Drug design and DNA structural research inspired by the Neidle laboratory: DNA minor groove binding and transcription factor inhibition by thiophene diamidines.

The understanding of sequence-specific DNA minor groove interactions has recently made major steps forward and as a result, the goal of development of compounds that target the minor groove is an active research area. In an effort to develop biologically active minor groove agents, we are preparing and exploring the DNA interactions of diverse diamidine derivatives with a 5'-GAATTC-3' binding site using a powerful array of methods including, biosensor-SPR methods, and X-ray crystallography. The benzimidazole-thiophene module provides an excellent minor groove recognition component. A central thiophene in a benzimidazole-thiophene-phenyl aromatic system provides essentially optimum curvature for matching the shape of the minor groove. Comparison of that structure to one with the benzimidazole replaced with an indole shows that the two structures are very similar, but have some interesting and important differences in electrostatic potential maps, the DNA minor groove binding structure based on x-ray crystallographic analysis, and inhibition of the major groove binding PU.1 transcription factor complex. The binding KD for both compounds is under 10 nM and both form amidine H-bonds to DNA bases. They both have bifurcated H-bonds from the benzimidazole or indole groups to bases at the center of the -AATT- binding site. Analysis of the comparative results provides an excellent understanding of how thiophene compounds recognize the minor groove and can act as transcription factor inhibitors.

Evaluation of plasma nucleosome concentrations in dogs with a variety of common cancers and in healthy dogs.

BACKGROUND: Cell free DNA, in the form of nucleosomes, is released into circulation during apoptosis and necrosis in a variety of diseases. They are small fragments of chromosomes that are composed of DNA wrapped around a histone core made of four duplicate histone proteins forming an octamer. The nucleosome compartment is a relatively uninvestigated area of circulating tumor biomarkers in dogs. The objectives of this study were to quantify and better characterize nucleosome concentrations in 528 dogs with various common malignancies and compare them to 134 healthy dogs. RESULTS: The sensitivity of increased circulating nucleosome concentrations for the detection of cancer in all dogs was 49.8% with a specificity of 97% with an area under the curve of 68.74%. The top 4 malignancies detected by the test included lymphoma, hemangiosarcoma, histiocytic sarcoma and malignant melanoma. The malignancies least likely to be detected were soft tissue sarcomas, osteosarcoma and mast cell tumors. CONCLUSIONS: A variety of tumor types may cause increased nucleosome concentrations in dogs. Tumors of hematopoietic origin are most likely to cause elevations and local tumors such as soft tissue sarcomas are least likely to cause elevations in plasma nucleosome concentrations.

Melanoma in women of childbearing age and in pregnancy in California, 1994-2015: a population-based cohort study.

BACKGROUND: Melanoma is one of the most common malignancies during pregnancy. There is debate regarding the impact of pregnancy on the prognosis of melanoma. Recent large population-based studies from the United States are lacking. OBJECTIVES: To determine the characteristics and survival of women with pregnancy-associated melanoma. METHODS: This population-based, retrospective cohort study used California Cancer Registry data linked with state-wide hospitalization and ambulatory surgery data to identify 15-44-year-old female patients diagnosed with melanoma in 1994-2015, including pregnant patients. Multivariable logistic regression compared demographic and clinical characteristics between pregnant and non-pregnant women with melanoma. Multivariable cox proportional hazards regression models assessed melanoma-specific and overall survival. RESULTS: We identified 13 108 patients, of which 1406 were pregnant. Pregnancy-associated melanoma was more frequent in Hispanic compared to non-Hispanic White women. Melanoma occurring post-partum was associated with greater tumour thickness (2.01-4.00 vs. 0.01-1.00 mm, odds ratio 1.75, 95% confidence interval: 1.03-2.98). There were otherwise no significant differences between pregnant and non-pregnant women. Worse survival was associated with Asian, Black and Native American race/ethnicity (vs. non-Hispanic White), lower neighbourhood socio-economic status, public insurance, tumour site, greater tumour thickness and lymph node involvement, but not pregnancy. CONCLUSIONS: Melanoma occurring post-partum was associated with greater tumour thickness, but pregnancy status did not affect survival after melanoma. Race/ethnicity, socio-economic status and health insurance impacted survival, emphasizing the importance of reducing health disparities.

Dissection of integrated readout reveals the structural thermodynamics of DNA selection by transcription factors.

Nucleobases such as inosine have been extensively utilized to map direct contacts by proteins in the DNA groove. Their deployment as targeted probes of dynamics and hydration, which are dominant thermodynamic drivers of affinity and specificity, has been limited by a paucity of suitable experimental models. We report a joint crystallographic, thermodynamic, and computational study of the bidentate complex of the arginine side chain with a Watson-Crick guanine (Arg×GC), a highly specific configuration adopted by major transcription factors throughout the eukaryotic branches in the Tree of Life. Using the ETS-family factor PU.1 as a high-resolution structural framework, inosine substitution for guanine resulted in a sharp dissection of conformational dynamics and hydration and elucidated their role in the DNA specificity of PU.1. Our work suggests an under-exploited utility of modified nucleobases in untangling the structural thermodynamics of interactions, such as the Arg×GC motif, where direct and indirect readout are tightly integrated.

Single GC base pair recognition by a heterocyclic diamidine: structures, affinities, and dynamics.

The recognition of specific genomic arrangements by rationally designed small molecules is fundamental for the expansion of targeted gene expression. Here, we report the first X-ray crystal structures that demonstrate single G (guanine) recognition by a highly selective diamidine (DB2447) in a mixed DNA sequence. The study presents detailed structural information on the mechanism of single G recognition by D2447 and its various interactions in the DNA minor groove. Molecular dynamics and binding studies were used to evaluate the details of our reported structures. The study provides structural insight and resources necessary for understanding single G selection in genomic sequences.

Structure of an RNA G-quadruplex from the West Nile virus genome.

Potential G-quadruplex sites have been identified in the genomes of DNA and RNA viruses and proposed as regulatory elements. The genus Orthoflavivirus contains arthropod-transmitted, positive-sense, single-stranded RNA viruses that cause significant human disease globally. Computational studies have identified multiple potential G-quadruplex sites that are conserved across members of this genus. Subsequent biophysical studies established that some G-quadruplexes predicted in Zika and tickborne encephalitis virus genomes can form and known quadruplex binders reduced viral yields from cells infected with these viruses. The susceptibility of RNA to degradation and the variability of loop regions have made structure determination challenging. Despite these difficulties, we report a high-resolution structure of the NS5-B quadruplex from the West Nile virus genome. Analysis reveals two stacked tetrads that are further stabilized by a stacked triad and transient noncanonical base pairing. This structure expands the landscape of solved RNA quadruplex structures and demonstrates the diversity and complexity of biological quadruplexes. We anticipate that the availability of this structure will assist in solving further viral RNA quadruplexes and provides a model for a conserved antiviral target in Orthoflavivirus genomes.

X-ray Structure Characterization of the Selective Recognition of AT Base Pair Sequences.

The rational design of small molecules that target specific DNA sequences is a promising strategy to modulate gene expression. This report focuses on a diamidinobenzimidazole compound, whose selective binding to the minor groove of AT DNA sequences holds broad significance in the molecular recognition of AT-rich human promoter sequences. The objective of this study is to provide a more detailed and systematized understanding, at an atomic level, of the molecular recognition mechanism of different AT-specific sequences by a rationally designed minor groove binder. The specialized method of X-ray crystallography was utilized to investigate how the sequence-dependent recognition properties in general, A-tract, and alternating AT sequences affect the binding of diamidinobenzimidazole in the DNA minor groove. While general and A-tract AT sequences give a narrower minor groove, the alternating AT sequences intrinsically have a wider minor groove which typically constricts upon binding. A strong and direct hydrogen bond between the N-H of the benzimidazole and an H-bond acceptor atom in the minor groove is essential for DNA recognition in all sequences described. In addition, the diamidine compound specifically utilizes an interfacial water molecule for its DNA binding. DNA complexes of AATT and AAAAAA recognition sites show that the diamidine compound can bind in two possible orientations with a preference for water-assisted hydrogen bonding at either cationic end. The complex structures of AAATTT, ATAT, ATATAT, and AAAA are bound in a singular orientation. Analysis of the helical parameters shows a minor groove expansion of about 1 Å across all the nonalternating DNA complexes. The results from this systematic approach will convey a greater understanding of the specific recognition of a diverse array of AT-rich sequences by small molecules and more insight into the design of small molecules with enhanced specificity to AT and mixed DNA sequences.

DNA selection by the master transcription factor PU.1.

The master transcriptional regulator PU.1/Spi-1 engages DNA sites with affinities spanning multiple orders of magnitude. To elucidate this remarkable plasticity, we have characterized 22 high-resolution co-crystallographic PU.1/DNA complexes across the addressable affinity range in myeloid gene transactivation. Over a purine-rich core (such as 5'-GGAA-3') flanked by variable sequences, affinity is negotiated by direct readout on the 5' flank via a critical glutamine (Q226) sidechain and by indirect readout on the 3' flank by sequence-dependent helical flexibility. Direct readout by Q226 dynamically specifies PU.1's characteristic preference for purines and explains the pathogenic mutation Q226E in Waldenström macroglobulinemia. The structures also reveal how disruption of Q226 mediates strand-specific inhibition by DNA methylation and the recognition of non-canonical sites, including the authentic binding sequence at the CD11b promoter. A re-synthesis of phylogenetic and structural data on the ETS family, considering the centrality of Q226 in PU.1, unifies the model of DNA selection by ETS proteins.

Nursing and Health Care Preferences Among Opioid and Stimulant Using Black Sexual Minority Men: An Exploratory Study.

ABSTRACT: Black sexual minority men (BSMM) experience the worst HIV treatment outcomes in the United States. Drug use increases HIV transmission risks and reduces health care engagement. Perceived health care provider stigma and medical mistrust minimizes treatment efforts. This study identified nursing and health care preferences among drug-using BSMM. In-depth qualitative interviews were conducted among 30 BSMM who reported drug use in Baltimore City, MD, from December 2018 to March 2019. Analysis identified themes as client preferences for nursing practices and gaps in clinical services. Participants' ages ranged from 23 to 63 years (M = 41.1). Most (91%) reported living with HIV. The following themes were identified as nursing and health care preferences: (a) being genuine, (b) knowing drug treatment and social services, (c) understanding drug use effects, (d) providing mental health services, and (e) clarifying treatment recommendations. Nurses and health care facilities can improve cultural competency for drug-using BSMM. Future research should identify the impact of these preferences on HIV care outcomes among BSMM.

Quasi-stellar objects: possible local origin.

Many difficulties face the conventional interpretation of the red shift of quasars as a Hubble shift, with associated immense distances. These objects are not of galactic size or nature, and are not associated with galaxies or clusters of galaxies. The continuing energy source for such enormous powers for a period of 10(6) to 10(7) years has not been clearly revealed. The absence of the expected absorption for the Lyman-alpha spectral line of hydrogen is a new difficulty. Because of the relativistic limit on the diameter which can produce rapid fluctuations of light output, there may not be enough surface to radiate the required light.A similar and perhaps more serious difficulty exists for the fluctuating radio output. Calculations given here for synchrotron radiation self-absorption lead to a reasonably accurate formula for the angular diameter of a radio source. For the quasar 3C 273B these relations indicate a conflict with the usually assumed distance. However, the discrepancy may be explained in terms of strong variation of radio diameter with frequency. For CTA 102 the conflict is more serious, and could be explained -for cosmological distance-only by rejecting the data of Sholomitskii. These difficulties are removed by the hypothesis that the observed quasars were ejected from a gravitational collapse at the center of our own galaxy, which may have occurred roughly 5 million years ago. The resultant distances, of the order of a million lightyears, reduce the energy problem by a factor of 10(6) or 10(7). On this basis the optical diameter would be less than a light-hour, about the size of the earth's orbit. A rotating mass of a few thousand solar masses with this diameter would account for the unusual line width, could easily produce the required radiated energy, and could readily account for observed short fluctuation periods and variations in spectrum. It is suggested that the radio output may be produced by high-speed passage of the quasar through intergalactic gas. This would probably correspond to a radio size of a few light-years or less, in agreement with the fluctuations. Since the radio power would be considerably less than that of radio galaxies, it is suggested that radio galaxies may have ejected groups of quasars. This would explain the peculiarly distant locations of the radio sources for many such galaxies. The objections to this model that have been raised are apparently not fatal. In particular, the receding hydrogen cloud discovered by Koehler to be in the line of sight to 3C 273 is more plausibly interpreted as having been ejected from our own galaxy, in the manner observed for other galaxies, than as being associated with the Virgo cluster of galaxies. The latter interpretation, which would place 3C 273 further away, is in conflict with Lyman-alpha absorption data for 3C 9 and other quasars. Thus the local model seems to give a reasonable explanation not only of quasars but also of radio galaxies, bothv of which seem largely to defy explanation on other grounds. Whether or not this model is valid, it is clear that an understanding of quasars will radically change our understanding of the universe.

Cigarettes: chemical effects of sodium nitrate content.

Although addition of sodium nitrate to cigarettes is reported to reduce the components and properties of cigarette smoke that are associated with tumorigenicity, we find that the additive nevertheless significantly increases the levels of certain vapor-phase constituents of smoke that are known to inhibit ciliary movement; and also it produces other effects of questionable value.

"Blocking" and "Filtering": a Commentary on Mobile Technology, Racism, and the Sexual Networks of Young Black MSM (YBMSM).

While research investigates the role and influence of geo-social networking (GSN) applications on HIV, less is known about the impact of GSN functions on disease transmission. In our formative research on young Black men who have sex with men's (YBMSM) technology use patterns and preferences for a smartphone-based HIV prevention intervention, we found that study participants used GSN "block" and "filter" functions as protective mechanisms against racism and racial sexual discrimination. Yet, we suggest that these functions may unintentionally create restrictive sexual networks that likely increase their risk for disease transmission. As such, we contend that attention to the unintended effects of these protective mechanisms against racism on GSN applications is fundamentally a public health issue that requires more research and explicit intervention. Ultimately, we use this work to hypothesize the role of blocking and filtering as a strategy to avoid racism on GSN applications that may partly explain HIV disparities among YBMSM.

Structure/function of the inhibition of human 3beta-hydroxysteroid dehydrogenase type 1 and type 2 by trilostane.

The human type 1 (placenta, breast tumors) and type 2 (gonads, adrenals) isoforms of 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD) are key enzymes in biosynthesis of all active steroid hormones. Human 3beta-HSD1 is a critical enzyme in the conversion of DHEA to estradiol in breast tumors and may be a major target enzyme for the treatment of breast cancer. 3beta-HSD2 participates in the production of cortisol and aldosterone in the human adrenal gland. The goals of this project are to evaluate the role of the 2alpha-cyano group on trilostane (2alpha-cyano-4alpha,5alpha-epoxy-17beta-ol-androstane-3-one) and determine which amino acids may be critical for 3beta-HSD1 specificity. Trilostane without the 2alpha-cyano group, 4alpha,5alpha-epoxy-testosterone, was synthesized. Using our structural model of 3beta-HSD1, trilostane or 4alpha,5alpha-epoxy-testosterone was docked in the active site using Autodock 3.0, and the potentially critical residues (Met187 and Ser124) were identified. The M187T and S124T mutants of 3beta-HSD1 were created, expressed and purified. Dixon analyses of the inhibition of wild-type 3beta-HSD1, 3beta-HSD2, M187T and S124T by trilostane and 4alpha,5alpha-epoxy-testosterone suggest that the 2alpha-cyano group of trilostane is anchored by Ser124 in both isoenzymes. Kinetic analyses of cofactor and substrate utilization as well as the inhibition kinetics of M187T and the wild-type enzymes suggest that the 16-fold higher-affinity inhibition of 3beta-HSD1 by trilostane may be related to the presence of Met187 in 3beta-HSD1 and Thr187 in 3beta-HSD2. This structure/function information may lead to the production of more highly specific inhibitors of 3beta-HSD1 to block the hormone-dependent growth of breast tumors.

Eight Americas: investigating mortality disparities across races, counties, and race-counties in the United States.

BACKGROUND: The gap between the highest and lowest life expectancies for race-county combinations in the United States is over 35 y. We divided the race-county combinations of the US population into eight distinct groups, referred to as the "eight Americas," to explore the causes of the disparities that can inform specific public health intervention policies and programs. METHODS AND FINDINGS: The eight Americas were defined based on race, location of the county of residence, population density, race-specific county-level per capita income, and cumulative homicide rate. Data sources for population and mortality figures were the Bureau of the Census and the National Center for Health Statistics. We estimated life expectancy, the risk of mortality from specific diseases, health insurance, and health-care utilization for the eight Americas. The life expectancy gap between the 3.4 million high-risk urban black males and the 5.6 million Asian females was 20.7 y in 2001. Within the sexes, the life expectancy gap between the best-off and the worst-off groups was 15.4 y for males (Asians versus high-risk urban blacks) and 12.8 y for females (Asians versus low-income southern rural blacks). Mortality disparities among the eight Americas were largest for young (15-44 y) and middle-aged (45-59 y) adults, especially for men. The disparities were caused primarily by a number of chronic diseases and injuries with well-established risk factors. Between 1982 and 2001, the ordering of life expectancy among the eight Americas and the absolute difference between the advantaged and disadvantaged groups remained largely unchanged. Self-reported health plan coverage was lowest for western Native Americans and low-income southern rural blacks. Crude self-reported health-care utilization, however, was slightly higher for the more disadvantaged populations. CONCLUSIONS: Disparities in mortality across the eight Americas, each consisting of millions or tens of millions of Americans, are enormous by all international standards. The observed disparities in life expectancy cannot be explained by race, income, or basic health-care access and utilization alone. Because policies aimed at reducing fundamental socioeconomic inequalities are currently practically absent in the US, health disparities will have to be at least partly addressed through public health strategies that reduce risk factors for chronic diseases and injuries.

Radiation concurrent with gemcitabine for locally advanced head and neck cancer: a phase I trial and intracellular drug incorporation study.

PURPOSE: To examine the feasibility and dose-limiting toxicity (DLT) of once-weekly gemcitabine at doses predicted in preclinical studies to produce radiosensitization, concurrent with a standard course of radiation for locally advanced head and neck cancer. Tumor incorporation of gemcitabine triphosphate (dFdCTP) was measured to assess whether adequate concentrations were achieved at each dose level. PATIENTS AND METHODS: Twenty-nine patients with unresectable head and neck cancer received a course of radiation (70 Gy over 7 weeks, 5 days weekly) concurrent with weekly infusions of low-dose gemcitabine. Tumor biopsies were performed after the first gemcitabine infusion (before radiation started), and the intracellular concentrations of dFdCTP were measured. RESULTS: Severe acute and late mucosal and pharyngeal-related DLT required de-escalation of gemcitabine dose in successive patient cohorts receiving dose levels of 300 mg/m(2)/wk, 150 mg/m(2)/wk, and 50 mg/m(2)/wk. No DLT was observed at 10 mg/m(2)/wk. The rate of endoscopy- and biopsy-assessed complete tumor response was 66% to 87% in the various cohorts. Tumor dFdCTP levels were similar in patients receiving 50 to 300 mg/m(2) (on average, 1.55 pmol/mg, SD 1.15) but were barely or not detectable at 10 mg/m(2). CONCLUSION: A high rate of acute and late mucosa-related DLT and a high rate of complete tumor response were observed in this regimen at the dose levels of 50 to 300 mg/m(2), which also resulted in similar, subcytotoxic intracellular dFdCTP concentrations. These results demonstrate significant tumor and normal tissue radiosensitization by low-dose gemcitabine. Different regimens of combined radiation and gemcitabine should be evaluated, based on newer preclinical data promising an improved therapeutic ratio.

Getting PrEPared for HIV Prevention Navigation: Young Black Gay Men Talk About HIV Prevention in the Biomedical Era.

Biomedical HIV prevention strategies, such as pre-exposure prophylaxis (PrEP) and post-exposure prophylaxis (PEP), represent new opportunities to reduce critically high HIV infection rates among young black men who have sex with men (YBMSM). We report results of 24 dyadic qualitative interviews (N=48), conducted in Los Angeles, CA, exploring how YBMSM and their friends view PrEP and PEP. Interviews were analyzed using a grounded theory approach. Participants had widely divergent levels of knowledge about these prevention methods. Misconceptions and mistrust regarding PrEP were common, and concerns were expressed about PrEP-related stigma and the potential for gossip among peers who might assume a person on PrEP was HIV-positive. Yet participants also framed PrEP and PEP as valuable new options within an expanded "tool kit" of HIV prevention strategies that created possibilities for preventing new HIV infections, dating men with a different HIV status, and decreased anxiety about exposure to HIV. We organized themes around four main areas: (1) information and misinformation about biomedical HIV prevention; (2) expectations about PrEP, sexual behavior, and stigma; (3) gossip, disclosure, and "spreading the word" about PrEP and PEP; and (4) the roles of PrEP and PEP in an expanded HIV prevention tool kit. The findings suggest a need for guidance in navigating the increasingly complex array of HIV-prevention options available to YBMSM. Such "prevention navigation" could counter misconceptions and address barriers, such as stigma and mistrust, while helping YBMSM make informed selections from among expanded HIV prevention options.

Insulin-like growth factor carrier proteins in neonatal and adult rat serum are immunologically different: demonstration using a new radioimmunoassay for the carrier protein from BRL-3A rat liver cells.

A carrier protein for insulin-like growth factors (IGFs) has been purified from serum-free medium conditioned by the Buffalo rat liver (BRL)-3A cell line and used to immunize rabbits. Purified carrier protein was 125I labeled and affinity purified on IGF-Sepharose. The major labeled protein had a mol wt of about 33,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (appropriate for the IGF carrier protein subunit) and gave a single predominant peak of radioactivity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and acid-urea gel electrophoresis that was immunoprecipitated by immune serum and comigrated with unlabeled proteins that bind [125I] IGF. A RIA was developed using affinity purified [125I]carrier protein and immune serum. Tracer binding was inhibited only by preparations containing IGF carrier proteins, but not by unrelated proteins or by the IGFs themselves. Carrier proteins from BRL-3A cells gave equivalent strong reactivity either after dissociation of endogenous IGF or as an IGF-carrier protein complex. The antiserum effectively recognized the approximately 40,000 mol wt (Mr approximately 40,000) carrier protein from neonatal rat serum, both as a native complex and after acid stripping. It did not effectively recognize the Mr approximately 150,000 carrier protein from adult rat serum either as endogenous complex or after acid stripping. These results suggest that the Mr approximately 40,000 carrier protein of neonatal rat serum and the Mr approximately 40,000 binding subunit of the Mr approximately 150,000 carrier protein in adult rat serum are immunologically distinct. These antisera to the BRL-3A carrier protein should be useful tools with which to dissect the relationships between different carrier protein species and to study the regulation of IGF carrier protein gene expression.

22-Mb integrated physical and genetic map based on YAC/STS content spanning the interval DXS1125-DXS95 in human Xq12-q21.31.

A YAC/STS map has been assembled spanning 22 Mb across Xq12-q21.31, between markers DXS1125 and DXS95. In addition to the landmark loci for the X-inactivation center XIST and the ATRX, ATP7A, phosphoglycerate kinase, POU3F4, and choroideremia genes, the candidate disease gene regions for torsion dystonia 3 and two X-linked mental retardation syndromes are included. Also, the human voltage-dependent anion channel gene (HVDAC1) has been placed near DXS986. The current map incorporates 211 YACs from five different libraries, formatted with 185 STSs that comprise 26 genetic linkage markers, 60 newly-developed YAC-end STSs, and eight ESTs. The multiple clone coverage and average resolution of one STS per 120 kb provide resources for disease gene searches and are facilitating complete sequencing of the region.

Xerostomia and its predictors following parotid-sparing irradiation of head-and-neck cancer.

PURPOSE: To assess long-term xerostomia in patients receiving parotid-sparing radiation therapy (RT) for head-and-neck cancer, and to find the patient and therapy-related factors that affect its severity. PATIENTS AND METHODS: From March 1994 through January 2000, 84 patients received comprehensive bilateral neck RT using conformal and multisegmental intensity-modulated RT (IMRT) aiming to spare the major salivary glands. Before RT and periodically through 2 years after the completion of RT, salivary flow rates from each of the major salivary glands were selectively measured. At the same time intervals, each patient completed an 8-item self-reported xerostomia-specific questionnaire (XQ). To gain a relative measure of the effect of RT on the minor salivary glands, whose output could not be measured, the surfaces of the oral cavity (extending to include the surface of the base of tongue) were outlined in the planning CT scans. The mean doses to the new organ ("oral cavity") were recorded. Forty-eight patients receiving unilateral neck RT were similarly studied and served as a benchmark for comparison. Factors predicting the XQ scores were analyzed using a random-effects model. RESULTS: The XQ was found to be reliable and valid in measuring patient-reported xerostomia. The spared salivary glands which had received moderate doses in the bilateral RT group recovered to their baseline salivary flow rates during the second year after RT, and the spared glands in the unilateral RT group, which had received very low doses, demonstrated increased salivary production beyond their pre-RT levels. The increase in the salivary flow rates during the second year after RT paralleled an improvement in xerostomia in both patient groups. The improvement in xerostomia was faster in the unilateral compared with the bilateral RT group, but the difference narrowed at 2 years. The major salivary gland flow rates had only a weak correlation with the xerostomia scores. Factors found to be independently associated with the xerostomia scores were the pre-RT baseline scores, the time since RT, and the mean doses to the major salivary glands (notably to the submandibular glands) and to the oral cavity. CONCLUSION: An improvement over time in xerostomia, occurring in tandem with rising salivary production from the spared major salivary glands, suggests a long-term clinical benefit from their sparing. The oral cavity mean dose, representing RT effect on the minor salivary glands, was found to be a significant, independent predictor of xerostomia. Thus, in addition to the major salivary glands, sparing the uninvolved oral cavity should be considered as a planning objective to further reduce xerostomia.