streptococcus mutans, an oral bacterium found on human tooth surfaces, is a primary causative agent of the disease dental caries. Dental caries is the result of the demineralization of the tooth surface, partially due to the production of acid by oral bacteria through carbohydrate metabolism. Rapid sugar metabolism in S. mutans depends on the availability of reduced nicotinamide dinucleotides (NADH). A key enzyme for the regeneration of NADH is the flavin-containing NADH oxidase (Nox). This enzyme oxidizes NADH to NAD+, while reducing diatomic oxygen to H2O. In this study, we characterized the role of the NADH oxidase in the oxidative and acid stress responses of S. mutans, determined specific regulatory controls of NADH oxidase and described the global regulatory effects from the loss of nox. The nox mutant strain exhibited reduced ability to metabolize environmental oxygen present in chemostat-grown S. mutans cultures resulting in activation of the oxygen and acid-mediated stress responses, as demonstrated by elevated activity of superoxide dismutase and glutathione oxidoreductase, elevated transcription of DNA repair genes and altered membrane fatty acid composition, independent of external pH. An Spx recognition site was identified within the first 120 bp upstream of the translational start site of the nox-coding region. Measurements of transcription rates from the nox promoter showed that SpxA activates nox and that SpxB slightly inhibits nox, indicating that nox is part of the Spx global regulon. The global effects of the nox mutation and the impacts of oxygen were characterized using cDNA microarrays. Transcriptional patterns were similar between the nox mutant and parent strain, UA159, when exposed to elevated oxygen concentrations, with some exceptions. Notably, in the nox mutant strain, the global regulator Rex, responsible for regulation of oxygen homeostasis, sugar metabolism, and biofilm formation, was differentially regulated, suggesting that Nox and Rex participate in a redox-sensing and signaling pathway. In conclusion, nox is a major consumer of environmental oxygen in S. mutans and the loss of NADH oxidase, or an increase in environmental oxygen, leads to global transcriptional changes, allowing the organism to respond rapidly to stress at the expense of overall growth rate