Fluconazole resistance in Candida albicans is induced by Pseudomonas aeruginosa quorum sensing.

Microorganisms employ quorum sensing (QS) mechanisms to communicate with each other within microbial ecosystems. Emerging evidence suggests that intraspecies and interspecies QS plays an important role in antimicrobial resistance in microbial communities. However, the relationship between interkingdom QS and antimicrobial resistance is largely unknown. Here, we demonstrate that interkingdom QS interactions between a bacterium, Pseudomonas aeruginosa and a yeast, Candida albicans, induce the resistance of the latter to a widely used antifungal fluconazole. Phenotypic, transcriptomic, and proteomic analyses reveal that P. aeruginosa's main QS molecule, N-(3-Oxododecanoyl)-L-homoserine lactone, induces candidal resistance to fluconazole by reversing the antifungal's effect on the ergosterol biosynthesis pathway. Accessory resistance mechanisms including upregulation of C. albicans drug-efflux, regulation of oxidative stress response, and maintenance of cell membrane integrity, further confirm this phenomenon. These findings demonstrate that P. aeruginosa QS molecules may confer protection to neighboring yeasts against azoles, in turn strengthening their co-existence in hostile polymicrobial infection sites.

Regulation of mammary gland sensitivity to thyroid hormones during the transition from pregnancy to lactation.

Thyroid hormones are galactopoietic and help to establish the mammary gland's metabolic priority during lactation. Expression patterns for genes that can alter tissue sensitivity to thyroid hormones and thyroid hormone activity were evaluated in the mammary gland and liver of cows at 53, 35, 20, and 7 days before expected parturition, and 14 and 90 days into the subsequent lactation. Transcript abundance for the three isoforms of iodothyronine deiodinase, type I (DIO1), type II (DIO2) and type III (DIO3), thyroid hormone receptors alpha1 (TRalpha1), alpha2 (TRalpha2) and beta1 (TRbeta1), and retinoic acid receptors alpha (RXRalpha) and gamma (RXRgamma), which act as coregulators of thyroid hormone receptor action, were evaluated by quantitative RT-PCR. The DIO3 is a 5-deiodinase that produces inactive iodothyronine metabolites, whereas DIO1 and DIO2 generate the active thyroid hormone, triiodothyronine, from the relatively inactive precursor, thyroxine. Low copy numbers of DIO3 transcripts were present in mammary gland and liver. DIO2 was the predominant isoform expressed in mammary gland and DIO1 was the predominant isoform expressed in liver. Quantity of DIO1 mRNA in liver tissues did not differ with physiological state, but tended to be lowest during lactation. Quantity of DIO2 mRNA in mammary gland increased during lactation (P < 0.05), with copy numbers at 90 days of lactation 6-fold greater than at 35 and 20 days prepartum. When ratios of DIO2/DIO3 mRNA were evaluated, the increase was more pronounced (>100-fold). Quantity of TRbeta1 mRNA in mammary gland increased with onset of lactation, whereas TRalpha1 and TRalpha2 transcripts did not vary with physiological state. Conversely, quantity of RXRalpha mRNA decreased during late gestation to low levels during early lactation. Data suggest that increased expression of mammary TRbeta1 and DIO2, and decreased RXRalpha, provide a mechanism to increase thyroid hormone activity within the mammary gland during lactation.

Acetophenone as an anti-attractant for the western pine beetle, Dendroctonus Brevicomis LeConte (Coleoptera: Scolytidae).

Host location and colonization by bark beetles is dependent upon the relative and absolute amounts of attractant and antiattractant compounds available. Many investigations have lead to use of antiattractants for the management of these pests and have been especially focused on verbenone. However, recent studies have identified new antiattractants for several species of bark beetles. We report results of recent investigations of the response of western pine beetle, Dendroctonus brevicomis LeConte, to two recently identified antiattractants, acetophenone, and fenchyl alcohol, with verbenone as a standard of comparison, in northern California. Release of both acetophenone and verbenone resulted in significantly lower trap catches of D. brevicomis in aggregation pheromone-baited traps, while fenchyl alcohol was inactive. Acetophenone was the only antiattractant that did not reduce numbers of the most abundant predator of D. brevicomis, Temnochila chlorodia (Mannerheim), responding to the attractant pheromone of its prey. Aggregation pheromone-baited traps with acetophenone also had the highest predator/prey ratio. Our results suggest that acetophenone may be part of the intra- and interspecific interactions among sympatric species of bark beetles and may have application in their control.

Response to host volatiles by native and introduced populations of Dendroctonus valens (Coleoptera: Curculionidae, Scolytinae) in North America and China.

Bark beetles (Coleoptera: Curculionidae, Scolytinae) have specialized feeding habits, and commonly colonize only one or a few closely related host genera in their geographical ranges. The red turpentine beetle, Dendroctonus valens LeConte, has a broad geographic distribution in North America and exploits volatile cues from a wide variety of pines in selecting hosts. Semiochemicals have been investigated for D. valens in North America and in its introduced range in China, yielding apparent regional differences in response to various host volatiles. Testing volatiles as attractants for D. valens in its native and introduced ranges provides an opportunity to determine whether geographic separation promotes local adaptation to host compounds and to explore potential behavioral divergence in native and introduced regions. Furthermore, understanding the chemical ecology of host selection facilitates development of semiochemicals for monitoring and controlling bark beetles, especially during the process of expansion into new geographic ranges. We investigated the responses of D. valens to various monoterpenes across a wide range of sites across North America and one site in China, and used the resulting information to develop an optimal lure for monitoring populations of D. valens throughout its Holarctic range. Semiochemicals were selected based on previous work with D. valens: (R)-(+)-alpha-pinene, (S)-(-)-alpha-pinene, (S)-(-)-beta-pinene, (S)-(+)-3-carene, a commercially available lure [1:1:1 ratio of (R)-(+)-alpha-pinene:(S)-(-)-beta-pinene:(S)-(+)-3-carene], and a blank control. At the release rates used, (+)-3-carene was the most attractive monoterpene tested throughout the native range in North America and introduced range in China, confirming results from Chinese studies. In addition to reporting a more effective lure for D. valens, we present a straightforward statistical procedure for analysis of insect trap count data yielding cells with zero counts, an outcome that is common but makes the estimation of the variance with a Generalized Linear Model unreliable because of the variability/mean count dependency.

Chromosomal mapping and quantitative analysis of estrogen-related receptor alpha-1, estrogen receptors alpha and beta and progesterone receptor in the bovine mammary gland.

Steroid receptors are key transcriptional regulators of mammary growth, development and lactation. Expression of estrogen receptors alpha (ERalpha) and beta (ERbeta), progesterone receptor (PR), and estrogen-related receptor alpha-1 (ERRbeta) have been evaluated in bovine mammary gland. The ERRalpha is an orphan receptor that, in other species and tissues, appears to function in the regulation of estrogen-response genes including lactoferrin and medium chain acyl-CoA dehydrogenase and in mitochondrial biogenesis. Expression of ERalpha, ERbeta, PR and ERRalpha was characterized in mammary tissue obtained from multiple stages of bovine mammary gland development using quantitative real-time RT-PCR. Expression was evaluated in prepubertal heifers, primigravid cows, lactating non-pregnant cows, lactating pregnant cows and non-lactating pregnant cows (n=4 to 9 animals/stage). In addition, ERalpha, ERbeta, PR and ERRalpha were mapped to chromosomes 9, 10, 15 and 29 respectively, by linkage and radiation hybrid mapping. Results indicated that expression of ERalpha, PR and ERRalpha was largely coordinately regulated and they were present in significant quantity during all physiological stages evaluated. In contrast, ERbeta transcripts were present at a very low concentration during all stages. Furthermore, no ERbeta protein could be detected in bovine mammary tissue by immunohistochemistry. The ERalpha and PR proteins were detected during all physiological states, including lactation. Our results demonstrate the presence of ERalpha, PR and ERRalpha during all physiological stages, and suggest a functional role for ERRalpha and a relative lack of a role for ERbeta in bovine mammary gland development and lactation.

Effect of bovine somatotropin and rumen-undegradable protein on mammary growth of prepubertal dairy heifers and subsequent milk production.

Rapid body growth during the prepubertal period may be associated with reductions in mammary parenchymal growth and subsequent milk yield. The objective of this study was to test effects of dietary rumen-undegradable protein (RUP) and administration of recombinant bovine somatotropin (bST) during the prepubertal period on mammary growth and milk yield of dairy heifers. Seventy-two Holstein heifers were used in the experiment. At 90 d of age, 8 heifers were slaughtered before initiation of treatment. Remaining heifers were assigned randomly to 1 of 4 treatments. Treatments consisted of a control diet (5.9% RUP, 14.9% CP, DM basis) or RUP-supplemented diet (control diet plus 2% added RUP) with or without 0.1 mg of bST/kg of BW per day applied in a 2 x 2 factorial design. A total of 6 heifers per treatment (3 each at 5 and 10 mo of age) were slaughtered for mammary tissue analysis. Remaining heifers were bred to evaluate impact of treatment on subsequent milk yield and composition. Mammary parenchymal growth was not affected by RUP or bST treatment. Total parenchymal mass increased from 16 to 364 g, and parenchymal DNA from 58 to 1022 mg from 3 to 10 mo of age, respectively. Furthermore, number of mammary epithelial cells likely was not affected by diet or bST because the epithelial cell proliferation index, assessed by Ki-67 labeling, was not affected by treatment, nor was total parenchymal DNA and lipid content. Neither deleterious effects of increased rates of gain nor positive effects of bST were evident in prepubertal mammary growth. Subsequent milk production and composition was not different among treatments.

Bovine somatotropin and rumen-undegradable protein effects in prepubertal dairy heifers: effects on body composition and organ and tissue weights.

The objectives of this study were to determine the effect of recombinant bovine somatotropin (bST) and added dietary rumen undegradable protein (RUP) on organ and tissue weights and body composition in growing dairy heifers. Thirty-two Holstein heifers were in the experiment, 8 killed initially at 3 mo of age, with the remaining 24 Holstein heifers randomly assigned to treatments (n = 6) consisting of 0.1 mg/kg of body weight per day of bST and 2% added dietary RUP (dry matter basis) applied in a 2 x2 factorial design. A total of 6 heifers per treatment group (3 each at 5 and 10 mo of age), were slaughtered to determine body composition and organ masses. Feed intake measured from group intakes were increased by 0.25 and 0.35 kg/d with bST and RUP, respectively. Administration of bST tended to increase the weights of visceral organs including heart, kidney, and spleen by 16, 16, and 38%, respectively. At 10 mo of age, there was a trend for increased empty body weights (EBW) and non-carcass components for heifers treated with bST, but there were no effects of RUP. Body components and organ weights, expressed as a percentage of BW were not affected by RUP or bST. Somatotropin increased ash weight at 10 mo without affecting amounts of protein, fat, and energy. Rates of ash deposition between 3 and 10 mo of age were increased 7 and 4 g/d by bST and RUP, respectively. There were no treatment effects on rates of body fat, protein, and energy deposition. Bovine somatotropin and RUP altered the metabolism of growing heifers in a manner that was consistent with increased rates of skeletal growth. This suggests that nutritional and endocrine manipulations could increase growth rates of skeletal tissues without increasing fat deposition in prepubertal dairy heifers.

Characterization of open reading frame-expressed sequence tags generated from Bos indicus and B. taurus mammary gland cDNA libraries.

Sequence-based gene expression data are used to interpret results from functional genomic and proteomics studies. Although more than 300 000 bovine-expressed sequence tags (ESTs) are available in public databases, a more thorough and directed sampling of the expressed genome is needed to identify new transcripts and improve assembly and annotation of existing transcript sequences. Accordingly, we examined the utility of constructing cDNA libraries synthesized by arbitrarily primed RT-PCR of mRNA from tissues not well represented in the publicly available bovine EST database. A total of 33 cDNA libraries were constructed from healthy and infected mammary gland tissues of Brazilian Gir and Holstein cattle. This series of libraries was used to generate 6481 open reading frame-expressed sequence tags (ORESTES) that assembled into 1798 unique sequence elements of which, 1157 did not significantly match sequence assemblies available in the Bos taurus gene index. However, a total of 264 of these 1157 sequence elements aligned with mouse and human expressed sequences demonstrating that ORESTES is an effective resource for discovery of novel expressed sequences in cattle. Furthermore, comparison of the alignment position of bovine ORESTES-derived sequence elements to human gene reference sequences suggested that the priming events for cDNA synthesis more often occurred at the central portion of a transcript, which may have contributed to the relatively high rate of novel sequence discovery.

Postnatal mammary ductal growth: three-dimensional imaging of cell proliferation, effects of estrogen treatment, and expression of steroid receptors in prepubertal calves.

Cows may provide insights into mammary development that are not easily obtained using mouse models. Mammary growth in control and estrogen-treated calves was investigated to evaluate general patterns of proliferation and relationship to estrogen receptor (ER) expression. After in vivo labeling with bromodeoxyuridine (BrdU), serial histological sections of mammary tissue were used to generate three-dimensional reconstructions. BrdU-labeled cells were present throughout the highly branched terminal ducts. ER and progesterone receptors (PR) were colocalized in nuclei of ductal epithelial cells. However, basal cells and epithelial cells that were located in the central region of epithelial cords and those that lined the lumen of patent ducts were ER- and PR-negative, as were stromal cells. Cells along the basal portion of the epithelium were not myoepithelial. ER in mammary epithelial cells but not stromal cells is analogous to patterns in human breast but contrasts with localization in murine mammary gland. After estrogen stimulation, 99% of BrdU-labeled (and Ki67-labeled) epithelial cells were ER-negative. Data suggest that proliferation in response to estrogen treatment was initiated within ER-positive epithelial cells of the developing mammary gland and the signal was propagated in paracrine fashion to stromal elements and ER-negative epithelial cells.

Mammary cell number, proliferation, and apoptosis during a bovine lactation: relation to milk production and effect of bST.

This investigation evaluated mammary cell loss and replacement during lactation and the impact of administration of bST on these processes. During lactation, a gradual decrease in number of mammary epithelial cells within the mammary glands occurs and largely accounts for the decline in milk production with advancing lactation. This decrease is not appreciably impacted by the loss of viable epithelial cells in milk. Rather, the net decline in cell number (approximately 50% during the entire lactation) results from continual death by apoptosis. Accompanying the decline in mammary cell number by apoptosis is a degree of cell renewal. Approximately 0.3% of mammary cells in lactating, nonpregnant cows were labeled by a 24-h in vivo treatment with the thymidine analog, bromodeoxyuridine. During the entire lactation, the number of new cells amounts to approximately 50% of the number of cells initially present. By the end of lactation, most cells present in the mammary gland were formed after calving. Increasing cell replacement or decreasing apoptosis during lactation may provide a means to increase persistency of lactation. Indeed, administration of bST to Holstein cows during midlactation increased the proportion of mammary epithelial cells expressing the nuclear proliferation antigen, Ki-67, from 0.5 to 1.6%. Bovine somatotropin appears to increase the rate of cell renewal in the lactating mammary gland. Knowledge of molecular regulation of apoptosis and cell proliferation should provide a means to modulate cell turnover in the mammary gland. A change in the ratio of epithelial proliferation to cell death during lactation will affect the persistency of lactation.

Escherichia coli induces apoptosis and proliferation of mammary cells.

Mammary cell apoptosis and proliferation were assessed after injection of Escherichia coli into the left mammary quarters of six cows. Bacteriological analysis of foremilk samples revealed coliform infection in the injected quarters of four cows. Milk somatic cell counts increased in these quarters and peaked at 24 h after bacterial injection. Body temperature also increased, peaking at 12 h postinjection. The number of apoptotic cells was significantly higher in the mastitic tissue than in the uninfected control. Expression of Bax and interleukin-1beta converting enzyme increased in the mastitic tissue at 24 h and 72 h postinfection, whereas Bcl-2 expression decreased at 24 h but did not differ significantly from the control at 72 h postinfection. Induction of matrix metalloproteinase-9, stromelysin-1 and urokinase-type plasminogen activator was also observed in the mastitic tissue. Moreover, cell proliferation increased in the infected tissue. These results demonstrate that Escherichia coli-induced mastitis promotes apoptosis and cell proliferation.

Innovative approaches to educating medical students for practice in a changing health care environment: the National UME-21 Project.

In today's continually changing health care environment, there is serious concern that medical students are not being adequately prepared to provide optimal health care in the system where they will eventually practice. To address this problem, the Health Resources and Services Administration (HRSA) developed a $7.6 million national demonstration project, Undergraduate Medical Education for the 21st Century (UME-21). This project funded 18 U.S. medical schools, both public and private, for a three-year period (1998-2001) to implement innovative educational strategies. To accomplish their goals, the 18 UME-21 schools worked with more than 50 organizations external to the medical school (e.g., managed care organizations, integrated health systems, Area Health Education Centers, community health centers). The authors describe the major curricular changes that have been implemented through the UME-21 project, discuss the challenges that occurred in carrying out those changes, and outline the strategies for evaluating the project. The participating schools have developed curricular changes that focus on the core primary care clinical clerkships, take place in ambulatory settings, include learning objectives and competencies identified as important to providing care in the future health care system, and have faculty development and internal evaluation components. Curricular changes implemented at the 18 schools include having students work directly with managed care organizations, as well as special demonstration projects to teach students the knowledge, skills, and attitudes necessary for successfully managing care. It is already clear that the UME-21 project has catalyzed important curricular changes within 12.5% of U.S. medical schools. The ongoing national evaluation of this project, which will be completed in 2002, will provide further information about the project's impact and effectiveness.

Principles of patient safety in pediatrics.

The American Academy of Pediatrics and its members are committed to improving the health care system to provide the best and safest health care for infants, children, adolescents, and young adults. In response to a 1999 Institute of Medicine report on building a safer health system, a set of principles was established to guide the profession in designing a health care system that maximizes quality of care and minimizes medical errors through identification and resolution. This set of principles provides direction on setting up processes to identify and learn from errors, developing performance standards and expectations for safety, and promoting leadership and knowledge.

Penetrating head injury: a prospective study of outcomes.

The purpose of our study was to describe the outcomes of persons with penetrating brain injury resulting from a gunshot wound to the head. It is a prospective study of 442 patients admitted with gunshot wounds to the head over a 7 year period to our University Trauma Center Emergency Department, an urban trauma center and an inpatient rehabilitation hospital with a specialized brain injury unit. Measures and factors described include initial Glasgow Coma Scale score, Revised Trauma Score, the Disability Rating Scale, Functional Independence Measure, levels of cognitive functioning, patient demographics, length of stay, hospital charges, and discharge disposition. Initially 36% of patients expired in or were dead upon arrival to the Emergency Department; 64% of patients survived to be admitted for inpatient care. Of those admitted, 41% expired within the first 48 h of admission. Fifty-two percent of those admitted had severe injuries, 7% moderate injuries, and 42% had mild head injuries. Sixty-two percent of the survivors were discharged from acute care to private residences. The remaining 38% were discharged to programs providing varying levels of care depending upon their level of functioning and care needs. Patients sustaining severe injuries following gunshot wound(s) to the head have high early mortality. Survivors able to participate in an inpatient rehabilitation program have good potential for functional improvement.

Severe penetrating head injury: a study of outcomes.

OBJECTIVE: To determine and describe the demographics and functional outcomes of persons who require inpatient rehabilitation for severe penetrating head injury resulting from a gunshot wound to the head. DESIGN: Data were collected prospectively from the time of admission to acute care through discharge from inpatient rehabilitation. SETTING: Two sites: an urban, level I, acute care, trauma center and an inpatient rehabilitation hospital with a specialized brain injury unit. PARTICIPANTS: Twenty-seven persons with severe penetrating head injury. MAIN OUTCOME MEASURES: The FIM instrument, the Disability Rating Scale (DRS), and the length of stay (LOS). RESULTS: Demographic data showed our population to be similar to other groups of persons at high risk for violent injury. Eighty-five percent of the subjects were men with a mean age of 34 years. The majority were African American (93%), reflective of our general patient population. Average acute care LOS was 31 days and average rehabilitation LOS was 44 days. Average FIM gain was 40.2 and, on average, DRS scores improved 7.6 points from rehabilitation admission to discharge. All study participants made enough progress to be discharged to private residences. CONCLUSION: Although the mortality rate is high among patients with penetrating head injury, those who survive to receive inpatient rehabilitation can achieve functional improvement.

The thersites complex in plastic surgical patients.

Body dysmorphic disorder describes the preoccupation with an imagined defect of appearance. A subgroup of patients suffer from the so-called Thersites complex, in which a minimal physical deformity causes excessive psychological disturbances and distress. Patients with body dysmorphic disorder tend primarily to visit a plastic surgeon for relief with distinct plans for surgical correction of their "deformity." Psychotherapy is generally refused or ineffective. The plastic surgeon should be familiar with this mental disorder and recognize these patients during consultation. Most of these patients should be excluded from surgery; however, patients of the Thersites complex category might be candidates for plastic surgical correction after careful selection. Only the experienced plastic surgeon should make the decision to operate in this situation. A successful treatment can relieve the patient from his or her distress and improve the quality of life substantially. A surgical result that is not accepted by the patient can end in a tragedy for either the patient or the doctor.

Effect of somatotropin on thyroid hormones and cytokines in lactating dairy cows during ad libitum and restricted feed intake.

Twelve Holsteins in first lactation were used to investigate the relationship between energy balance and effects of bovine somatotropin (bST) on thyroid hormone metabolism and cytokine concentrations in serum. Six cows were fed for ad libitum intake and six cows were feed restricted to induce negative energy balance during two treatment periods of 6 d. During treatment periods, cows were administered vehicle or 40 mg of bST/d according to a crossover design. Between treatment periods was a 15-d recovery period, during which all cows were fed ad libitum. Cows that were fed ad libitum remained in positive energy balance during control and bST treatments, whereas cows that were fed for restricted intake were in negative energy balance during control and bST treatment periods. In both dietary groups, bST decreased energy balance. Milk production and the fat percentage of milk increased during bST treatment in both dietary groups. Fat-corrected milk yield was increased 13% by bST treatment. Serum concentrations of IGF-I did not differ between dietary groups but were greater during bST than control periods. Serum thyroxine concentration was decreased by bST treatment. Serum triiodothyronine and reverse-triiodothyronine were not altered by hormone treatment, but circulating concentrations of thyroid hormones were apparently reduced by dietary restriction. Neither hepatic nor mammary thyroxine 5'-deiodinase was affected by bST treatment. Plasma concentration of tumor necrosis factor-alpha, a potential regulator of thyroxine 5'-deiodinase, was not affected by bST treatment. Short-term treatment with bST did not influence thyroid hormone metabolism in lactating cows in positive or negative energy balance.