Assessment of Bacillus subtilis Plasmid pLS20 Conjugation in the Absence of Quorum Sensing Repression.

Bacillus subtilis conjugative plasmid pLS20 uses a quorum-sensing mechanism to control expression levels of its conjugation genes, involving the repressor RcopLS20, the anti-repressor RappLS20, and the signaling peptide Phr*pLS20. In previous studies, artificial overexpression of rappLS20 in the donor cells was shown to enhance conjugation efficiency. However, we found that the overexpression of rappLS20 led to various phenotypic traits, including cell aggregation and death, which might have affected the correct determination of the conjugation efficiency when determined by colony formation assay. In the current study, conjugation efficiencies were determined under different conditions using a two-color fluorescence-activated flow cytometry method and measuring a single-round of pLS20-mediated transfer of a mobilizable plasmid. Under standard conditions, the conjugation efficiency obtained by fluorescence-activated flow cytometry was 23-fold higher than that obtained by colony formation. Furthermore, the efficiency difference increased to 45-fold when rappLS20 was overexpressed.

Comparison of radiation dermatitis between hypofractionated and conventionally fractionated postoperative radiotherapy: objective, longitudinal assessment of skin color.

This study aimed to quantitatively compare radiation dermatitis due to hypofractionated (Hypo) and conventionally fractionated (Conv) external-beam radiotherapy in patients who underwent postoperative radiotherapy after breast-conserving surgery. Skin color changes, in terms of L* (brightness, white-black), a* (red-green), and b* (yellow-blue) values, due to external-beam radiotherapy were examined at alternate fractions using an objective method. Twenty-six patients were included in the Hypo group (42.56 Gy/16 fractions) and 46 in the Conv group (50 Gy/25 fractions). Radiotherapy decreased the L* value (darker) and increased the a* value (redder) gradually. These color alterations progressed linearly according to elapsed fractions and were similar between Hypo and Conv per fraction. The Hypo group showed significantly milder alterations in L* and a* values than the Conv group. The maximal dosage was significantly correlated to alterations in L* and a* values. Common Terminology Criteria for Adverse Events v4 assessment did not show a statistically significant difference between the Hypo (Grade 0:1:2 = 2:24:1) and Conv (1:39:6, p = 0.25) groups. The results of our objective analysis revealed that patients undergoing Hypo show milder color alteration than those undergoing Conv and that the maximal dosage is a useful predicator of color alteration.

Development and verification of a taxonomy of assessment metrics for surgical technical skills.

PURPOSE: To create and empirically verify a taxonomy of metrics for assessing surgical technical skills, and to determine which types of metrics, skills, settings, learners, models, and instruments were most commonly reported in the technical skills assessment literature. METHOD: In 2011-2012, the authors used a rational analysis of existing and emerging metrics to create the taxonomy, and used PubMed to conduct a systematic literature review (2001-2011) to test the taxonomy's comprehensiveness and verifiability. Using 202 articles identified from the review, the authors classified metrics according to the taxonomy and coded data concerning their context and use. Frequencies (counts, percentages) were calculated for all variables. RESULTS: The taxonomy contained 12 objective and 4 subjective categories. Of 567 metrics identified in the literature, 520 (92%) were classified using the new taxonomy. Process metrics outnumbered outcome metrics by 8:1. The most frequent metrics were "time," "manual techniques" (objective and subjective), "errors," and "procedural steps." Only one new metric, "learning curve," emerged. Assessments of basic motor skills and skills germane to laparoscopic surgery dominated the literature. Novices, beginners, and intermediate learners were the most frequent subjects, and box trainers and virtual reality simulators were the most frequent models used for assessing performance. CONCLUSIONS: Metrics convey what is valued in human performance. This taxonomy provides a common nomenclature. It may help educators and researchers in procedurally oriented disciplines to use metrics more precisely and consistently. Future assessments should focus more on bedside tasks and open surgical procedures and should include more outcome metrics.

Polysaccharide-degrading thermophiles generated by heterologous gene expression in Geobacillus kaustophilus HTA426.

Thermophiles have important advantages over mesophiles as host organisms for high-temperature bioprocesses, functional production of thermostable enzymes, and efficient expression of enzymatic activities in vivo. To capitalize on these advantages of thermophiles, we describe here a new inducible gene expression system in the thermophile Geobacillus kaustophilus HTA426. Six promoter regions in the HTA426 genome were identified and analyzed for expression profiles using ß-galactosidase reporter assay. This analysis identified a promoter region upstream of a putative amylose-metabolizing gene cluster that directed high-level expression of the reporter gene. The expression was >280-fold that without a promoter and was further enhanced 12-fold by maltose addition. In association with a multicopy plasmid, this promoter region was used to express heterologous genes. Several genes, including a gene whose product was insoluble when expressed in Escherichia coli, were successfully expressed as soluble proteins, with yields of 0.16 to 59 mg/liter, and conferred new functions to G. kaustophilus strains. Remarkably, cellulase and α-amylase genes conferred the ability to degrade cellulose paper and insoluble starch at high temperatures, respectively, generating thermophiles with the potential to degrade plant biomass. Our results demonstrate that this novel expression system expands the potential applications of G. kaustophilus.

Assessment of planning target volume margin for a small number of vertebral metastatic lesions using image-guided intensity-modulated radiation therapy by helical tomotherapy.

AIM: To evaluate an appropriate planning target volume (PTV) margin in for one to three vertebral metastases using megavolt computed tomography (MVCT) images during the course of image-guided and stereotactic intensity-modulated radiotherapy (IGRT-IMRT) by use of helical tomotherapy. PATIENTS AND METHODS: A total of 25 lesions in 24 patients with vertebral metastases who received IGRT-IMRT were analyzed. MVCT images were acquired before and after radiation therapy. Intra-fractional movement and PTV margin were calculated by comparing treatment planning images and these 310 MVCT images for right-left (RL), superior-inferior (SI), and anteroposterior (AP) dimensions. Five patients were treated by 35 Gy/5 fractions, 17 by 30 Gy/5 fractions, one by 25 Gy/5 fractions, and one by 60 Gy/30 fractions. A margin to compensate for these variations was calculated with the formula of vanHerk's equation. RESULTS: The intra-fractional motion was 0.02 (-1.3 to 1.4) ± 0.34 mm in the RL direction, -0.09 (-1.8 to 0.28) ± 0.44 mm in the SI direction, and 0.20 (-1.8 to 1.8) ± 0.36 mm in the AP direction. The required PTV margin was 0.98 mm in the RL direction, 0.69 mm in the SI direction, and 1.26 mm in the AP direction. No patient showed a deviation greater than 2 mm. CONCLUSION: The PTV margin in hypofractionated IGRT-IMRT, using helical tomotherapy for a few vertebral metastases, was 2 mm or less and our tentative PTV margin of 5 mm was sufficient and reducible.

Genome mining and motif modifications of glycoside hydrolase family 1 members encoded by Geobacillus kaustophilus HTA426 provide thermostable 6-phospho-ß-glycosidase and ß-fucosidase.

Members of glycoside hydrolase family 1 (GH1) hydrolyze various glycosides and are widely distributed in organisms. With the aim of producing thermostable GH1 catalysts with potential applications in biotechnology, three GH1 members encoded by the thermophile Geobacillus kaustophilus HTA426 (GK1856, GK2337, and GK3214) were characterized using 24 p-nitrophenyl glycosides as substrates. GK1856 and GK3214 exhibited 6-phospho-ß-glycosidase activity, while GK2337 did not. GK3214 was extremely thermostable and retained most of its activity during 7 days of incubation at 60 °C. GK3214 was found to have transglycosylation activity, a dimeric structure, and a possible motif that governed its substrate specificity. Substitution of the GK3214 motif with that of a ß-glucosidase resulted in the unexpected generation of a thermostable, highly specific ß-fucosidase, concomitant with large increases in ß-glucosidase, ß-cellobiosidase, α-arabinofuranosidase, ß-mannosidase, ß-glucuronidase, ß-xylopyranosidase, and ß-fucosidase activities and a dramatic decline in 6-phospho-ß-glycosidase activity. This is the first report to identify a gene encoding thermostable 6-phospho-ß-glycosidase and to generate a thermostable ß-fucosidase. These results provided thermostable enzyme catalysts and also suggested a promising approach to develop novel GH1 biocatalysts.

Counterselection system for Geobacillus kaustophilus HTA426 through disruption of pyrF and pyrR.

Counterselection systems facilitate marker-free genetic modifications in microbes by enabling positive selections for both the introduction of a marker gene into the microbe and elimination of the marker from the microbe. Here we report a counterselection system for Geobacillus kaustophilus HTA426, established through simultaneous disruption of the pyrF and pyrR genes. The pyrF gene, essential for pyrimidine biosynthesis and metabolization of 5-fluoroorotic acid (5-FOA) to toxic metabolites, was disrupted by homologous recombination. The resultant MK54 strain (ΔpyrF) was auxotrophic for uracil and resistant to 5-FOA. MK54 complemented with pyrF was prototrophic for uracil but insensitive to 5-FOA in the presence of uracil. To confer 5-FOA sensitivity, the pyrR gene encoding an attenuator to repress pyrimidine biosynthesis by sensing uracil derivatives was disrupted. The resultant MK72 strain (ΔpyrF ΔpyrR) was auxotrophic for uracil and resistant to 5-FOA. MK72 complemented with pyrF was prototrophic for uracil and 5-FOA sensitive even in the presence of uracil. The results suggested that pyrF could serve as a counterselection marker in MK72, which was demonstrated by efficient marker-free integrations of heterologous ß-galactosidase and α-amylase genes. The integrated genes were functionally expressed in G. kaustophilus and conferred new functions on the thermophile. This report describes the first establishment of a pyrF-based counterselection system in a Bacillus-related bacterium, along with the first demonstration of homologous recombination and heterologous gene expression in G. kaustophilus. Our results also suggest a new strategy for establishment of counterselection systems.

Genetic transformation of Geobacillus kaustophilus HTA426 by conjugative transfer of host-mimicking plasmids.

We established an efficient transformation method for thermophile Geobacillus kaustophilus HTA426 using conjugative transfer from Escherichia coli of host-mimicking plasmids that imitate DNA methylation of strain HTA426 to circumvent its DNA restriction barriers. Two conjugative plasmids, pSTE33T and pUCG18T, capable of shuttling between E. coli and Geobacillus spp., were constructed. The plasmids were first introduced into E. coli BR408, which expressed one inherent DNA methylase gene (dam) and two heterologous methylase genes from strain HTA426 (GK1380-GK1381 and GK0343-GK0344). The plasmids were then directly transferred from E. coli cells to strain HTA426 by conjugative transfer using pUB307 or pRK2013 as a helper plasmid. pUCG18T was introduced very efficiently (transfer efficiency, 10(-5)-10(-3) recipient(-1)). pSTE33T showed lower efficiency (10(-7)-10(-6) recipient(-1)) but had a high copy number and high segregational stability. Methylase genes in the donor substantially affected the transfer efficiency, demonstrating that the host-mimicking strategy contributes to efficient transformation. The transformation method, along with the two distinguishing plasmids, increases the potential of G. kaustophilus HTA426 as a thermophilic host to be used in various applications and as a model for biological studies of this genus. Our results also demonstrate that conjugative transfer is a promising approach for introducing exogenous DNA into thermophiles.

Three inositol dehydrogenases involved in utilization and interconversion of inositol stereoisomers in a thermophile, Geobacillus kaustophilus HTA426.

Geobacillus kaustophilus HTA426, a thermophilic Bacillus-related species, utilizes some inositol stereoisomers, including myo-, d-chiro- and scyllo-inositols (MI, DCI and SI), as sole carbon sources. Within its genome are three paralogous genes that possibly encode inositol dehydrogenase. These genes are located in tandem within a large gene cluster containing an almost complete set of iol genes homologous to genes involved in inositol catabolism in Bacillus subtilis. Each of the three plausible inositol dehydrogenases was purified as a His(6)-tag fusion. The enzymes exhibited thermophilic activity, each with its own characteristic specificity for the inositol stereoisomers and cofactors. Northern blot and primer extension analyses revealed that the three enzymes were encoded by the same 5 kb polycistronic transcript and were induced simultaneously in the presence of MI. HTA426 was subjected to ethyl methanesulfonate (EMS) mutagenesis to isolate a mutant strain, PS8, which was not able to utilize MI. In PS8, inositol dehydrogenase activity was abolished along with the 5 kb transcript, suggesting that any of the three enzymes supports MI-dependent growth. Analysis of metabolites in HTA426 cells grown in the presence of MI revealed that substantial amounts of DCI and SI appeared intracellularly during the stationary phase, while only MI was present in PS8 cells, suggesting that interconversion of inositol stereoisomers may involve these three enzymes.

Assessment of daily needle applicator displacement during high-dose-rate interstitial brachytherapy for prostate cancer using daily CT examinations.

To improve treatment conformity for prostate cancer, we investigated daily applicator displacement during high-dose-rate interstitial brachytherapy (HDR-ISBT). Thirty patients treated with HDR-ISBT as monotherapy were examined. All patients received a treatment dosage of 49 Gy per 7 fractions over 4 days. For dose administration, we examined 376 flexible applicators (1128 points) using our unique ambulatory implant technique. Using CT images with a 3-mm slice thickness, we calculated the relative coordinates of the titanium markers and the tips of the applicators. We calculated the distance between the center of gravity of the markers and the tips of the catheters, and compared the distances measured on the day of implantation and the second, third, and fourth treatment days. The mean displacement distance for all applicators was 4.3 ± 3.4 mm, 4.6 ± 4.1 mm, and 5.8 ± 4.5 mm at 21, 45, and 69 hours after initial planning CT. We used a 15-mm margin for needle displacement and only 2 points of 2 patients (16 mm and 18 mm at 69 hours, 2/1128 = 0.2%) exceeded this range. Almost patients (87%) showed the largest displacement within the first 21 hours. The relative doses that covered 100% of CTV (D100(CTV)) values compared with the initial treatment plan were reduced to 0.96 ± 0.08, 0.96 ± 0.08 and 0.94 ± 0.1 at 21, 45 and 69 hours. However, the relative D90(CTV) values kept acceptable levels (1.01 ± 0.02, 1.01 ± 0.03 and 1.01 ± 0.03). Cranial margin of 15 mm seems to be effective to keep D90(CTV) level if we do not do corrective action.

Assessment of radiation dermatitis using objective analysis for patients with breast cancer treated with breast-conserving therapy: influence of body weight.

PURPOSE: To evaluate the effect of patient factors on radiation dermatitis for patients with breast cancer who underwent postoperative radiotherapy after breast-conserving surgery. MATERIALS AND METHODS: The study population comprised 87 patients who underwent breast-conserving surgery followed by 50 Gy/25 fractions (median) of radiotherapy with or without boost radiation (10 Gy/5 fractions). We examined their treated and contralateral breast skin color by use of an objective analyzer, and expressed findings as L, a, b ratios by dividing by pre-RT values. Next, we examined correlation between patient factors (age, height, body weight, and body mass index, BMI) and change of L and a values by use of correlation coefficients. RESULTS: Radiation therapy caused changes in a and L ratios (p < 0.0001) but not in b ratio. The a ratio (reddish) increased 1.4-fold and peaked after radiotherapy. The L ratio (darkening) decreased by 10 % and reached a minimum value between completion of radiotherapy and 1 month after treatment. Although, age and height did not affect Δ value, body weight and BMI correlated significantly with Δa value (p = 0.0012 and 0.0017) not with ΔL value. CONCLUSION: Body weight and BMI predict degree of radiation dermatitis, and more reddish dermatitis was observed for heavier patients than for their lighter counterparts.

Objective and longitudinal assessment of dermatitis after postoperative accelerated partial breast irradiation using high-dose-rate interstitial brachytherapy in patients with breast cancer treated with breast conserving therapy: reduction of moisture deterioration by APBI.

PURPOSE: To objectively evaluate the radiation dermatitis caused by accelerated partial breast irradiation (APBI) using high-dose-rate interstitial brachytherapy. PATIENTS AND METHODS: The skin color and moisture changes were examined using a newly installed spectrophotometer and corneometer in 22 patients who had undergone APBI using open cavity implant high-dose-rate interstitial brachytherapy (36 Gy in six fractions) and compared with the corresponding values for 44 patients in an external beam radiotherapy (EBRT) control group (50-60 Gy in 25-30 fractions within 5-6 weeks) after breast conserving surgery. RESULTS: All values changed significantly as a result of APBI. The extent of elevation in a∗ (reddish) and reduction in L∗ (black) values caused by APBI were similar to those for EBRT, with slightly delayed recovery for 6-12 months after treatment owing to the surgical procedure. In contrast, only APBI caused a change in the b∗ values, and EBRT did not, demonstrating that the reduction in b∗ values (yellowish) depends largely on the surgical procedure. The changes in moisture were less severe after APBI than after EBRT, and the recovery was more rapid. The toxicity assessment using the Common Toxicity Criteria, version 3, showed that all dermatitis caused by APBI was Grade 2 or less. CONCLUSION: An objective analysis can quantify the effects of APBI procedures on color and moisture cosmesis. The radiation dermatitis caused by APBI using the present schedule showed an equivalent effect on skin color and a less severe effect on moisture than the effects caused by standard EBRT.

Objective assessment of dermatitis following post-operative radiotherapy in patients with breast cancer treated with breast-conserving treatment.

PURPOSE: To evaluate radiation dermatitis objectively in patients with breast cancer who had undergone post-operative radiotherapy after breast-conserving surgery. PATIENTS AND METHODS: Skin color (L*, a*, and b* values) and moisture analyses were performed for both breasts (before, after, 1 month, 6 months, and 1 year after radiotherapy) to examine irradiated and non-irradiated skin divided into four quadrants in 118 patients. These patients underwent breast conservative surgery followed by 50 Gy/25 fractions (median) of radiotherapy with or without boost irradiation (10 Gy/5 fractions). RESULTS: L*, a*, and moisture values were changed by irradiation and maximized at completion or 1 month after radiotherapy. One year after radiotherapy, the skin color had returned to the range observed prior to radiotherapy. However, moisture did not return to previous values even 1 year after treatment. The lateral upper side (quadrant C) showed greater changes than other quadrants in the L* value (darker) at the end of radiotherapy. The Common Toxicity Criteria version 3 scores were found to correlate well with a* and L* values at the completion and 1 month after radiotherapy. Boost radiotherapy intensified reddish and darker color changes at the completion of radiotherapy, while chemotherapy did not intensify the skin reaction caused by radiotherapy. CONCLUSION: Moisture impairment as a result of irradiation lasts longer than color alterations. Objective assessments are useful for analyzing radiation dermatitis.

Assessment of influence of smoking, drinking, leukoplakia and dental irritation on local control of early oral tongue carcinoma treated with brachytherapy: age and dental factors are potential prognostic factors.

BACKGROUND: To examine the background characteristics of elderly patients (65 years or older) with node-negative mobile tongue cancer (T1-2N0M0) who showed worse local control than a younger group. MATERIALS AND METHODS: We retrospectively analyzed background data for 265 patients treated with brachytherapy with or without external radiotherapy between 1967 and 1999. We examined dental factors (such as irritation by prosthesis), leukoplakia, tobacco smoking and alcohol consumption for comparisons between the elderly (age > or = 65 years; n = 83) and a control group (64 years or younger; n = 182). RESULTS: The elderly patients showed a worse outcome than the control group (respectively 86% and 70% at 5 years; P < 0.05). Incidence of dental factors tended to be higher for elderly patients (53%) than the control group (40%, P = 0.07). Dental factors proved to have prognostic importance for local control. Five-year local control rate was 85% for patients with and 76% for patients without dental factors (P = 0.04). The elderly group positive for dental factors showed a lower 5-year local control rate (61%) than the other three groups [(elderly without the dental factor (-) group (80%), control with the dental factor (+) group (84%), and control without the dental factor (-) group (87%)] (P < 0.05). Leukoplakia was found more frequently in the control (23%) than in the elderly group (5%) (P = 0.006) but had no effect on treatment outcome. CONCLUSIONS: Age and dental factors (including prosthesis irritation) are potentially important prognostic factors for local control of oral tongue cancer treated with brachytherapy, especially for elderly patients.

Non-invasive assessment of plaque rupture by 64-slice multidetector computed tomography--comparison with intravascular ultrasound.

BACKGROUND: Plaque rupture and secondary thrombus formation play key roles in the onset of acute coronary syndrome (ACS). Multidetector computed tomography (MDCT) allows the non-invasive assessment of coronary artery stenosis and plaque properties. In this study, we investigated whether 64-slice MDCT could non-invasively detect a plaque rupture in patients with de novo angina. METHODS AND RESULTS: The study population comprised 67 patients with de novo angina. All patients underwent contrast-enhanced 64-slice MDCT and intravascular ultrasound (IVUS). Patients were divided into a plaque rupture group (n=27) and a non-rupture group (n=40) based on the IVUS. The 64-slice MDCT revealed that the prevalence of an ulcer-like enhancement space (37% vs 5%, p<0.01), a ring-like sign (41% vs 18%, p=0.04), in the plaque rupture group was higher than those in the non-rupture group. Maximum plaque thickness (2.1+/-0.9 mm vs 1.6+/-1.0 mm, p=0.04), outer vessel area (17.6+/-4.9 mm2 vs 13.4+/-5.0 mm2, p<0.01), percentage plaque area (82.3+/-9.1% vs 73.4+/-15.7%, p=0.01), and remodeling index (1.11+/-0.18 vs 1.01+/-0.15, p=0.04) of the plaque rupture group were all significantly larger than those of the non-rupture group. CONCLUSIONS: The 64-slice MDCT can identify differences in lesion morphologies between ruptured plaques and non-ruptured plaques. From our results, the 64-slice MDCT might provide a useful tool for the non-invasive detection of plaque rupture.

The simultaneous assessment of aortic valve area and coronary artery stenosis using 16-slice multidetector-row computed tomography in patients with aortic stenosis comparison with echocardiography.

BACKGROUND: Recent advancements in 16-slice multidetector-row computed tomography (16-slice MDCT) provide for non-invasive assessment of not only coronary artery disease (CAD), but also myocardial properties and the anatomy of the whole heart. The purpose of the present study was to investigate whether the aortic valve area (AVA) in patients with aortic stenosis (AS) assessed by 16-slice MDCT corresponds to echocardiographic assessment and to evaluate simultaneously the clinical accuracy in detecting CAD with 16-slice MDCT. METHODS AND RESULTS: The AVA of 29 consecutive AS patients with transthoracic echocardiography (TTE) and 16-slice MDCT were analyzed. The AVA was estimated by means of the continuity equation method in 2-dimensional echocardiography (DE) and the quantitative planimetric method after multi-planar reformation in 16-slice MDCT. Concomitantly, the severity of the coronary artery stenosis was assessed by 16-slice MDCT. In the present study, the AVA assessed by TTE and 16-slice MDCT was 1.34+/-0.32 cm(2) and 1.38+/-0.32 cm(2), respectively. Regression analysis showed that the AVA in patients with AS determined by 16-slice MDCT correlated well with those determined by 2-DE (r=0.96, p<0.001). Significant coronary artery stenosis of more than 50% diameter reduction was present in 48% of the study population. CONCLUSIONS: In patients with AS, the analysis of the severity of the AVA by 16-slice MDCT provides a feasible and accurate estimation with the concomitant evaluation of CAD.

Assessment of biocompatibility of chronically implanted polyimide and platinum intrafascicular electrodes.

Longitudinal intrafascicular electrodes (LIFEs) are electrodes designed to be placed inside the peripheral nerve to improve stimulation selectivity and to increase the recording signal-to-noise ratio. We evaluated the functional and morphological effects of either Pt wire LIFEs or polyimide-based thin-film LIFEs implanted in the rat sciatic nerve for 3 mo. The newly designed thin-film LIFEs are more flexible, can be micromachined and allow placement of more active electrode sites than conventional Pt LIFEs. Functional results at 1 mo indicated an initial decline in the nerve conduction velocity and in the amplitude of muscle responses, which recovered during the following 2 mo towards normal values. Morphological results showed that both types of LIFEs induced a mild scar response and a focal but chronic inflammatory reaction, which were limited to a small area around the electrode placed in the nerve. Both types of LIFEs can be considered biocompatible and cause reversible, minimal nerve damage.