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Biosystems Engineering and Control Group 20

15545559

Comparison of a new fibrin sealant with standard topical hemostatic agents.

Bleeding following liver resection continues to be a significant morbidity of the procedure. Fibrin sealants represent an improvement over conventional topical hemostatic agents, because they contain components that actively form clot. However, most available agents contain nonhuman protein, which represents an immunologic risk. An investigational surgical fibrin sealant (Crosseal; American Red Cross, Washington, DC) composed of human clottable proteins and human thrombin is more effective than standard topical hemostatic agents in reducing the time required to achieve hemostasis after liver resection. Prospective, randomized, controlled trial. Fifteen major referral centers in the United States and the United Kingdom. After liver resection using standard surgical techniques, 121 patients seen between May 1999 and May 2000 were randomized to treatment with a 2-component fibrin sealant (n=58) or to standard topical hemostatic agents, used singly or in combination (n=63). Up to 10 mL of Crosseal was administered by a spray applicator, as recommended by the manufacturer, whereas agents used in the control group were applied according to their instructions for use. The primary outcome measured was time to hemostasis. Secondary outcomes measured included blood loss between application of the hemostatic agent and closure of the abdomen, duration of postoperative biliary drainage, and the occurrence of complications, defined a priori as reoperation for any reason, development of abdominal fluid collections, or bilious appearance of drained fluid for at least 1 day postoperatively. The mean time to hemostasis was 282 seconds with Crosseal, compared with 468 seconds with standard agents (2-sided; P =.06), for the 116 efficacy-evaluable patients. Hemostasis was achieved within 10 minutes in 53 patients (91.4%) treated with the study fibrin sealant and in 44 control patients (69.8%) (2-sided; P =.003). Intraoperative blood loss was similar in the 2 groups. In the Crosseal group, the percentage of patients developing postoperative complications was 17.2%, compared with 36.5% in the control group (2-sided; P =.02). Compared with the use of standard topical hemostatic agents, Crosseal fibrin sealant significantly reduced the time to achieve hemostasis following liver resection. Patients treated with the new fibrin sealant also experienced significantly fewer postoperative complications.

15545548

The safety of office-based surgery: review of recent literature from several disciplines.

To review recent literature pertaining to adverse outcomes and mortality associated with office-based surgery. Representative articles from the general and plastic surgery, medical, health regulatory, and dermatology literature. Information regarding which surgical treatments should be performed, which specialties should perform them, what level of anesthesia is appropriate, and who should administer it was assessed, with particular attention to issues of patient safety. Office-based surgery is safe and cost-effective. We caution against attempts to prohibit or severely restrict this important aspect of medical care.

15545547

Nonablative facial remodeling: erythema reduction and histologic evidence of new collagen formation using a 300-microsecond 1064-nm Nd:YAG laser.

A variety of nonablative lasers have been used to improve skin color and toning. Evidence of new collagen has been seen. Using blinded observer analysis of electron microscopic changes, we have documented the effect of a nonablative Nd:YAG laser on collagen production and its relationship to patient age. Ultrastructural analysis of 9 patients showed a decrease in overall collagen fiber diameter in the papillary dermis at 1 month and 3 months after 3 treatment sessions. This is consistent with the formation of new collagen. Younger patients had a greater decrease in collagen fiber diameter compared with older patients. The change in collagen fiber diameter with time as well as the relationship between that change and the patient's age were statistically significant (P<.001). Photographic evaluation showed that those patients with preexisting erythema showed improvement in erythema along with an associated improvement in skin quality. There were no adverse events. Microsecond Nd:YAG lasers appear to be safe for nonablative laser remodeling. Our study indicates that microsecond Nd:YAG lasers can produce new collagen formation in the papillary dermis. In addition, the condition of patients with erythema may be improved. Younger patients may form more new collagen compared with older patients with photodamage.

15545546

Teaching and evaluation of surgical skills in dermatology: results of a survey.

To assess how the surgical skills of residents are taught and evaluated within dermatology residency programs in the United States; to assess which surgical techniques training directors and residents consider important for residents to perform or at least understand by the end of residency training. A 126-question survey was sent to all 106 of the US dermatology residency programs accredited by the Accreditation Council for Graduate Medical Education. Contact was initially made via e-mail. Surveys were addressed to the program director, surgical training director, and chief resident of each program. A follow-up survey was mailed to nonresponders. Ninety-five surveys were returned representing 71 (67%) of 106 programs. Eighty-nine percent of programs (n=63) reported having a formal curriculum in dermatologic surgery. Among programs represented, 97% (n=69) taught surgical skills in the procedure room, 84% (n=57) used pigs' feet, and fewer than 10% (n=6) used human cadavers. Ninety-four percent of programs (n=61) scheduled surgical lectures; two thirds (n=41) formally assigned surgical reading, and over half (n=36) used Web-based lectures to teach skills. To assess training, most programs (86%; n=50) used subjective global evaluation at the end of a surgery rotation. Fewer than 30% (n=15) discussed specific objectives prior to the rotation. Only about 25% of programs (n=17) reported the use of written or oral examinations to assess resident surgery skills. Traditional biopsy and simple surgical procedures were reported as most important to know and perform. Interest by both faculty members and residents in more advanced surgical techniques was more limited and variable. Cosmetic surgery techniques were most likely to be viewed as unimportant. Most dermatology programs teach surgical skills by traditional apprenticeship methods supplemented by work in pigs' feet laboratory classes and regularly scheduled lectures. Skill assessment is mainly done through subjective means. Almost all respondents thought that basic biopsy and excisional skills were essential for residents to know and perform. More complex surgical techniques and the use of lasers were considered less important. Cosmetic techniques were those most frequently viewed as unimportant.

15545545

An education theory-based method to teach a procedural skill.

To determine the effectiveness of an education theory-based method to teach students to place and tie a simple interrupted stitch. A teaching intervention before-after trial. Dermatology department, academic university. Fourth-year medical students and dermatology residents. Scores on a 12-criterion grading instrument before and after instruction. The scores for medical students and residents in each class showed significant improvement. The mean score for all participants (N = 23) rose by 24% after instruction (P< .001). Scores in 9 of the 12 graded performance areas improved significantly after instruction, including scores in tissue damage/teeth marks (P<.001), needle dulled/bent (P< .001), needle loaded properly and knots square (P = .01), throws done correctly (P = .01), stitch tension and needle entry/exit angle (P = .02), amount of suture used (P = .03), and correct number of throws (P = .04). In addition, participants' confidence increased significantly after instruction (P<.001). No difference was noted between men and women in preinstruction vs postinstruction score improvement. This teaching method can be effectively used to teach students to place and tie a simple interrupted stitch. Once validated and expanded, it may prove useful in shortening and standardizing procedural skill training and in objectively documenting competency.

15545544

Effect of volume and concentration on the diffusion of botulinum exotoxin A.

To investigate whether the volume of solution used to inject equivalent units of botulinum exotoxin A affects the diffusion of toxin and areas of rhytid diminution in the treatment of dynamic forehead lines. Ten volunteers with dynamic forehead lines were included. Each study patient received a single injection at a point 2.5 cm above the orbital rim on either side of the forehead with equivalent units, but in different volumes, of botulinum exotoxin A. The sides of injection were randomized; one side of the patient's forehead was injected first with 5 U of botulinum exotoxin A in 0.25 mL (2 U/0.1 mL) of preserved saline in the midpupillary line, followed by injection of the other side with 5 U in 0.05 mL of preserved saline (2 U/0.02 mL). There was a 5-fold difference in volume injected. Subjects were evaluated 14 days later for total area affected during visual inspection of the subjects' foreheads during active muscle contraction. Private dermatology office. Visual inspection to measure the area of rhytid effacement in both height and width. The area affected by the botulinum exotoxin A injection was 50% greater in the side with the larger volume in 9 of 10 subjects. The average area affected was 6.05 cm(2) for the injection of the larger volume compared with 4.12 cm(2) for the injection with the smaller volume. The shape of rhytid effacement was oval, rather than round, with the average width longer than the average height. In this prospective, randomized, controlled study, we found that injection of botulinum exotoxin A in low concentration and higher volume resulted in greater diffusion and a larger affected area. The pattern of toxin spread is altered by muscular contraction in the injected sites. These results show that the dilution has implications on the desired effect of botulinum exotoxin A.

15545543

Laser treatment of rosacea: a pathoetiological study.

To study the effect of laser treatment on rosacea, a common facial skin disease with symptoms of blushing, redness, telangiectasis, papules, pustules, and diffuse swelling of the skin, we focused on the stinging sensation and performed immunohistochemical evaluation of nerve density and neuropeptide expression. Clinical investigation as well as the lactic acid (stinger) test was performed before and 3 months after the treatment with flashlamp pulsed dye laser, when skin biopsy specimens were also taken. University hospital. Thirty-two patients with rosacea, all with positive results from the lactic acid "stinger" test, were treated by flashlamp pulsed dye laser. The biopsy specimens were taken from the stinger-positive areas in the nasolabial folds, fixed in Lanas fixative (10% formalin and 0.4% picric acid), and analyzed for the expression of protein gene product 9.5 (general nerve marker), substance P, calcitonin gene-related peptide, and vasoactive intestinal polypeptide, using a biotinylated streptavidin technique. Thirty-one patients who were stinger positive before treatment showed decreased scores after treatment, and 1 patient had the same stinger test score before and after treatment. The number of protein gene product 9.5-positive fibers in the epidermis (P< .05) as well as the papillary dermis (P< .01) was decreased. This was also the case for substance P in the papillary dermis (P< .001), whereas no evident difference was noted for vasoactive intestinal polypeptide and calcitonin gene-related peptide. No difference was found for contact between nerves and vessels (factor VIII positive). Laser treatment of rosacea that destroys small vessels has a good medical relevance because it reduces the unpleasant symptoms of the sensitive skin. A neurogenic etiology of stinging may be possible.

15545542

Treatment of atrophic facial acne scars with the 1064-nm Q-switched Nd:YAG laser: six-month follow-up study.

To quantitatively assess improvement in acne scarring after a series of nonablative laser treatments and to determine efficacy at 1-, 3-, and 6-month follow-up after treatment. Before-after trial of consecutively selected patients. Private practice at the Laser and Skin Surgery Center of New York, New York. Eleven patients with mild to moderate atrophic acne scarring were treated. A 3-dimensional optical profiling imaging system was used to assess skin topography before, during, and after treatment. Patients were treated with a 1064-nm Q-switched Nd:YAG laser and reassessed after 3 treatment sessions and at 1, 3, and 6 months after the fifth treatment session. The skin roughness analysis was quantified at baseline and at each follow-up interval. Pain, erythema, and petechiae formation were assessed on 3-point scales. At midtreatment (1 month after the third treatment session), an 8.9% improvement in roughness analysis was seen. This improvement increased to 23.3%, 31.6%, and 39.2% at 1, 3, and 6 months after the fifth treatment, respectively. Patients reported mild to moderate pain with treatment. The only adverse effects noted were transient erythema and mild pinpoint petechiae. Treatment with the nonablative 1064-nm Q-switched Nd:YAG laser results in significant quantitative improvements in skin topography in patients with mild to moderate atrophic acne scars. Continued incremental improvements were noted at 1-, 3-, and 6-month follow-up, indicating ongoing dermal collagen remodeling after the treatment.

15545541

Single-pass carbon dioxide laser skin resurfacing combined with cold-air cooling: efficacy and patient satisfaction of a prospective side-by-side study.

Ablative skin resurfacing with carbon dioxide (CO(2)) and erbium:YAG lasers is still considered the gold standard for treating rhytides, photodamage, and acne scars. However, the prolonged downtime and undesired concomitant effects that are involved have sent dermatologists looking for less invasive nonablative laser techniques to rejuvenate skin. To combine cold-air cooling with single-pass CO(2) laser skin resurfacing to generate as much benefit as possible while minimizing the spectrum of adverse effects. The efficacy of single-pass CO(2) laser skin resurfacing on perioral and periorbital wrinkles was examined prospectively during a 6-month follow-up period. In a side-by-side comparison, the influence of simultaneous cold-air cooling on concomitant effects, pain tolerance, therapeutic success, and patient satisfaction was also studied. Eight patients with perioral and/or periorbital wrinkles underwent the procedure. During laser treatment, only the right half of each face was cooled using a cold-air system. Six months after treatment, a mild improvement of the wrinkles was observed in all cases. The use of cold-air cooling did not have any impact on the long-term results, although in a direct comparison between sides, it was observed that cooling reduced the recovery period from 3.9 +/- 1.5 (mean +/- SD) days to 3.5 +/- 1.4 days (P = .09) and helped postoperative erythema fade more quickly, from an average of 21.3 +/- 17.9 days to 11.7 +/- 3.9 days (P = .17). The reduction of pain was significant, which led to a much higher level of patient acceptance: on a numerical analog scale of 1 to 10, the rate decreased from an average of 6.8 +/- 1.8 (mean +/- SD) to 3.6 +/- 1.7 (P = .006). Given the clear decline in demand for invasive laser technologies, single-pass CO(2) laser skin resurfacing in conjunction with cold-air cooling is a worthwhile alternative both to conventional resurfacing and to conventional subsurfacing. The use of cold-air cooling not only minimizes intraoperative and postoperative adverse effects, it also contributes strongly to patient satisfaction.

15545540

Connective tissue remodeling induced by carbon dioxide laser resurfacing of photodamaged human skin.

To quantitatively examine the dynamics of molecular alterations involved in dermal remodeling after carbon dioxide (CO(2)) laser resurfacing of photodamaged human skin. Serial in vivo biochemical analyses after laser therapy. Academic referral center, Department of Dermatology, University of Michigan, Ann Arbor. Subjects Volunteer sample of 28 adults, 48 to 76 years old, with clinically evident photodamage of the forearms. Intervention Focal CO(2) laser resurfacing of photodamaged forearms and serial biopsies at baseline and various times after treatment. Reverse transcriptase real-time polymerase chain reaction technology and immunohistochemistry were used to assess levels of type I and type III procollagens; matrix metalloproteinases (MMPs) 1, 3, 9, and 13; tropoelastin; fibrillin; primary cytokines interleukin 1beta and tumor necrosis factor alpha; and profibrotic cytokine transforming growth factor beta1. Production of type I procollagen and type III procollagen messenger RNA peaked at 7.5 and 8.9 times baseline levels, respectively, 21 days after treatment and remained elevated for at least 6 months. Increases in messenger RNA levels of several cytokines (interleukin 1beta, tumor necrosis factor alpha, and transforming growth factor beta1) preceded and/or accompanied changes in collagen levels. Marked increases in messenger RNA levels of MMP-1 (39 130-fold), MMP-3 (1041-fold), MMP-9 (75-fold), and MMP-13 (767-fold) were noted. Levels of fibrillin and tropoelastin rose in a delayed fashion several weeks after treatment. The biochemical changes seen after CO(2) laser resurfacing proceed through a well-organized and highly reproducible wound healing response that results in marked alterations in dermal structure. These quantitative changes may serve as a means for comparison as other therapeutic modalities meant to improve the appearance of photodamaged skin are evaluated.

15545535

Nonablative laser and light therapies for skin rejuvenation.

Multiple modalities have been described for skin rejuvenation, including ablative and nonablative therapies. Because of the prolonged recovery period associated with ablative procedures that injure the epidermis, nonablative skin treatments have grown increasingly popular. Various laser- and light-based systems have been designed or applied for promoting skin remodeling without damage to the epidermis. Studies investigating the use of nonablative procedures for facial rhytids or acne scarring with clinical, histological, and objective quantitative measurements are systematically reviewed. Nonablative treatments are associated with clinical and objective improvements for the treatment of facial rhytids and acne scarring. Dermal remodeling seems to occur as a result of thermal injury, leading to dermal fibrosis without epidermal disruption. Although results are not as impressive as those of ablative treatments, nonablative procedures are effective in the treatment of photoaging and acne scarring. As technology in nonablative therapies continues to evolve, future laser and light sources may yield even more favorable results.

15545534

The use of custom-designed midfacial and submalar implants in the treatment of facial wasting syndrome.

Facial wasting syndrome is part of a lipodystrophy that occurs as a complication of highly active antiretroviral therapy. The loss of subcutaneous fat in the cheeks and temples results in a hollow-eyed, bony, emaciated appearance that is characteristic of the results of treatment of human immunodeficiency virus. Cessation of therapy results in a rebound in viral load and subsequent morbidity. The appearance of facial wasting syndrome is optimally treated with custom-designed implants that are made using high-resolution computed tomography combined with surgeon input and computer-aided design and manufacturing technology. Twenty-two patients with facial wasting syndrome were treated using either submalar implants (in more moderate cases) or custom-designed implants (in more severe cases). In each patient, the appearance of volumetric soft tissue restoration was successfully achieved, returning a permanent and more healthful appearance to the face.

15545533

Endoscopic forehead-lift using a bioabsorbable fixation device.

Endoscopic brow-lift is the method of upper face rejuvenation preferred by facial plastic surgeons and patients alike. The technique has undergone significant scrutiny regarding methods of fixation. Many techniques have been described, including external bolster dressings, tissue adhesives, Kirschner-wire fixation, cortical tunnels and troughs, and permanent or temporary screw and plate fixation. Many of these techniques are associated with postoperative alopecia, poor reliability, possible intracranial complications, and palpable hardware. Thus, the search for a safe, effective, reliable, and straightforward procedure continues. We describe our experience with an easy-to-insert bioabsorbable device that is fixed to the calvaria and provides multipoint fixation to the soft tissue.

15545532

Subcutaneous superficial musculoaponeurotic system grafting of the aging melolabial furrow.

To describe a technique of subcutaneous superficial musculoaponeurotic system grafting during rhytidectomy to treat the aging melolabial furrow. Two hundred seventeen patients received bilateral superficial musculoaponeurotic system grafts from December 1, 1996, through June 30, 2003 (a 78-month period). The patients' charts were reviewed for demographic data, follow-up dates, and complications. Selected preoperative and postoperative photographs were viewed. In the 217 patients, 434 grafts were performed, with a mean follow-up of 23.2 months. Follow-up postoperative photographs showed effective long-term effacement of the melolabial furrow. There was a 0.9% complication rate involving 4 cases of unilateral graft infection that resolved with oral antibiotic therapy. Subcutaneous superficial musculoaponeurotic system grafting of the melolabial furrow is a safe, effective technique of treating the prominent melolabial furrow. It adds very little time to the rhytidectomy procedure, is well tolerated by patients, and shows durable results.

15545531

Nonanimal stabilized hyaluronic acid for lip augmentation and facial rhytid ablation.

To evaluate the effectiveness of nonanimal stabilized hyaluronic acid as an injectable filling agent. Nonrandomized, retrospective, interventional case series. A total of 1446 consecutive patients (1029 women and 417 men) underwent intradermal injection of commercially available nonanimal stabilized hyaluronic acid (2242 treatments) for the enhancement of lip volume and contour and the reduction of visible facial rhytids. Almost 61% of all patients remained satisfied with their results after 9 months. The effect was longest in the glabellar and nasolabial fold areas. Minimal transient sequelae were noted. Nonanimal stabilized hyaluronic acid is an effective and safe facial soft tissue expander. Its duration varies with each facial area treated.

15545530

Dose response of combination photorejuvenation using intense pulsed light-activated photodynamic therapy and radiofrequency energy.

To report the results of a dose-response study using a novel photorejuvenation regimen consisting of intense pulsed light-activated photodynamic therapy and radiofrequency energy in patients with Fitzpatrick skin types I through III. A combination intense pulsed light and radiofrequency device (Syneron Medical Ltd, Yokneam, Israel) was used in combination with the topical photosensitizer aminolevulinic acid (ALA) (Levulan Kerastick; DUSA Pharmaceuticals Inc, Wilmington, Mass). Duplicate titration trials were performed on the dorsal forearm skin of 3 patients with Fitzpatrick skin types I through III. Multiple treatments, with varying times of application of ALA (30 minutes and 1, 2, and 3 hours) and intense pulsed light fluence (24-30 J/cm(2)), were completed to determine a minimal erythema dose and a maximal tolerated dose based on epidermal reaction (erythema and crusting) and patient discomfort. Radiofrequency energy levels were constant in all treatment groups. The ALA application time of 1 to 2 hours for all skin types and fluence levels of 26 to 28 and 24 to 26 J/cm(2) for Fitzpatrick skin types I/II and III, respectively, were determined to be the minimal erythema dose. The ALA application times of 2 to 3 hours and fluence levels of 28 to 30 and 26 to 28 J/cm(2) for skin types I/II and III, respectively, were determined to be the maximal tolerated dose and resulted in severe erythema and crusting. The presence of severe erythema and discomfort precluded longer ALA exposure and higher fluence levels. No epithelial breakdown was observed at any treatment levels. The dose-response results defining the minimal erythema dose and the maximal tolerated dose of intense pulsed light-activated photodynamic therapy and radiofrequency energy have led to the development of 2 photorejuvenation protocols based on skin type and severity of photodamage.

15545529

Radiofrequency treatment for middle and lower face laxity.

To compare the effectiveness of 1 and 2 radiofrequency (RF) treatments with the ThermaCool TC system (Thermage Inc, Hayward, Calif) on middle and lower face laxity. Twenty patients with mild to moderate laxity of the middle and lower face were randomly assigned to receive either a single RF treatment or 2 treatments spaced 1 month apart. Treatment energy levels were titrated to patient tolerance and ranged from 85 to 135 J/cm(2). Acute clinical response was recorded after each session. Standardized photographs were taken before treatment and at 1 and 4 months after the last treatment. Using a percentage scale, 4 blinded physicians experienced in dermatologic laser therapy independently rated improvement in nasolabial folds, marionette lines, jowls, laxity under the chin, and overall appearance. In addition, subjects completed quality-of-life surveys 1 and 4 months after treatment. Each patient paid the same fee for involvement in the study. Eleven patients received a single RF treatment, and 9 patients underwent 2 treatments. All subjects experienced mild edema and mild to moderate erythema as an acute clinical response; no patients experienced burns, skin breakdown, or scarring. At 4-month follow-up, patients in the 2-treatment group received higher scores in all categories of photographic analysis; the difference in improvement in the nasolabial folds was statistically significant (P = .04). In self-assessment ratings, individuals receiving 2 treatments reported more improvement than subjects in the single-treatment group 4 months after treatment (P = .03). In both treatment groups, physician photographic assessment demonstrated continued improvement in all subsites between the 1-month and 4-month assessments (P<.05). Although the overall change noted by both patients and physicians was modest in most patients, 75% of subjects (n = 15) stated they would consider paying for additional treatments. Two RF treatments yielded significantly better improvement than a single treatment in the nasolabial folds. Significant improvement in laxity after treatment was seen between the 1- and 4-month follow-up visits in both single- and 2-treatment groups. Although overall improvements were modest in both groups, patient satisfaction was relatively high.

15545522

Directed vascular expression of human cysteinyl leukotriene 2 receptor modulates endothelial permeability and systemic blood pressure.

The proinflammatory and vascular actions of cysteinyl leukotrienes (CysLTs) are mediated by 2 receptors: cysteinyl leukotriene 1 receptor (CysLT1R) and cysteinyl leukotriene 2 receptor (CysLT2R). However, the distinct contribution of CysLT2R to the vascular actions of CysLTs has not been addressed. We generated an endothelial cell-specific human CysLT2R (EC-hCysLT2R) transgenic (TG) mouse model using the Tie2 promoter/enhancer. Strong expression of hCysLT2R in TG lung and endothelial cells, detected by real-time polymerase chain reaction, markedly enhanced CysLT-stimulated intracellular calcium mobilization compared with endogenous expression in cells from nontransgenic mice. The permeability response to exogenous LTC4 and to endogenous CysLTs evoked by passive cutaneous anaphylaxis was augmented in TG mice. The rapid, systemic pressor response to intravenous LTC4 was also diminished in TG mice coincidentally with augmented production of nitric oxide. The development of EC-hCysLT2R mice has permitted detection of distinct vascular effects of CysLTs, which can be mediated via the CysLT2R in vivo.

15545521

High-density lipoprotein stimulates myocardial perfusion in vivo.

Several clinical studies have demonstrated a close association between plasma HDL cholesterol levels and endothelium-dependent vasodilation in peripheral arteries. In isolated arteries, HDL has been shown to mediate vasodilation via NO release. In vivo, administration of reconstituted HDL restored abnormal endothelial function of the brachial artery in hypercholesterolemic patients. However, no data are currently available on the effect of HDL on myocardial perfusion. In this study, administration of human HDL enhanced incorporation of the perfusion tracer 99mTc-methoxyisobutylisonitrile (99mTc-MIBI) into the murine heart in vivo by approximately 18%. This increase was completely abolished in mice deficient for endothelial NO synthase. Because we have recently identified sphingosine 1-phosphate (S1P) as an important vasoactive component contained in HDL, we measured myocardial perfusion after administration of S1P in vivo. We observed an approximately 25% decrease in myocardial MIBI uptake, which was abolished in mice deficient for the S1P receptor S1P3. In S1P3-/- mice, the stimulatory effect of HDL on myocardial perfusion was preserved. HDL increased myocardial perfusion under basal conditions in vivo via NO-dependent mechanisms, whereas S1P inhibited myocardial perfusion through the S1P3 receptor. Thus, HDL may reduce coronary risk via direct NO-mediated vasodilatory effects on the coronary circulation.

15545520

Enhanced arteriogenesis and wound repair in dystrophin-deficient mdx mice.

The absence of functional dystrophin in Duchenne muscular dystrophy (DMD) patients and in mdx mice results in progressive muscle degeneration associated with necrosis, fibrosis, and inflammation. Because vascular supply plays a key role in tissue repair, we examined whether new blood vessel development was altered in mdx mice. In a model of hindlimb ischemia on femoral artery dissection, hindlimb perfusion, measured by laser Doppler imaging, was higher in mdx mice (0.67+/-0.26) than in wild-type (WT) mice (0.33+/-0.18, P<0.03). In keeping with these data, a significant increase in arteriole length density was found in mdx mice (13.6+/-8.4 mm/mm3) compared with WT mice (7.8+/-4.6 mm/mm3, P<0.03). Conversely, no difference was observed in capillary density between mice of the 2 genotypes. The enhanced regenerative response was not limited to ischemic skeletal muscle, because in a wound-healing assay, mdx mice showed an accelerated wound closure rate compared with WT mice. Moreover, a vascularization assay in Matrigel plugs containing basic fibroblast growth factor injected subcutaneously revealed an increased length density of arterioles in mdx (46.9+/-14.7 mm/mm3) versus WT mice (19.5+/-5.8 mm/mm3, P<0.001). Finally, serum derived from mdx mice sustained formation of endothelium-derived tubular structures in vitro more efficiently than WT serum. These results demonstrate that arteriogenesis is enhanced in mdx mice both after ischemia and skin wounding and in response to growth factors.

15545519

Resistin promotes smooth muscle cell proliferation through activation of extracellular signal-regulated kinase 1/2 and phosphatidylinositol 3-kinase pathways.

Resistin, a novel adipokine, is elevated in patients with type 2 diabetes and may play a role in the vascular complications of this disorder. One recent study has shown that resistin has a proinflammatory effect on endothelial cells. However, there is no information on whether resistin could also affect vascular smooth muscle cells (SMCs). Thus, the purpose of this study was to assess whether resistin could induce SMC proliferation and to study the mechanisms whereby resistin signals in SMCs. Human aortic smooth muscle cells (HASMCs) were stimulated with increasing concentrations of resistin for 48 hours. Cell proliferation was induced by resistin in a dose-dependent manner as assessed by direct cell counting. To gain more insights into the mechanism of action of resistin, we investigated the extracellular signal-regulated kinase (ERK) and/or phosphatidylinositol 3-kinase (PI3K) signaling pathways. Transient phosphorylation of the p42/44 mitogen-activated protein kinase (ERK 1/2) occurred after addition of resistin to HASMCs. U0126, a specific inhibitor of ERK phosphorylation, significantly inhibited ERK 1/2 phosphorylation and reduced resistin-simulated proliferation of HASMCs. LY294002, a specific PI3K inhibitor, also significantly inhibited HASMC proliferation after resistin stimulation. Our results demonstrate that resistin induces HASMC proliferation through both ERK 1/2 and Akt signaling pathways. The proliferative action exerted by resistin on HASMCs may account in part for the increased incidence of restenosis in diabetes patients.

15545518

Temporal and spatial variations in structural protein expression during the progression from stunned to hibernating myocardium.

Dysfunctional and normally perfused remote regions show equal myolysis and glycogen accumulation in pig hibernating myocardium. We tested the hypothesis that these arose secondary to elevations in preload rather than ischemia. Expression of structural protein (desmin, desmoplakin, titin, cardiotin, alpha-smooth muscle actin, lamin-A/C, and lamin-B2) in viable dysfunctional myocardium was analyzed by immunohistochemistry. We performed blinded analysis of paired dysfunctional left anterior descending coronary artery and normal remote subendocardial samples from stunned (24 hours; n=6), and hibernating (2 weeks; n=6) myocardium versus sham controls pigs (n=7). Within 24 hours, cardiac myocytes globally reexpressed alpha-smooth muscle actin. In stunned myocardium, cardiotin was globally reduced, whereas reductions in desmin were restricted to the dysfunctional region. Alterations progressed with the transition to hibernating myocardium, in which desmin, cardiotin, and titin were globally reduced. A qualitatively similar reorganization of cytoskeletal proteins occurred 3 hours after transient elevation of left ventricular end-diastolic pressure to 33+/-3 mm Hg. Qualitative cardiomyocyte remodeling similar to that in humans with chronic hibernation occurs rapidly after a critical coronary stenosis is applied, as well as after transient elevations in left ventricular end-diastolic pressure in the absence of ischemia. Thus, reorganization of cytoskeletal proteins in patients with viable dysfunctional myocardium appears to reflect chronic and/or cyclical elevations in preload associated with episodes of spontaneous regional ischemia.

15545517

Dynamin-2 regulates oxidized low-density lipoprotein-induced apoptosis of vascular smooth muscle cell.

On exposure to oxidized low-density lipoprotein (oxLDL), vascular cells generally undergo apoptosis, which is one of the major pathogenic factors of atherosclerosis. In this study, we examined the role of dynamin (a crucial GTPase protein in endocytosis) in oxLDL-induced apoptosis of vascular smooth muscle cells (VSMC). After oxLDL stimulation, dynamin-2 colocalized with LOX-1 around the cell surface, as well as oxLDL in the cytoplasm, suggesting that dynamin-2 was involved in scavenger receptor-mediated oxLDL endocytosis. Downregulation of dynamin-2 induced by dynamin-2 dominant negative plasmid (K44A) resulted in a decrease of oxLDL uptake and thereby in a reduction of apoptosis. These data demonstrated that dynamin-2 was involved in oxLDL-induced apoptosis via the oxLDL endocytotic pathway. On the other hand, dynamin-2 wild-type plasmid transfection promoted oxLDL-induced apoptosis without increasing oxLDL uptake. Interestingly, the p53 inhibitor pifithrin-alpha (PFT) significantly reduced apoptosis promoted by wild-type dynamin-2 (78% reduction compared with the PFT[-] condition). These results indicated that dynamin-2 enhanced oxLDL-induced apoptosis of VSMC by participating in the p53 pathway, probably as a signal transducer. Moreover, we demonstrated that, in advanced plaques of apolipoprotein E-/- mice, dynamin-2 expression was often enhanced in apoptotic VSMC, suggesting that dynamin-2 might participate in apoptosis of VSMC even in vivo. Our data demonstrated that dynamin-2 at least partially regulated oxLDL-induced apoptosis of VSMC by participating in 2 independent pathways: the oxLDL endocytotic pathway and the p53 pathway. These findings suggest that dynamin-2 may serve as a new research or therapeutic target in vascular disease.

15545516

Overexpression of brain natriuretic peptide facilitates neutrophil infiltration and cardiac matrix metalloproteinase-9 expression after acute myocardial infarction.

Recent clinical trials have shown that systemic infusion of nesiritide, a recombinant human brain natriuretic peptide (BNP), improves hemodynamic parameters in acutely decompensated hearts. This suggests that BNP exerts a direct cardioprotective effect and might thus be a useful therapeutic agent with which to treat acute myocardial infarction (MI). In the present study, we used BNP-transgenic (BNP-Tg) mice with elevated plasma BNP to determine whether and how BNP contributes to left ventricular remodeling and healing after MI. We examined the accumulation of neutrophils and the expression and activation of matrix metalloproteinase (MMP)-9 in the ventricles of male BNP-Tg mice and their nontransgenic (non-Tg) littermates during the early phase after acute MI. The numbers of neutrophils infiltrating the infarcted area were significantly increased in BNP-Tg mice 3 days after MI. In addition, both the gene expression and zymographic activity of MMP-9, but not MMP-2, were significantly higher in BNP-Tg than non-Tg mice. Double immunostaining revealed that neutrophils are the main source of the MMP-9, although doxycycline, an MMP inhibitor, had no effect on neutrophil infiltration of the infarcted area in BNP-Tg mice. These results demonstrate that elevated plasma BNP facilitates neutrophil infiltration of the infarcted area after MI and increases the activity of the MMP-9 they produce. This suggests that BNP plays a key role in the processes of extracellular matrix remodeling and wound-healing during the early phase after acute MI.

15545515

Elastin degradation and calcification in an abdominal aorta injury model: role of matrix metalloproteinases.

Elastin calcification is a widespread feature of vascular pathology, and circumstantial evidence exists for a correlation between elastin degradation and calcification. We hypothesized that matrix metalloproteinase (MMP)-mediated vascular remodeling plays a significant role in elastin calcification. In the present studies, we determined that short-term periadventitial treatment of the rat abdominal aorta with low concentrations of calcium chloride (CaCl2) induced chronic degeneration and calcification of vascular elastic fibers in the absence of aneurysm formation and inflammatory reactions. Furthermore, the rate of progression of calcification depended on the application method and concentration of CaCl2 applied periarterially. Initial calcium deposits, associated mainly with elastic fibers, were persistently accompanied by elastin degradation, disorganization of aortic extracellular matrix, and moderate levels of vascular cell apoptosis. Application of aluminum ions (known inhibitors of elastin degradation) before the CaCl2-mediated injury significantly reduced elastin calcification and abolished both extracellular matrix degradation and apoptosis. We also found that MMP-knockout mice were resistant to CaCl2-mediated aortic injury and did not develop elastin degeneration and calcification. Collectively, these data strongly indicate a correlation between MMP-mediated elastin degradation and vascular calcification.

15545514

Inhibition of sphingomyelin synthesis reduces atherogenesis in apolipoprotein E-knockout mice.

In clinical studies, sphingomyelin (SM) plasma levels correlated with the occurrence of coronary heart disease independently of plasma cholesterol levels. We hypothesized that inhibition of SM synthesis would have antiatherogenic effects. To test this hypothesis, apolipoprotein E (apoE)-knockout (KO) mice were treated with myriocin, a potent inhibitor of serine palmitoyltransferase, the rate-limiting enzyme in SM biosynthesis. Diet-admix treatment of apoE-KO mice with myriocin in Western diet for 12 weeks lowered SM and sphinganine plasma levels. Decreases in sphinganine and SM concentrations were also observed in the liver and aorta of myriocin-treated animals compared with controls. Inhibition of de novo sphingolipid biosynthesis reduced total cholesterol and triglyceride plasma levels. Cholesterol distribution in lipoproteins demonstrated a decrease in beta-VLDL and LDL cholesterol and an increase in HDL cholesterol. Oil red O staining of total aortas demonstrated reduction of atherosclerotic lesion coverage in the myriocin-treated group. Atherosclerotic plaque area was also reduced in the aortic root and brachiocephalic artery. Inhibition of de novo SM biosynthesis in apoE-KO mice lowers plasma cholesterol and triglyceride levels, raises HDL cholesterol, and prevents development of atherosclerotic lesions.

15545513

Cardiovascular, renal, and metabolic responses to chronic central administration of agouti-related peptide.

Although excess hypothalamic agouti-related peptide (AGRP), an endogenous antagonist of the melanocortin 3/4 receptor, causes hyperphagia and obesity, its role in regulating cardiovascular function is unclear. This study examined control of mean arterial pressure (MAP), heart rate (HR), and metabolism during chronic central administration of AGRP in rats. A cannula was placed in the lateral ventricle for intracerebroventricular infusion, and arterial and venous catheters were implanted for monitoring MAP and HR 24 hours per day, as well as intravenous infusions. After a 5-day control period, rats received AGRP (n=6; 0.02 nmol per hour ICV) or artificial cerebrospinal fluid (aCSF; n=9; 0.02 nmol per hour ICV) for 12 days, followed by a 5-day recovery period. A third group was infused intracerebroventricularly with AGRP and pair-fed to match food intake of control rats (n=7). AGRP produced a peak decrease in MAP and HR of -7+/-2 mm Hg and -68+/-7 bpm, respectively, despite increased food intake (from 23+/-0.5 to 36+/-3 g per day) and weight gain (from 350+/-8 to 454+/-5 g). AGRP also increased glomerular filtration rate, plasma insulin, glucose, and leptin. AGRP infusion in pair-fed rats produced a peak decrease in HR of -70+/-8 bpm but did not alter MAP or other variables. The metabolic effects of AGRP may be secondary to hyperphagia because they were abolished in pair-fed rats. aCSF infusion did not change any of the variables studied. These results demonstrate that increased central nervous system AGRP levels produce chronic reductions in MAP and HR despite marked increases in food intake and weight gain that normally tend to raise arterial pressure.

15545512

Novel approach to examining first cardiovascular events after hypertension onset.

Hypertension confers risk for multiple types of cardiovascular events, but competing risks for these outcomes are unknown. We estimated the competing risks over 12 years after hypertension onset among cases and age-, sex-, and examination-matched controls using competing Cox cumulative incidence and proportional hazards models. We included all Framingham Heart Study subjects examined after 1977 with new-onset hypertension who were free of cardiovascular disease. There were 645 men and 702 women with new-onset hypertension (mean age: men, 55+/-12 years; women, 59+/-12 years). Compared with matched nonhypertensive controls, subjects with new-onset hypertension were more likely to experience a cardiovascular event first rather than noncardiovascular death. Among new-onset hypertensives, the 12-year competing cumulative incidence of any cardiovascular end point as a first event in men was 24.7%, compared with 9.8% for noncardiovascular death (hazards ratio [HR], 2.53; 95% confidence interval [CI], 1.83 to 3.50); in women, the competing incidences were 16.0% versus 10.1%, respectively (HR, 1.58; 95% CI, 1.13 to 2.20). The most common first major cardiovascular events were hard coronary disease (8.2%) in men and stroke (5.2%) in women. Type and incidence of first cardiovascular events varied by age and severity of hypertension at onset, with stroke predominating among older subjects with new-onset hypertension. After hypertension onset, cardiovascular events are more likely to occur first as opposed to noncardiovascular death. Types of initial events differ by gender, age, and severity of hypertension at onset. These results represent a novel approach to understanding the complications of hypertension and may help target therapies for patients with new-onset hypertension to optimize prevention strategies.

15545511

Role of podocytes for reversal of glomerulosclerosis and proteinuria in the aging kidney after endothelin inhibition.

The cause of focal-segmental glomerulosclerosis as a consequence of physiological aging, which is believed to be inexorable, is unknown. This study investigated whether inhibition of endothelin-1, a growth-promoting peptide contributing to renal injury in hypertension and diabetes, affects established glomerulosclerosis and proteinuria in the aged kidney. We also determined the role of endothelin receptors for podocyte injury in vivo and in vitro. Aged Wistar rats, a model of spontaneous age-dependent glomerulosclerosis, were treated with the orally active endothelin subtype A (ET(A)) receptor antagonist darusentan, and evaluation of renal histology, renal function studies, and expression analyses were performed. In vitro experiments using puromycin aminonucleoside to induce podocyte injury investigated the role of ET(A) receptor signaling for apoptosis, cytoskeletal injury, and DNA synthesis. In aged Wistar rats, established glomerulosclerosis and proteinuria were reduced by >50% after 4 weeks of darusentan treatment, whereas blood pressure, glomerular filtration rate, or tubulo-interstitial renal injury remained unaffected. Improvement of structural injury in glomeruli and podocytes was accompanied by a reduction of the expression of matrix metalloproteinase-9 and p21Cip1/WAF1. In vitro experiments blocking ET(A) receptors using specific antagonists or RNA interference prevented apoptosis and structural damage to podocytes induced by puromycin aminonucleoside. In conclusion, these results support the hypothesis that endogenous endothelin contributes to glomerulosclerosis and proteinuria in the aging kidney. The results further suggest that age-dependent glomerulosclerosis is not merely a "degenerative" but a reversible process locally confined to the glomerulus involving recovery of podocytes from previous injury.

15545510

Angiotensin-(1-7) attenuates neointimal formation after stent implantation in the rat.

Angiotensin-(1-7) is an endogenous, biologically active peptide of the renin-angiotensin system with vasodilatory, antithrombotic, and antiproliferative properties. This study examined the effects of angiotensin-(1-7) infusion on neointimal formation after stent placement in the rat. Male Wistar rats underwent stent implantation in the abdominal aorta or sham surgery. Subsequently, an osmotic minipump was placed for angiotensin-(1-7) (24 microg/kg per hour) or saline administration. After 4 weeks, histomorphometric and histological analyses were performed, and the endothelial function was measured in isolated thoracic aortic rings. Stent implantation resulted in equal mean injury scores within the groups. The angiotensin-(1-7)-treated group displayed a significant reduction in neointimal thickness (112+/-8 versus 141+/-11 microm; P<0.05), neointimal area (0.51+/-0.05 versus 0.70+/-0.07 mm2; P<0.05), and percentage stenosis (10.4+/-1.0 versus 14.0+/-1.3%; P<0.05) compared with the saline-treated group. Furthermore, angiotensin-(1-7) infusion attenuated the stenting-induced impairment in endothelium-dependent relaxation (42.6+/-3.0 versus 64.5+/-6.0% of phenylephrine maximal contraction; P<0.001). In conclusion, angiotensin-(1-7) treatment attenuates neointimal formation after stent implantation in the rat, combined with an improvement of endothelial function.

15545509

Pulse pressure and coronary atherosclerosis progression in postmenopausal women.

Pulse pressure, an index of large artery stiffness, has been associated with coronary events. However, mechanisms for this association remain unclear. In this study, we examined the relationship between pulse pressure and the progression of coronary atherosclerosis and the effects of hormone replacement therapy (HRT) on pulse pressure in postmenopausal women with angiographically confirmed coronary disease followed for 3.2 years in the Estrogen Replacement in Atherosclerosis (ERA) trial. In the ERA trial, 309 postmenopausal women (mean age 66+/-7 years) with coronary disease were randomized to estrogen, estrogen plus progestin, or placebo, and followed for 3.2 years. Ten standardized epicardial segments were measured for minimal diameter values at baseline and follow-up using quantitative coronary angiography. For this study, mixed-model analysis of covariance was used to: (1) test the association between pulse pressure and change in mean minimum diameter (MMD) adjusted for baseline MMD and (2) the effect of HRT on follow-up pulse pressure. After adjustment for potential confounders, there was a significant graded increase in progression of coronary stenosis with increasing quartiles of baseline pulse pressure (P test for trend=0.0001). The progression rate in women with the highest quartile of baseline pulse pressure was 5-fold higher than in women in the lowest quartile (P<0.01). In postmenopausal women with coronary disease, increased levels of baseline pulse pressure are associated with subsequent progression of coronary atherosclerosis in postmenopausal women. HRT had no detectable effect on pulse pressure.

15545508

Treating osteoarthritis with cyclooxygenase-2-specific inhibitors: what are the benefits of avoiding blood pressure destabilization?

Osteoarthritis and hypertension are highly prevalent among older Americans. Anti-inflammatory medications can destabilize blood pressure control. We estimated the decreased cardiovascular risk, premature mortality, and direct health care costs that could be avoided if blood pressure control is not destabilized among hypertensive Americans taking cyclooxygenase-2 (COX-2)-specific inhibitors for osteoarthritis. Data from the Third National Health and Nutrition Examination Survey (NHANES III) provided the distribution of cardiovascular risk factors among American adults with osteoarthritis and hypertension. The Cardiovascular Disease Life Expectancy Model was used to estimate the impact of a 2.26% increase in systolic blood pressure on the basis of results of a randomized trial comparing COX-2-specific inhibitors. A similar analysis was completed for American adults with osteoarthritis and untreated hypertension (> or =140/90 mm Hg). Among 7.3 million Americans with treated hypertension, maintaining blood pressure control would avoid >30,000 stroke deaths and 2,000 coronary deaths resulting in >449,000 person years of life saved and 1.4 billion dollars in direct health care cost savings. When an additional 3.8 million Americans with untreated hypertension are considered, maintaining blood pressure control could prevent >47,000 stroke deaths, 39,000 coronary deaths, and result in 668,000 person years of life saved and >2.4 billion dollars in direct health care cost savings. We conclude that even a small increase in systolic blood pressure among hypertensive Americans with osteoarthritis may substantially increase the clinical and economic burden of cardiovascular disease. Maintaining blood pressure control may be associated with substantial benefits.

15545507

Transcriptional regulation of renin: an update.

Renin, as a component of the renin-angiotensin system, plays important roles in the regulation of blood pressure, electrolyte homeostasis, and mammalian renal development. Transcription of renin genes is subject to complex developmental and tissue-specific regulation. Progress has been made recently in elucidating the molecular mechanisms involved in renin gene expression. Using mouse As4.1 cells, which have many features characteristic of the renin-expressing juxtaglomerular cells of kidney, a proximal promoter region (-197 to -50 bp) and an enhancer (-2866 to -2625 bp) have been identified in the mouse renin gene, Ren-1(c), that are critical for its expression. The proximal promoter region contains at least 7 transcription factor-binding sites, including a binding site for the products of Hox, developmental control genes. The enhancer consists of at least 11 transcription factor-binding sites and is responsive to various signal transduction pathways, including cAMP, retinoic acid, endothelin-1, and cytokines, to alter renin mRNA levels. Sequence highly homologous to the mouse enhancer is also found in the human and rat renin genes. How these regulatory regions function in vivo will be the focus of future study.

15545506

Extracorporeal high intensity focused ultrasound ablation in the treatment of patients with large hepatocellular carcinoma.

High intensity focused ultrasound (HIFU) is a noninvasive treatment modality that induces complete coagulative necrosis of a deep tumor through the intact skin. The current study was conducted to determine the safety, efficacy, and feasibility of extracorporeal HIFU in the treatment of patients with hepatocellular carcinoma (HCC). A total of 55 patients with HCC with cirrhosis were enrolled in this prospective, nonrandomized clinical trial. Among them, 51 patients had unresectable HCC. Tumor size ranged from 4 to 14 cm in diameter with mean diameter of 8.14 cm. According to tumor, node, metastasis (TNM) classification, 15 patients corresponded to stage II, 16 to stage IIIA, and 24 to IIIC. All patients had HIFU, and the median number of HIFU session was 1.69. Safety and efficacy of HIFU were assessed in this trial. No severe side effect was observed in the patients treated with HIFU. Follow-up imaging showed an absence of tumor vascular supply and the shrinkage of treated lesions. Serum alpha-fetoprotein returned to normal level in 34% of patients. The overall survival rates at 6, 12, and 18 months were 86.1%, 61.5%, and 35.3%, respectively. The survival rates were significantly higher in patients in stage II than those in stage IIIA (P = .0132) and in stage IIIC (P = .0265). As a noninvasive therapy, HIFU appears to be effective, safe, and feasible in the treatment of patients with HCC. It may play an important role in the ablation of large tumors.

15545505

Prediction of lymph node metastasis with use of artificial neural networks based on gene expression profiles in esophageal squamous cell carcinoma.

The aim of the study was (1) to detect candidate genes involved in lymph node metastasis in esophageal cancers and (2) to investigate whether we can estimate and predict occurrence of lymph node metastasis by analyzing artificial neural networks (ANNs) using these gene subsets. Twenty-eight primary esophageal squamous cell carcinomas were used. Gene expression profiles of all primary tumors were obtained by cDNA microarray. Lymph node metastasis-related genes were extracted with use of Significance Analysis of Microarrays (SAM). Predictive accuracy for lymph node metastasis was calculated by evaluation of 28 cases by ANNs with leave-one-out cross-n. The results were compared with those of other analyses such as clustering or predictive scoring (LMS). Our ANN model could predict lymph node metastasis most accurately with 60 clones. The highest predictive accuracy for lymph node metastasis by ANN was 10 of 13 (77%) in newly added cases that were not used for gene selection by SAM and 24 of 28 (86%) in all cases (sensitivity: 15/17, 88%; specificity: 9/11, 82%). Predictive accuracy of LMS was 9 of 13 (69%) in newly added cases and 24 of 28 (86%) in all cases (sensitivity: 17/17, 100%; specificity: 7/11, 67%). It was difficult to extract useful information for the prediction of lymph node metastasis by clustering analysis. ANN had superior potential in comparison with other methods of analysis for the prediction of lymph node metastasis. This systematic analysis combining SAM with ANN was very useful for the prediction of lymph node metastasis in esophageal cancers and could be applied clinically in the near future.

15545504

Assessment of the current Medicare reimbursement system for breast cancer operations.

Medicare determines procedural reimbursement by means of formulas considering physician work, practice, and liability expenses. Since no mechanism exists to consider outcomes in calculating reimbursements, we hypothesized that Medicare reimbursements do not correlate with outcomes for different breast cancer operations. We prospectively studied 240 patients with T1, 2N0M0 breast cancer in three surgical treatment arms: segmental mastectomy with axillary node dissection (SM&ALND ; n = 42); SM with sentinel node dissection (SM&SLND ; n = 96); and mastectomy without reconstruction (MRM; n = 102). Outcome parameters of complications, hospital stay, analgesic usage, and days to return to work were correlated with procedure reimbursements. Median follow-up was 26 months. SM&SLND patients rarely required hospital stays (14%) in comparison with either SM&ALND (96%) or MRM patients (99%) (P < 0.001). SM&ALND and MRM patients required 9 and 10 median days of narcotics, respectively, versus 1 day in the SLND group (P < 0.001). SM&SLND patients returned to work at a median of 3 days, in comparison with 19 for SM&ALND and 26 for MRM patients (P < 0.001). Complications were more common in the MRM group (67% numbness/10% pain) and the SM&ALND group (56%/9%) than in the SM&SLND group (0%/1%). Reimbursements were inversely correlated with outcomes. MRM was reimbursed the highest, at an average of 1,075.03 dollars, with SM&ALND at 882.72 dollars. SM&SLND was reimbursed at 642.00 dollars. Medicare reimbursements for breast cancer operations do not correlate with outcomes. Less-invasive procedures are paid for at lower rates despite better outcomes and fewer complications. The data from this study raise the question of the impact of reimbursement on breast procedure selection.

15545503

Sentinel lymph node metastases detected by immunohistochemistry only do not mandate complete axillary lymph node dissection in breast cancer.

The significance of breast cancer sentinel lymph node (SLN) metastases detected only by immunohistochemistry staining (IHC) remains poorly understood. This study attempted to quantify the risk of non-SLN metastases. A prospectively collected database of 750 consecutive SLN biopsy procedures in breast cancer patients was reviewed. Medical records were reviewed to supplement the database. SLNs were identified in 738 (98.4%) of these procedures in 723 patients. Of these, 151 patients (20.5%) had metastases detected by hematoxylin and eosin staining (H&E), and 33 (4.6%) of the 718 with known IHC staining results had metastases detected by IHC only. Twenty-eight (84.8%) of 33 patients with IHC-detected metastases underwent complete axillary lymph node dissection (CALND). The median primary tumor size was 2.0 cm among those undergoing CALND and 0.9 cm among the five patients treated without CALND (P = .10). Two of the 28 patients (7.1%) had additional metastases detected with CALND. These patients had a T3 or T4 invasive lobular primary tumor. Of 24 patients with T1 or T2 primary tumors and IHC-detected metastases who underwent CALND, none had additional metastases detected. Median follow-up was 14.5 months. All patients with IHC-detected SLN metastases were treated with adjuvant systemic therapy. None of the five patients with IHC-detected metastases not undergoing CALND has subsequently manifested clinical axillary disease. CALND could have been or was safely omitted in 29 of 29 patients with T1 or T2 primary tumors and metastases detected by IHC. Such patients should be counseled about this low risk before CALND is recommended.

15545502

In vivo optical imaging of pleural space drainage to lymph nodes of prognostic significance.

Understanding the spatial and temporal drainage patterns of the pleural space could have profound impact on the treatment of lung cancer and mesothelioma. The purpose of this study was to identify the in vivo pattern of drainage from the pleural space to prognostic lymph node stations. Fifty-six rats underwent pleural space injection of a novel lymph tracer composed of recombinant human serum albumin (HSA) covalently conjugated to the near-infrared (NIR) fluorophore IRDye78 via an amide bond (HSA-78). Nodal uptake was imaged at 10, 20, 30, and 60 minutes and 4, 12, and 24 hours after injection with a custom system that simultaneously acquires color video, NIR fluorescence of HSA-78, and a merged picture of the two. Six pigs underwent the same procedure with imaging at 30 minutes, 1 hour, and 24 hours. In both the rat model and the pig model, HSA-78 drained from the pleural space to superior mediastinal lymph nodes first, followed by other intrathoracic and then extrathoracic lymph nodes over the course of 24 hours. NIR fluorescence imaging in two species shows that the superior mediastinal lymph nodes are the first to drain the pleural space. Over the course of 24 hours, the pleural space also communicates with other intrathoracic and then extrathoracic lymph nodes. This study also demonstrates an intraoperative method for identifying nodes communicating with the pleural space, with potential utility in the staging and/or resection of lung cancer and mesothelioma.

15545499

Intraspecies sequence comparisons for annotating genomes.

Analysis of sequence variation among members of a single species offers a potential approach to identify functional DNA elements responsible for biological features unique to that species. Due to its high rate of allelic polymorphism and ease of genetic manipulability, we chose the sea squirt, Ciona intestinalis, to explore intraspecies sequence comparisons for genome annotation. A large number of C. intestinalis specimens were collected from four continents, and a set of genomic intervals were amplified, resequenced, and analyzed to determine the mutation rates at each nucleotide in the sequence. We found that regions with low mutation rates efficiently demarcated functionally constrained sequences: these include a set of noncoding elements, which we showed in C. intestinalis transgenic assays to act as tissue-specific enhancers, as well as the location of coding sequences. This illustrates that comparisons of multiple members of a species can be used for genome annotation, suggesting a path for the annotation of the sequenced genomes of organisms occupying uncharacterized phylogenetic branches of the animal kingdom. It also raises the possibility that the resequencing of a large number of Homo sapiens individuals might be used to annotate the human genome and identify sequences defining traits unique to our species.

15545498

Extensive and breed-specific linkage disequilibrium in Canis familiaris.

The 156 breeds of registered dogs in the United States offer a unique opportunity to map genes important in disease susceptibility, morphology, and behavior. Linkage disequilibrium (LD) is of current interest for its application in whole genome association mapping, since the extent of LD determines the feasibility of such studies. We have measured LD at five genomic intervals, each 5 Mb in length and composed of five clusters of sequence variants spaced 800 kb-1.6 Mb apart. These intervals are located on canine chromosomes 1, 2, 3, 34, and 37, and none is under obvious selective pressure. Approximately 20 unrelated dogs were assayed from each of five breeds: Akita, Bernese Mountain Dog, Golden Retriever, Labrador Retriever, and Pekingese. At each genomic interval, SNPs and indels were discovered and typed by resequencing. Strikingly, LD in canines is much more extensive than in humans: D' falls to 0.5 at 400-700 kb in Golden Retriever and Labrador Retriever, 2.4 Mb in Akita, and 3-3.2 Mb in Bernese Mountain Dog and Pekingese. LD in dog breeds is up to 100x more extensive than in humans, suggesting that a correspondingly smaller number of markers will be required for association mapping studies in dogs compared to humans. We also report low haplotype diversity within regions of high LD, with 80% of chromosomes in a breed carrying two to four haplotypes, as well as a high degree of haplotype sharing among breeds.

15545497

Coelacanth genome sequence reveals the evolutionary history of vertebrate genes.

The coelacanth is one of the nearest living relatives of tetrapods. However, a teleost species such as zebrafish or Fugu is typically used as the outgroup in current tetrapod comparative sequence analyses. Such studies are complicated by the fact that teleost genomes have undergone a whole-genome duplication event, as well as individual gene-duplication events. Here, we demonstrate the value of coelacanth genome sequence by complete sequencing and analysis of the protocadherin gene cluster of the Indonesian coelacanth, Latimeria menadoensis. We found that coelacanth has 49 protocadherin cluster genes organized in the same three ordered subclusters, alpha, beta, and gamma, as the 54 protocadherin cluster genes in human. In contrast, whole-genome and tandem duplications have generated two zebrafish protocadherin clusters comprised of at least 97 genes. Additionally, zebrafish protocadherins are far more prone to homogenizing gene conversion events than coelacanth protocadherins, suggesting that recombination- and duplication-driven plasticity may be a feature of teleost genomes. Our results indicate that coelacanth provides the ideal outgroup sequence against which tetrapod genomes can be measured. We therefore present L. menadoensis as a candidate for whole-genome sequencing.

15545496

Noncoding regulatory sequences of Ciona exhibit strong correspondence between evolutionary constraint and functional importance.

We show that sequence comparisons at different levels of resolution can efficiently guide functional analyses of regulatory regions in the ascidians Ciona savignyi and Ciona intestinalis. Sequence alignments of several tissue-specific genes guided discovery of minimal regulatory regions that are active in whole-embryo reporter assays. Using the Troponin I (TnI) locus as a case study, we show that more refined local sequence analyses can then be used to reveal functional substructure within a regulatory region. A high-resolution saturation mutagenesis in conjunction with comparative sequence analyses defined essential sequence elements within the TnI regulatory region. Finally, we found a significant, quantitative relationship between function and sequence divergence of noncoding functional elements. This work demonstrates the power of comparative sequence analysis between the two Ciona species for guiding gene regulatory experiments.

15545495

A survey of RNA editing in human brain.

We have conducted a survey of RNA editing in human brain by comparing sequences of clones from a human brain cDNA library to the reference human genome sequence and to genomic DNA from the same individual. In the RNA sample from which the library was constructed, approximately 1:2000 nucleotides were edited out of >3 Mb surveyed. All edits were adenosine to inosine (A-->I) and were predominantly in intronic and in intergenic RNAs. No edits were found in translated exons and few in untranslated exons. Most edits were in high-copy-number repeats, usually Alus. Analysis of the genome in the vicinity of edited sequences strongly supports the idea that formation of intramolecular double-stranded RNA with an inverted copy underlies most A-->I editing. The likelihood of editing is increased by the presence of two inverted copies of a sequence within the same intron, proximity of the two sequences to each other (preferably within 2 kb), and by a high density of inverted copies in the vicinity. Editing exhibits sequence preferences and is less likely at an adenosine 3' to a guanosine and more likely at an adenosine 5' to a guanosine. Simulation by BLAST alignment of the double-stranded RNA molecules that underlie known edits indicates that there is a greater likelihood of A-->I editing at A:C mismatches than editing at other mismatches or at A:U matches. However, because A:U matches in double-stranded RNA are more common than all mismatches, overall the likely effect of editing is to increase the number of mismatches in double-stranded RNA.

15545494

Proposal to list ATCC 43642 as the type strain of Leptospira interrogans in the Approved Lists of Bacterial Names. Request for an opinion.

ATCC 23581(T) has been cited as the type strain of Leptospira interrogans since 1980. In 1986, the Centers for Disease Control informed the ATCC that this strain was Leptospira interrogans serovar budapest, not serovar icterohaemorrhagiae as deposited originally. An authentic culture of the Leptospira interrogans serovar icterohaemorrhagiae RGA(T) strain was then submitted and assigned ATCC 43642, which was designated as the type strain of Leptospira interrogans in an article by Yasuda et al. [Yasuda et al. (1987). Int J Syst Bacteriol 37, 407-415]. In this Request for an Opinion to the Judicial Commission, it is proposed that a correction be made in the Approved Lists of Bacterial Names to acknowledge ATCC 43642 as the type strain of Leptospira interrogans.

15545493

Proposal to acknowledge Beijerinck as the original author of the species Pantoea agglomerans. Request for an opinion.

The name 'Bacillus agglomerans' was first published by Beijerinck in 1888. Ewing and Fife changed the name to Enterobacter agglomerans in 1972 as a new combination, acknowledging that Beijerinck had been the original author and making the new full name Enterobacter agglomerans (Beijerinck 1888) Ewing and Fife 1972. Beijerinck's name was omitted from the Approved Lists of Bacterial Names in 1980, which listed only Ewing and Fife as the authors. The current listings in the List of Bacterial Names with Standing in Nomenclature follow the Approved Lists and omit Beijerinck from the names for both Enterobacter agglomerans and Pantoea agglomerans. It is proposed that Beijerinck be acknowledged as the original author of this species by having his name reinstated in its full name, Pantoea agglomerans (Beijerinck 1888) Gavini et al. 1989, and a Request for an Opinion is put forward to the Judicial Commission.

15545492

Kazachstania aerobia sp. nov., an ascomycetous yeast species from aerobically deteriorating corn silage.

In an investigation of the yeast biota involved in silage deterioration, a considerable number of strains belonging to Saccharomyces and related genera were isolated from aerobically deteriorating corn silage in Tochigi, Japan. Analysis of sequences of the internal transcribed spacer and the large-subunit rRNA gene D1/D2 domain and electrophoretic karyotyping indicated that two of the strains, NS 14(T) and NS 26, represent a novel species with close phylogenetic relationships to Kazachstania servazzii and Kazachstania unispora. It is proposed that the novel species be named Kazachstania aerobia sp. nov., with NS 14(T) (=AS 2.2384(T)=CBS 9918(T)) as the type strain.

15545491

Expansion of the Candida tanzawaensis yeast clade: 16 novel Candida species from basidiocarp-feeding beetles.

A major clade of new yeast taxa from the digestive tract of basidiocarp-feeding beetles is recognized based on rRNA gene sequence analyses. Almost 30 % of 650 gut isolates formed a statistically well-supported clade that included Candida tanzawaensis. The yeasts in the clade were isolated from 11 families of beetles, of which Tenebrionidae and Erotylidae were most commonly sampled. Repeated isolation of certain yeasts from the same beetle species at different times and places indicated strong host associations. Sexual reproduction was never observed in the yeasts. Based on comparisons of small- and large-subunit rRNA gene sequences and morphological and physiological traits, the yeasts were placed in Candida ambrosiae and in 16 other undescribed taxa. In this report, the novel species in the genus Candida are described and their relationships with other taxa in the Saccharomycetes are discussed. The novel species and their type strains are as follows: Candida guaymorum (NRRL Y-27568(T)=CBS 9823(T)), Candida bokatorum (NRRL Y-27571(T)=CBS 9824(T)), Candida kunorum (NRRL Y-27580(T)=CBS 9825(T)), Candida terraborum (NRRL Y-27573(T)=CBS 9826(T)), Candida emberorum (NRRL Y-27606(T)=CBS 9827(T)), Candida wounanorum (NRRL Y-27574(T)=CBS 9828(T)), Candida yuchorum (NRRL Y-27569(T)=CBS 9829(T)), Candida chickasaworum (NRRL Y-27566(T)=CBS 9830(T)), Candida choctaworum (NRRL Y-27584(T)=CBS 9831(T)), Candida bolitotheri (NRRL Y-27587(T)=CBS 9832(T)), Candida atakaporum (NRRL Y-27570(T)=CBS 9833(T)), Candida panamericana (NRRL Y-27567(T)=CBS 9834(T)), Candida bribrorum (NRRL Y-27572(T)=CBS 9835(T)), Candida maxii (NRRL Y-27588(T)=CBS 9836(T)), Candida anneliseae (NRRL Y-27563(T)=CBS 9837(T)) and Candida taliae (NRRL Y-27589(T)=CBS 9838(T)).

15545490

Candida leandrae sp. nov., an asexual ascomycetous yeast species isolated from tropical plants.

The novel yeast species Candida leandrae is described based on eight isolates from decaying fruits of Leandra reversa Cogn. (Melastomataceae) in an Atlantic rainforest site in Brazil, one from a Convolvulaceae flower in Costa Rica and one from a drosophilid in Hawai'i. The strains differed in their colony morphology, one being butyrous and smooth and the other being filamentous and rugose. Sequences of the D1/D2 domains of the large-subunit rRNA gene from both morphotypes were identical. C. leandrae belongs to the Kodamaea clade and is closely related to Candida restingae. The two species can be separated on the basis of growth at 37 degrees C and the assimilation of melezitose, negative in the novel species. The type culture of C. leandrae is strain UNESP 00-64R(T) (=CBS 9735(T)=NRRL Y-27757(T)).

15545489

Phylum-specific environmental DNA analysis reveals remarkably high global biodiversity of Cercozoa (Protozoa).

This study presents the first 18S rRNA multi-library environmental PCR survey of a single protozoan phylum, Cercozoa Cavalier-Smith 1998, from a range of different habitats. Phylogenetic analysis reveals at least nine novel clades within the phylum, several possibly at the level of order or above. Further experiments are described to ascertain the true ecological and geographical distributions of some clades that might be inferred from the tree to be restricted in either or both ways. These results suggest that the diversity of cercozoan taxa may run into thousands of lineages, making it comparable in diversity to the largest better-characterized protozoan phyla, e.g. Ciliophora (ciliates and suctorians) and Foraminifera. New sequences of cultured Spongomonas, Metromonas and Metopion are also presented. In the light of these additions, and the increased taxon sampling from the environmental libraries, some revisions of cercozoan classification are made: the transfer of Spongomonadea from Reticulofilosa to Monadofilosa; the removal of Metopiida from Sarcomonadea; and the creation of the new order Metromonadida, currently containing the single genus Metromonas. Although Metromonas groups with weak to moderate support with Chlorarachnea, it is here placed in superclass Monadofilosa, to which it is morphologically more similar.

15545488

Mycobacterium cosmeticum sp. nov., a novel rapidly growing species isolated from a cosmetic infection and from a nail salon.

Four isolates of a rapidly growing Mycobacterium species had a mycolic acid pattern similar to that of Mycobacterium smegmatis, as determined by HPLC analyses. Three of the isolates were from footbath drains and a sink at a nail salon located in Atlanta, GA, USA; the fourth was obtained from a granulomatous subdermal lesion of a female patient in Venezuela who was undergoing mesotherapy. By random amplified polymorphic DNA electrophoresis and PFGE of large restriction fragments, the three isolates from the nail salon were shown to be the same strain but different from the strain from the patient in Venezuela. Polymorphisms in regions of the rpoB, hsp65 and 16S rRNA genes that were shown to be useful for species identification matched for the two strains but were different from those of other Mycobacterium species. The 16S rRNA gene sequence placed the strains in a taxonomic group along with Mycobacterium frederiksbergense, Mycobacterium hodleri, Mycobacterium diernhoferi and Mycobacterium neoaurum. The strains produced a pale-yellow pigment when grown in the dark at the optimal temperature of 35 degrees C. Biochemical testing showed that the strains were positive for iron uptake, nitrate reduction and utilization of d-mannitol, d-xylose, iso-myo-inositol, l-arabinose, citrate and d-trehalose. The strains were negative for d-sorbitol utilization and production of niacin and 3-day arylsulfatase, although arylsulfatase activity was observed after 14 days. The isolates grew on MacConkey agar without crystal violet but not on media containing 5 % (w/v) NaCl or at 45 degrees C. They were susceptible to ciprofloxacin, amikacin, tobramycin, cefoxitin, clarithromycin, doxycycline, sulfamethoxazole and imipenem. The name Mycobacterium cosmeticum sp. nov. is proposed for this novel species; two strains, LTA-388(T) (=ATCC BAA-878(T)=CIP 108170(T)) (the type strain) and 2003-11-06 (=ATCC BAA-879=CIP 108169) have been designated, respectively, for the strains of the patient in Venezuela and from the nail salon in Atlanta, GA, USA.

15545487

Alkalibacterium psychrotolerans sp. nov., a psychrotolerant obligate alkaliphile that reduces an indigo dye.

A psychrotolerant, obligately alkaliphilic bacterium, IDR2-2(T), which is able to reduce indigo dye was isolated from a fermented polygonum indigo (Polygonum tinctorium Lour.) produced in Date, Hokkaido, using a traditional Japanese method. The isolate grew at pH 9-12 but not at pH 7-8. It was a Gram-positive, facultatively anaerobic, straight rod-shaped bacterium with peritrichous flagella. The isolate grew in 0-17 % (w/v) NaCl but not at NaCl concentrations higher than 18 % (w/v). Its major cellular fatty acids were C(14 : 0), C(16 : 0), C(16 : 1)9c and C(18 : 1)9c, and its DNA G+C content was 40.6 mol%. dl-lactic acid was the major end-product from d-glucose. No quinones could be detected. The peptidoglycan type was A4beta, Orn-d-Glu. A phylogenetic analysis based on 16S rRNA gene sequence data indicated that strain IDR2-2(T) is a member of the genus Alkalibacterium. DNA-DNA hybridization revealed low relatedness (less than 25 %) between the isolate and two phylogenetically related strains, Alkalibacterium olivapovliticus and Marinilactibacillus psychrotolerans. On the basis of phenotypic characteristics, phylogenetic data and DNA-DNA relatedness data, the isolate merits classification as a novel species, for which the name Alkalibacterium psychrotolerans sp. nov. is proposed. The type strain is IDR2-2(T) (=JCM 12281(T)=NCIMB 13981(T)).

15545486

Arthrobacter gangotriensis sp. nov. and Arthrobacter kerguelensis sp. nov. from Antarctica.

Two coryneform bacteria were isolated from a penguin rookery soil sample collected in Antarctica, near the Indian station Dakshin Gangotri (strain Lz1y(T)), and from sea water from Kerguelen island, Antarctica (strain KGN15(T)). They have morphological and chemotaxonomic properties (peptidoglycan A4alpha type; major menaquinones MK-8, MK-9 and MK-10; predominant fatty acids anteiso-C(15 : 0) and anteiso-C(17 : 0)) that are characteristic of members of the genus Arthrobacter. The isolates shared 97.8 % 16S rRNA gene sequence similarity to each other and were most closely related to Arthrobacter sulfureus (about 98.5 % sequence similarity). DNA-DNA hybridization experiments revealed 50 % relatedness between the isolates, while the levels of DNA-DNA relatedness between strains Lz1y(T) and KGN15 (T) and their phylogenetic relative, A. sulfureus, were respectively 54 and 12 %. Based on the above data and distinct phenotypic differences between the isolates and A. sulfureus, two novel species are proposed, Arthrobacter gangotriensis sp. nov. (type strain Lz1y(T)=DSM 15796(T)=JCM 12166(T)) and Arthrobacter kerguelensis sp. nov. (type strain KGN15(T)=DSM 15797(T)=JCM 12165(T)).

15545485

Campylobacter insulaenigrae sp. nov., isolated from marine mammals.

Phenotypic and phylogenetic studies were performed on four Campylobacter-like organisms recovered from three seals and a porpoise. Comparative 16S rRNA gene sequencing studies demonstrated that the organisms represent a hitherto unknown subline within the genus Campylobacter, associated with a subcluster containing Campylobacter jejuni, Campylobacter coli and Campylobacter lari. DNA-DNA hybridization studies confirmed that the bacteria belonged to a single species, for which the name Campylobacter insulaenigrae sp. nov. is proposed. The type strain of Campylobacter insulaenigrae sp. nov. is NCTC 12927(T) (=CCUG 48653(T)).

15545484

Classification of isolates from locations in Austria and Yellowstone National Park as Geobacillus tepidamans sp. nov.

Two moderately thermophilic, Gram-positive, spore-forming bacteria were isolated from different geographical locations and sources; strain GS5-97(T) from a beet sugar factory in Leopoldsdorf, Lower Austria, and strain YNP10 from a geothermally heated soil, Yellowstone National Park, USA. The sequences of their 16S rRNA genes were found to be 99.8% identical, and DNA-DNA hybridization experiments revealed that strains GS5-97(T) and YNP10 share 89.9 mol% similarity to each other, but only 34.3 and 39.2 mol% similarity, respectively, to Geobacillus caldoxylosilyticus DSM 12041(T), which is their closest related type strain. A polyphasic analysis showed that these two isolates were more similar to each other than to other characterized geobacilli. Their DNA G+C content was 43.2 and 42.4 mol%, respectively, and they were identical with respect to many phenotypic features (e.g. T(opt) 55 degrees C; pH(opt) 7.0). Both strains clearly displayed best growth when cultured aerobically. They differed slightly in their cellular fatty acid profiles and polar lipid pattern, and genotypically they could also be distinguished based on randomly amplified polymorphic DNA fingerprints and internal transcribed spacer analysis. Freeze-etching experiments revealed oblique surface layer (S-layer) lattices in both strains, and biochemical analyses of the purified S-layer proteins indicated the occurrence of glycosylation. Based on the properties of these organisms relative to those currently documented for the genus Geobacillus and for the various sister genera in the Bacillus radiation, a novel species is proposed, Geobacillus tepidamans sp. nov., with GS5-97(T) (=ATCC BAA-942(T)=DSM 16325(T)) as the type strain. Strain YNP10 has been deposited in the American Type Culture Collection as ATCC BAA-943.

15545483

Thermosinus carboxydivorans gen. nov., sp. nov., a new anaerobic, thermophilic, carbon-monoxide-oxidizing, hydrogenogenic bacterium from a hot pool of Yellowstone National Park.

A new anaerobic, thermophilic, facultatively carboxydotrophic bacterium, strain Nor1(T), was isolated from a hot spring at Norris Basin, Yellowstone National Park. Cells of strain Nor1(T) were curved motile rods with a length of 2.6-3 microm, a width of about 0.5 microm and lateral flagellation. The cell wall structure was of the Gram-negative type. Strain Nor1(T) was thermophilic (temperature range for growth was 40-68 degrees C, with an optimum at 60 degrees C) and neutrophilic (pH range for growth was 6.5-7.6, with an optimum at 6.8-7.0). It grew chemolithotrophically on CO (generation time, 1.15 h), producing equimolar quantities of H(2) and CO(2) according to the equation CO+H(2)O-->CO(2)+H(2). During growth on CO in the presence of ferric citrate or amorphous ferric iron oxide, strain Nor1(T) reduced ferric iron but produced H(2) and CO(2) at a ratio close to 1 : 1, and growth stimulation was slight. Growth on CO in the presence of sodium selenite was accompanied by precipitation of elemental selenium. Elemental sulfur, thiosulfate, sulfate and nitrate did not stimulate growth of strain Nor1(T) on CO and none of these chemicals was reduced. Strain Nor1(T) was able to grow on glucose, sucrose, lactose, arabinose, maltose, fructose, xylose and pyruvate, but not on cellobiose, galactose, peptone, yeast extract, lactate, acetate, formate, ethanol, methanol or sodium citrate. During glucose fermentation, acetate, H(2) and CO(2) were produced. Thiosulfate was found to enhance the growth rate and cell yield of strain Nor1(T) when it was grown on glucose, sucrose or lactose; in this case, acetate, H(2)S and CO(2) were produced. In the presence of thiosulfate or ferric iron, strain Nor1(T) was also able to grow on yeast extract. Lactate, acetate, formate and H(2) were not utilized either in the absence or in the presence of ferric iron, thiosulfate, sulfate, sulfite, elemental sulfur or nitrate. Growth was completely inhibited by penicillin, ampicillin, streptomycin, kanamycin and neomycin. The DNA G+C content of the strain was 51.7+/-1 mol%. Analysis of the 16S rRNA gene sequence revealed that strain Nor1(T) belongs to the Bacillus-Clostridium phylum of the Gram-positive bacteria. On the basis of the studied phenotypic and phylogenetic features, we propose that strain Nor1(T) be assigned to a new genus, Thermosinus gen. nov. The type species is Thermosinus carboxydivorans sp. nov. (type strain, Nor1(T)=DSM 14886(T)=VKM B-2281(T)).

15545482

Shewanella marisflavi sp. nov. and Shewanella aquimarina sp. nov., slightly halophilic organisms isolated from sea water of the Yellow Sea in Korea.

Two Gram-negative, motile, non-spore-forming, rod-shaped organisms, strains SW-117(T) and SW-120(T), were isolated from sea water of the Yellow Sea in Korea and subjected to a polyphasic taxonomic study. Strains SW-117(T) and SW-120(T) simultaneously contained both menaquinones (MK) and ubiquinones (Q) as isoprenoid quinones; the predominant menaquinone was MK-7 and the predominant ubiquinones were Q-7 and Q-8. The major fatty acid detected in the two strains was iso-C(15 : 0). The DNA G+C content of strains SW-117(T) and SW-120(T) was 51 and 54 mol%, respectively. Phylogenetic analyses based on 16S rRNA gene sequences showed that strains SW-117(T) and SW-120(T) fall within the radiation of the cluster comprising Shewanella species. Strains SW-117(T) and SW-120(T) showed a 16S rRNA gene sequence similarity of 97.4 % and a DNA-DNA relatedness level of 10.1 %. Strains SW-117(T) and SW-120(T) exhibited 16S rRNA gene sequence similarity levels of 93.8-98.5 % and 92.4-97.0 %, respectively, to Shewanella species. Strain SW-117(T) exhibited DNA-DNA relatedness levels of 8.3-20.3 % to the type strains of six phylogenetically related Shewanella species. On the basis of phenotypic, phylogenetic and genetic data, strains SW-117(T) and SW-120(T) were classified in the genus Shewanella as two distinct novel species, for which the names Shewanella marisflavi sp. nov. (type strain, SW-117(T)=KCCM 41822(T)=JCM 12192(T)) and Shewanella aquimarina sp. nov. (type strain, SW-120(T)=KCCM 41821(T)=JCM 12193(T)) are proposed, respectively.

15545481

Emended description of the genus Glycomyces and description of Glycomyces algeriensis sp. nov., Glycomyces arizonensis sp. nov. and Glycomyces lechevalierae sp. nov.

A polyphasic taxonomic evaluation of presumptive strains representative of the genus Glycomyces held within the Agricultural Research Service Culture Collection resulted in the discovery of three novel species. Analysis of the whole cell sugar content of these novel species, as well as of two species presently assigned to the genus, revealed that the whole cell sugar pattern was different from that reported in the formal description of the genus Glycomyces. The sugars present in all strains studied included ribose, xylose, mannose and galactose rather than xylose and arabinose as reported in the original description of the genus. Moreover, the menaquinone patterns observed for the novel species also deviated from the original genus description. The formal description of the genus Glycomyces is emended to reflect these new data. The novel species proposed and described are Glycomyces algeriensis sp. nov. (type strain NRRL B-16327(T)=DSM 44727(T)), Glycomyces arizonensis sp. nov. (type strain NRRL B-16153(T)=DSM 44726(T)) and Glycomyces lechevalierae sp. nov. (type strain NRRL B-16149(T)=DSM 44724(T)).

15545480

Aquiflexum balticum gen. nov., sp. nov., a novel marine bacterium of the Cytophaga-Flavobacterium-Bacteroides group isolated from surface water of the central Baltic Sea.

A bacterial isolate from the Baltic Sea, BA160(T), was characterized for its physiological and biochemical features, fatty acid profile, G+C content and phylogenetic position based on 16S rRNA gene sequences. The strain was isolated from the surface water of the central Baltic Sea during the decay of a plankton bloom. Phylogenetic analyses of the 16S rRNA gene sequence revealed a clear affiliation with the family 'Flexibacteraceae', and showed the closest phylogenetic relationship with the species Belliella baltica and Cyclobacterium marinum. The G+C content of the DNA was 38.4 mol%. The strain was red-coloured due to carotenoids, Gram-negative, rod-shaped, and catalase- and oxidase-positive. Growth was observed at salinities from 0 to 6 %, with an optimum around 1.5 %. Temperature for growth ranged from 4 to 40 degrees C, with an optimum around 30 degrees C. The fatty acids were dominated by branched-chain fatty acids (>87 %), with a high abundance of iso-C(15 : 0) (23 %) and anteiso-C(15 : 0) (19 %). According to its morphology, physiology, fatty acid composition, G+C content and 16S rRNA gene sequence, strain BA160(T) is considered to represent a new genus of the family 'Flexibacteraceae'. Due to its aquatic origin, the name Aquiflexum balticum gen. nov, sp. nov. is suggested for the type species (type strain, BA160(T)=DSM 16537(T)=LMG 22565(T)=CIP 108445(T)) of the new genus.

15545479

Thiomicrospira thermophila sp. nov., a novel microaerobic, thermotolerant, sulfur-oxidizing chemolithomixotroph isolated from a deep-sea hydrothermal fumarole in the TOTO caldera, Mariana Arc, Western Pacific.

A novel thermotolerant bacterium, designated strain I78(T), was isolated from a self-temperature-recording in situ colonization system deployed in a hydrothermal diffusing flow (maximal temperature 78 degrees C) at the TOTO caldera in the Mariana Arc, Western Pacific. Cells were highly motile curved rods with a single polar flagellum. Growth was observed at 15-55 degrees C (optimum 35-40 degrees C; 60 min doubling time) and pH 5.0-8.0 (optimum pH 6.0). The isolate was a microaerobic chemolithomixotroph capable of using thiosulfate, elemental sulfur or sulfide as the sole energy source, and molecular oxygen as the sole electron acceptor. The isolate was able to grow chemolithoautotrophically with carbon dioxide. Various organic substrates such as complex proteinaceous compounds, carbohydrates, organic acids, amino acids and sugars could also support growth as the carbon source instead of carbon dioxide with sulfur oxidation. The G+C content of the genomic DNA was 43.8 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolate belonged to the genus Thiomicrospira and was most closely related to Thiomicrospira crunogena strain TH-55(T) and Thiomicrospira sp. strain L-12, while DNA-DNA hybridization demonstrated that the novel isolate could be genetically differentiated from previously described strains of Thiomicrospira. On the basis of its physiological and molecular properties the isolate is representative of a novel Thiomicrospira species, for which the name Thiomicrospira thermophila sp. nov. is proposed (type strain, I78(T)=JCM 12397(T)=DSM 16397(T)).

15545478

Kaistella koreensis gen. nov., sp. nov., a novel member of the Chryseobacterium-Bergeyella-Riemerella branch.

Gram-negative, non-spore-forming, rod-shaped, yellow-pigmented bacteria isolated from a freshwater stream in Korea were investigated to determine their taxonomic position. Complete 16S rRNA gene sequence analysis indicated that the organisms should be placed in the Chryseobacterium-Bergeyella-Riemerella branch in the family Flavobacteriaceae. Phylogenetically, the strains were most closely related to Chryseobacterium balustinum ATCC 33487(T) and Chryseobacterium scophthalmum LMG 13028(T) (94.3 and 94.1 % 16S rRNA gene sequence similarity, respectively) and they clustered on a separate well-supported branch. The strains contained menaquinone MK-6 as the predominant respiratory quinone and showed higher G+C contents (41.7 mol%) than other species in the Chryseobacterium-Bergeyella-Riemerella branch and i-C(15 : 0) as a major fatty acid (47-52 %). The phylogenetic distances from any species with validly published names and their phenotypic properties confirmed that the strains constitute a separate species in a new genus, for which the name Kaistella koreensis gen. nov., sp. nov. is proposed (type strain Chj707(T)=KCTC 12107(T)=IAM 15050(T)).

15545477

Mycobacterium pyrenivorans sp. nov., a novel polycyclic-aromatic-hydrocarbon-degrading species.

The taxonomic position of a polycyclic-aromatic-hydrocarbon-degrading bacterium, strain 17A3(T), isolated from contaminated soil was determined using a combination of phenotypic and genotypic properties. The isolate showed phenotypic properties that were diagnostic for species of the genus Mycobacterium. Comparative 16S rRNA gene sequence analysis assigned 17A3(T) to the 16S rRNA gene subgroup that contains Mycobacterium aurum, Mycobacterium austroafricanum, Mycobacterium vaccae and Mycobacterium vanbaalenii, but it could clearly be distinguished from these species using a combination of physiological, chemotaxonomic markers and internal rRNA gene spacer analyses. The data showed that strain 17A3(T) (=DSM 44605(T)=NRRL B-24244(T)) merits recognition as the type strain of a novel species of the genus Mycobacterium. The name Mycobacterium pyrenivorans sp. nov. is proposed for the species because of its ability to use pyrene as a sole source of carbon and energy.

15545476

Pseudovibrio denitrificans gen. nov., sp. nov., a marine, facultatively anaerobic, fermentative bacterium capable of denitrification.

Two denitrifying strains of heterotrophic, facultatively anaerobic, marine bacteria, designated DN34(T) and DN33, were isolated from sea-water samples collected in Nanwan Bay, Kenting National Park, Taiwan. They were Gram-negative. Cells in late exponential to early stationary phase of growth were predominantly straight or curved rods, but Y- or V-shaped forms were also observed. They were motile by means of one to several lateral or subpolar flagella. Both strains required NaCl for growth and exhibited optimal growth at about 30 degrees C, pH 8 and 3 % NaCl. They were capable of anaerobic growth by carrying out denitrifying metabolism using nitrate, nitrite or nitrous oxide as terminal electron acceptors or, alternatively, by fermenting glucose, mannose, sucrose or trehalose as substrates. Anaerobic fermentative growth on glucose resulted in formation of various organic acids, including formate, lactate, acetate, pyruvate and fumarate. The major cellular fatty acids were 2-OH-14 : 0, 3-OH-14 : 0 and 16 : 0. DN34(T) and DN33 had DNA G+C contents of 51.7 and 51.6 mol%, respectively. Physiological characterization, together with phylogenetic analysis based on 16S rRNA gene sequence analysis, revealed that the two denitrifying strains could be accommodated in a novel genus, for which the name Pseudovibrio gen. nov. is proposed. Pseudovibrio denitrificans sp. nov. is the type species, with DN34(T) (=BCRC 17323(T)=JCM 12308(T)) as the type strain.

15545475

Nocardia xishanensis sp. nov., a novel actinomycete isolated from soil.

The taxonomic position of a soil isolate, strain 276(T), was established using a polyphasic approach. The organism showed a range of chemical and morphological properties consistent with its classification in the genus Nocardia. An almost complete 16S rRNA gene sequence determined for the strain was aligned with corresponding sequences of representatives of the genus Nocardia and related taxa using three tree-making algorithms. The organism formed a distinct phyletic line within the evolutionary radiation occupied by the genus Nocardia and was most closely related to the type strain of Nocardia abscessus. However, the two strains shared a low DNA-DNA relatedness value and were readily distinguished using a combination of phenotypic properties. The combined genotypic and phenotypic data show that strain 276(T) should be assigned to the genus Nocardia as a novel species. The name proposed for this new taxon is Nocardia xishanensis sp. nov. The type strain is 276(T) (=CGMCC 4.1860(T)=JCM 12160(T)).

15545474

Sulfuricurvum kujiense gen. nov., sp. nov., a facultatively anaerobic, chemolithoautotrophic, sulfur-oxidizing bacterium isolated from an underground crude-oil storage cavity.

A facultatively anaerobic, chemolithoautotrophic, sulfur-oxidizing bacterium, strain YK-1(T), was isolated from an underground crude-oil storage cavity at Kuji in Iwate, Japan. The cells were motile, curved rods and had a single polar flagellum. Optimum growth occurred in a low-strength salt medium at pH 7.0 and 25 degrees C. It utilized sulfide, elemental sulfur, thiosulfate and hydrogen as the electron donors and nitrate as the electron acceptor under anaerobic conditions, but it did not use nitrite. Oxygen also served as the electron acceptor under the microaerobic condition (O(2) in the head space 1 %). It did not grow on sugars, organic acids or hydrocarbons as carbon and energy sources. The DNA G+C content of strain YK-1(T) was 45 mol%. Phylogenetic analysis, based on the 16S rRNA gene sequence, showed that its closest relative was Thiomicrospira denitrificans in the 'Epsilonproteobacteria', albeit with low homology (90 %). On the basis of physiological and phylogenetic data, strain YK-1(T) should be classified into a novel genus and species, for which the name Sulfuricurvum kujiense gen. nov., sp. nov. is proposed. The type strain is YK-1(T) (=JCM 11577(T)=MBIC 06352(T)=ATCC BAA-921(T)).

15545473

Streptococcus equi subsp. ruminatorum subsp. nov., isolated from mastitis in small ruminants.

Six isolates of an unknown Gram-positive, catalase-negative, chain-forming, coccus-shaped organism isolated from ovine and caprine mastitis were characterized by phenotypic and molecular taxonomic methods. On the basis of cellular morphology and the results of biochemical tests, the organism was tentatively identified as a streptococcal species. Comparative 16S rRNA gene sequencing studies confirmed that the organism is a member of the genus Streptococcus, with Streptococcus equi as its closest phylogenetic relative (98.8 % similarity). DNA-DNA pairing studies showed that the unidentified organism displayed more than 70 % relatedness to the type strains of S. equi subsp. equi and subsp. zooepidemicus. Despite the relatively high DNA-DNA reassociation values, biotyping and ribotyping allowed clear differentiation of the unknown bacterium from the two recognized subspecies of S. equi. On the basis of phenotypic and molecular genetic evidence, it is proposed that the unknown Streptococcus isolates from ovine and caprine mastitis be classified as a novel subspecies, Streptococcus equi subsp. ruminatorum subsp. nov. The type strain is CECT 5772(T) (=CCUG 47520(T)=Mt 167(T)).

15545472

Taxonomy of the genus Cupriavidus: a tale of lost and found.

DNA-DNA hybridization experiments and an evaluation of phenotypic characteristics, DNA base ratios and 16S rRNA gene sequences demonstrated that Wautersia eutropha (Davies 1969) Vaneechoutte et al. 2004, the type species of the genus Wautersia, is a later synonym of Cupriavidus necator Makkar and Casida 1987, the type species of the genus Cupriavidus. In conformity with Rules 15, 17, 23a and 37a(1) of the International Code of Nomenclature of Bacteria, the genus name Cupriavidus has priority over the genus name Wautersia, and all other members of the genus Wautersia are reclassified into Cupriavidus as Cupriavidus basilensis comb. nov. (type strain LMG 18990(T)=DSM 11853(T)), Cupriavidus campinensis comb. nov. (type strain LMG 19282(T)=CCUG 44526(T)), Cupriavidus gilardii comb. nov. (type strain LMG 5886(T)=CCUG 38401(T)), Cupriavidus metallidurans comb. nov. (type strain LMG 1195(T)=DSM 2839(T)), Cupriavidus oxalaticus comb. nov. (type strain LMG 2235(T)=CCUG 2086(T)=DSM 1105(T)), Cupriavidus pauculus comb. nov. (type strain LMG 3244(T)=CCUG 12507(T)), Cupriavidus respiraculi comb. nov. (type strain LMG 21510(T)=CCUG 46809(T)) and Cupriavidus taiwanensis comb. nov. (type strain LMG 19424(T)=CCUG 44338(T)).

15545471

Loktanella hongkongensis sp. nov., a novel member of the alpha-Proteobacteria originating from marine biofilms in Hong Kong waters.

A Gram-negative, non-motile, non-spore-forming, short rod-shaped bacterium (UST950701-009P(T)) was isolated from a marine biofilm in Hong Kong waters. Colonies are pink in colour, convex with a smooth surface and entire edge. Brown diffusible pigment is produced. Whitish colonies, with otherwise identical morphology, emerge from every culture upon ageing. The white colonies can be maintained as separate cultures (UST950701-009W) without turning pink. UST950701-009P(T) and UST950701-009W have identical 16S rRNA gene sequences and similar G+C (65.9-66.2 mol%) and fatty acid (86.22-88.52 % 18 : 1omega7c) contents. Phylogenetic analysis of the 16S rRNA gene sequence places UST950701-009P(T) within the Rhodobacter group of the alpha-subclass of the Proteobacteria. The nearest neighbours belong to the genus Loktanella, with similarity values ranging from 94.5 to 95.5 %. Data on G+C and fatty acid contents support the affiliation to the genus Loktanella. UST950701-009P(T) and -009W are heterotrophic, strictly aerobic and require NaCl for growth (2.0-14.0 %). Both grow in pH 5.0-10.0 and at 8-44 degrees C. Both are positive in oxidase, catalase and beta-galactosidase tests, but they differ in the pattern of carbohydrate oxidation and assimilation. Molecular evidence together with phenotypic characteristics shows that UST950701-009P(T) constitutes a novel species within the genus Loktanella. The name Loktanella hongkongensis sp. nov. is proposed; the type strain is UST950701-009P(T) (=NRRL B-41039(T)=JCM 12479(T)) and a morphovar is UST950701-009W (=NRRL B-41040=JCM 12480).

15545470

Haloferax sulfurifontis sp. nov., a halophilic archaeon isolated from a sulfide- and sulfur-rich spring.

A pleomorphic, extremely halophilic archaeon (strain M6(T)) was isolated from a sulfide- and sulfur-rich spring in south-western Oklahoma (USA). It formed small (0.8-1.0 mm), salmon pink, elevated colonies on agar medium. The strain grew in a wide range of NaCl concentrations (6 % to saturation) and required at least 1 mM Mg(2+) for growth. Strain M6(T) was able to reduce sulfur to sulfide anaerobically. 16S rRNA gene sequence analysis indicated that strain M6(T) belongs to the family Halobacteriaceae, genus Haloferax; it showed 96.7-98.0 % similarity to other members of the genus with validly published names and 89 % similarity to Halogeometricum borinquense, its closest relative outside the genus Haloferax. Polar lipid analysis and DNA G+C content further supported placement of strain M6(T) in the genus Haloferax. DNA-DNA hybridization values, as well as biochemical and physiological characterization, allowed strain M6(T) to be differentiated from other members of the genus Haloferax. A novel species, Haloferax sulfurifontis sp. nov., is therefore proposed to accommodate the strain. The type strain is M6(T) (=JCM 12327(T)=CCM 7217(T)=DSM 16227(T)=CIP 108334(T)).

15545469

Methylobacterium nodulans sp. nov., for a group of aerobic, facultatively methylotrophic, legume root-nodule-forming and nitrogen-fixing bacteria.

Data on 72 non-pigmented bacterial strains that specifically induce nitrogen-fixing root nodules on the legume species Crotalaria glaucoides, Crotalaria perrottetii and Crotalaria podocarpa are reviewed. By SDS-PAGE analysis of total protein patterns and by 16S rRNA PCR-RFLP, these strains form a homogeneous group that is separate from other legume root-nodule-forming bacteria. The 16S rRNA gene-based phylogeny indicates that these bacteria belong to the genus Methylobacterium. They can grow on C(1) compounds such as methanol, formate and formaldehyde but not methylamine as sole carbon source, and carry an mxaF gene, encoding methanol dehydrogenase, which supports their methylotrophic metabolism. Presence of a nodA nodulation gene, and ability to nodulate plants of Crotalaria species and to fix nitrogen are features that separate the strains currently included in this group from other members of the genus Methylobacterium. The present study includes additional genotypic and phenotypic characterization of this novel Methylobacterium species, i.e. nifH gene sequence, morphology, physiology, enzymic and carbon source assimilation tests and antibiotic resistance. The name Methylobacterium nodulans sp. nov. (type strain, ORS 2060(T)=CNCM I 2342(T)=LMG 21967(T)) is proposed for this group of root-nodule-forming bacteria.