Lung cancer, especially non-small cell lung cancer (NSCLC), represents enormous challenges in continuously achieving treatment improvements. Besides cancer, obesity is becoming ever more prevalent. Obesity is increasingly acknowledged as a major risk factor for several types of common cancers. Significant mechanisms overlap in the pathobiology of obesity and tumorigenesis. One of these mechanisms involves oxidized low density lipoprotein receptor 1 (OLR1), as a link between obesity and cancer. Additionally, body mass index (BMI) has been widely used in exploiting the role of obesity on a series of diseases, including cancer. Significantly, squamous NSCLC revealed to be divergent clinical and molecular phenotypes compared with non-squamous NSCLC. Consequently, OLR1 immunostaining score and BMI were assessed by Fisher's linear discriminant analysis to discriminate if progression-free survival (PFS) would exceed 2 years. In addition, the final model was utilized to calculate the discriminant score in each study participant. Finally, 131 patients with squamous NCSLC were eligible for analysis. And a prediction model was established for PFS based on these 2 markers and validated in a second set of squamous NCSLC patients. The model offers a novel tool for survival prediction and could establish a framework for future individualized therapy for patients with squamous NCSLC. Lung cancer, with an increasing incidence, is the leading cause of cancer-related death worldwide . ThereinThe above phenomenon suggested a mechanistic overlap in the pathobiology of obesity and tumorigenesis . OxidizeNowadays, treatment failures still represent enormous challenges and it is doubtful if these standard treatment modalities could continuously achieve substantial improvements . AdditioSignificantly, combining application of appropriate biomarkers in prognosis prediction is emerging its high importance in cancer research . ConsequTotally, 131 patients with squamous NCSLC were eligible for the final analysis. The mean age was 59.56 years ; 116 patients were male (88.5%) and 15 female (11.5%). 103 (78.6%) patients were smokers. Moreover, stage IA disease occurred in 19 (14.5%) patients, IB in 36 (27.5%), IIA in 17 (13.0%), IIB in 17 (13.0%), IIIA in 40 (30.5%), and IIIB in 2 (1.5%). In addition, locations of tumor were 37 (28.2%) in left upper lobe, 27 (20.6%) in left lower lobe, 27 (20.6%) in right upper lobe, 11 (8.4%) in right middle lobe, and 29 (22.1%) in right lower lobe. Meantime, pathological analysis reported 7 patients (5.3%) with well differentiated, 36 (27.5%) with moderately differentiated, and 88 (67.2%) with poorly differentiated.The mean follow-up for survivors as of December 2014 was 47.23 months . Furthermore, mean PFS, due to last follow-up, was 724 days and the overall 1-, 2- and 3-year PFS rates were 87.8%, 47.3% and 39.7%, respectively Figure .OLR1 expressed on tumor cells. Among all these 131 specimens of squamous NCSLC, OLR1 expressed in either one or both of the cell membrane and cytoplasm, in a focal or scattered pattern Figure .n = 87) and validation (n = 44) cohorts in patients' sex, age, smoking habit, tumor size, tumor location, differentiation, pathological stage, follow-up, OLR1 immunostaining score (P > 0.1) (Table There was no significant difference between the training (P < 0.001).Based on training cohorts, BMI and OLR1 immunostaining score were used in setting a prediction model by employing Fisher's linear discriminant analysis (FLDA) with stepwise variant-selection. The clinical classifying model was described by the following equation: Y = −5.811 + 1.285 ×OLR1 immunostaining score + 0.152 ×BMI  0.884 –; 0.993] /2 = −0.2225. When the discriminant score Y was calculated to be > −0.2225, the case was predicted to be a PFS <= 2 years case; otherwise, the case was classified as a PFS > 2 years. For the training set of 87 leave-one-out-cross-validated cases, 49 of 52 PFS > 2 years (94.2% sensitivity) and 30 of 35 PFS <= 2 years (85.7% specificity) were correctly classified with an overall accuracy of 90.8% (79 of 87) and an area under the curve (AUC) of 0.938 [3] Table  and 3B.P < 0.001, 95% CI 0.806–1) was achieved  were applied to the validation set of 44 patients (18 PFS > 2 years and 26 PFS <= 2 years)  and validation group (n = 44).Chart review was performed on 1286 consecutive patients who suffered from squamous NSCLC with between November 2004 and March 2008. 131 of the 1286 patients were enrolled in the final analysis, while other patients with squamous NSCLC were excluded from analysis because of incomplete clinical or pathological data, such as unavailable formalin-fixed paraffin-embedded blocks. These 131 patients were randomly assigned (2:1) centrally by computer into training group (2) = weight (kg)/height (m2). Surgically resected or biopsied specimens were fixed in formalin and embedded in paraffin for routine histopathological diagnosis and immunohistochemical analysis. Then, PFS was defined as the time from the first documentation to the time of tumor progression or death. Notably, all data were reviewed and confirmed by two independent pathologists based on WHO classification of lung cancer [Characteristics of patients and tumors were collected. The weight and height were recorded the time of admission for all patients as routine clinical practice in SYSUCC. BMI was calculated as follow: BMI  antibody at 4°C overnight. After washing with phosphate-buffered saline (PBS), sections were incubated with horseradish peroxidase -conjugated secondary antibody  at room temperature for 30 min. After washing thrice with PBS, antibody complexes were colored with 3, 3′ -diamino benzidine and then counterstained with hematoxylin. Slides were dehydrated and evaluated.Isolated tumors were fixed in 10% neutral buffered formalin for 48 h and embedded in paraffin according to standard protocols. Sections  were deparaffinized and rehydrated in a graded series of alcohol solutions. For antigen retrieval, slides were immersed in ethylenediamine tetra-acetic acid  and boiled for 15 min in a microwave oven. Endogenous peroxidase activity was blocked in 3% HThe total OLR1 immunostaining score was calculated as the sum of the positively stained tumor cells and staining intensity. Briefly, the percentage of positive staining was scored as “0” , “1” , “2” , or “3” . Staining intensity was scored as “0” (no staining), “1” (weak staining), “2” (moderate staining), or “3” (strong staining). Both the percentage of positive cells and the staining intensity were evaluated under double-blind conditions. The total immunostaining score was calculated as the value of percent positivity score × staining intensity score, and ranged from 0 to 9. We defined OLR1 expression levels as: “0” (score 0–1), “1” (2–3), “2” (4–6) and “3” (>6). The score assessment was performed independently by two independent pathologists blinded to the clinical parameters.2 test and Fisher's exact test were used for categorical data, and an independent sample t-test or the Mann-Whitney U test were used for numerical data. Prognostic role of OLR1 and BMI for PFS were assessed by univariate analysis with log rank test and multivariate analysis with Cox proportional hazards regression.The data are presented as the number (%) or median (range) unless otherwise stated. The Pearson χP <0.05 was considered statistically significant). We internally validated the model using a cross-validation procedure, which enabled us to use the full data set for model development. P values <0.05 were considered statistically significant. Data analysis was performed using Predictive Analytics Software (PASW) Statistics 18.0 for Windows .First, the final model was used to calculate the discriminant score in each study participant. Second, the comparison between the discriminant score with the PFS was used to construct a receiver operating characteristic curve. In the meantime, the AUC and its 95% CI were also reported to describe the accuracy of the model for identifying metastases in our study participants. And the eigenvalue and canonical correlation was used to evaluate model fit (


Holmium laser enucleation (HoLEP) in surgical treatment of benign prostate hyperplasia (BPH) potentially offers advantages over transurethral resection of the prostate (TURP).Published randomized controlled trials (RCTs) were identified from PubMed, EMBASE, Science Citation Index, and the Cochrane Library up to October 10, 2013 . After methodological quality assessment and data extraction, meta-analysis was performed using STATA 12.0 and Trial Sequential Analysis (TSA) 0.9 software.Fifteen studies including 8 RCTs involving 855 patients met the criteria. The results of meta-analysis showed that: a) efficacy indicators: there was no significant difference in quality of life between the two groups (P>0.05), but compared with the TURP group, Qmax was better at 3 months and 12 months, PVR was less at 6, 12 months, and IPSS was lower at 12 months in the HoLEP, b) safety indicators: compared with the TURP, HoLEP had less blood transfusion , but there was no significant difference in early and late postoperative complications (P>0.05), and c) perioperative indicators: HoLEP was associated with longer operation time , shorter catheterization time  and hospital stay .In conventional meta-analyses, there is no clinically relevant difference in early and late postoperative complications between the two techniques, but HoLEP is preferable due to advantage in the curative effect, less blood transfusion rate, shorter catheterization duration time and hospital stay. However, trial sequential analysis does not allow us to draw any solid conclusion in overall clinical benefit comparison between the two approaches. Further large, well-designed, multicentre/international RCTs with long-term data and the comparison between the two approaches remain open. The latest American Urological Association's (AUA) Guideline defines transurethral resection of the prostate (TURP) as the “gold standard” surgical treatment for benign prostate hyperplasia (BPH) www.crd.york.ac.uk/PROSPERO).We reported this systematic review and meta-analysis based on the methodology recommended by the Cochrane Collaboration and according to the Preferred Reporting items for Systematic Review and Meta-analysis (PRISMA) statement Studies were eligible for inclusion if they met the following criteria: (1) study participants were clearly diagnosed as BPH and needed surgical treatment ; (2) randomized controlled studies which used HoLEP and TURP as the intervention and control arms, respectively; (3) at least reported one of the efficacy, safety or perioperative outcomes, which consisted of the International Prostate Symptom Score (IPSS), maximum flow rate (Qmax) (ml/s), quality of life (QoL), postvoid residual volume (PVR) (ml), the International Index of Erectile Function (IIEF), blood transfusion, TUR syndrome, urethral stricture, bladder neck contracture, secondary treatment, acute urinary retention (AUR), urinary tract infection (UTI), and transient hematuria, operating time (min), catheterization time (h), hospital stay (h), reduction of haemoglobin (g/dl) and serum sodium (mmol/L).We searched PubMed, EMBASE, Science Citation Index, and the Cochrane Library for relevant published studies up to October 10, 2013 . The search strategy was summarized in Our systematic search approach yielded titles and abstracts of published articles according to the above eligibility criteria and we excluded the clearly irrelevant results. The remaining trails were evaluated in full text. Information of each included trial was extracted using a pre-made data extraction form. We extracted the following trial characteristics: first author's name, publication year, country, and the detailed information of PICOS . For any missing data, we contacted the corresponding authors. Two authors independently selected study and extracted data, any disagreement was resolved by discussion.The methodological quality of included studies was evaluated using the Cochrane collaboration's tool for assessing risk of bias 2 statistic. Studies with I2 values of less than 40% were considered as having acceptable level of statistical heterogeneity All data were pooled using STATA version 12.0 (Stata Corp). For binary outcomes, relative risks (RRs) and corresponding 95% confidence intervals (CIs) were calculated; for continuous outcomes, weighted mean differences (WMDs) and their 95% CIs were calculated. The Cochran Q test was used to explore statistical heterogeneity with P<0.1 for statistical significance; a quantitative measure of heterogeneity across studies was also investigated using the I2 underestimate the required information size http://www.ctu.dk/tsa/) and provided the 95% confidence intervals adjusted for sparse data or repetitive testing.Cumulative meta-analyses of trials are at risk of yielding random errors because of sparse data and repetitive testing of accumulated data Our initial search yielded 1065 potential publications and finally 8 trials The risk of bias could be fully assessed in only one trial 2 = 0.0%, TSA adjusted 95% CI, −5.46 to 6.40; 6 months: WMD −0.61, 95% CI, −1.36 to 0.14, heterogeneity I2 = 66.4%, TSA adjusted 95% CI, −3.6 to 2.46)   . Howeverto 2.21) . Trial sto 2.21) .2 = 21.5%, TSA adjusted 95% CI, −0.62 to 2.02 ml/s). But the results showed significant differences favoring HoLEP at 3 and 12 months based on a random effects model   . Trial s91 ml/s) .2 = 0.0%), 6 months  and 12 months  all showed no significant difference between HoLEP and TURP based on a random effects model   . Trial s= 46.6%) , Fig. S5Only one trial 2 = 0.0%). Application of a constant continuity correction of 1.0 in the zero event trial did not change the result. TSA showed that 14.8% (755) of the required information size of 5112 patients were accrued to detect or reject a 35% reduction in relative risk, the cumulative Z-curve surpassed the futility boundary, but it did not cross the trial sequential monitoring boundary , but results of trial sequential analysis suggested evidence was not sufficient enough for the effect. Hence, we were only able to infer that HoLEP had the potential advantage in the curative effect.In the outcome of blood transfusion, HoLEP approach was obviously better than TURP and it might be associated with better laser coagulation technology; however, trial sequential analysis did not allow us to draw any solid conclusion on safety. Only one trial reported there were no significant difference of TUR syndrome and injury of mucosa rate between HoLEP and TURP. For early and late postoperative complications, we found no significant difference in AUR/re-catheterization, UTI, transient hematuria, urethral stricture, urinary incontinence, re-operation, transient dysuria, or bladder neck stenosis. In perioperative indicators, HoLEP was associated with longer operation time. This may be due to the fact that morcellation in HoLEP requires a much longer time than traditional TURP. However, Holmium laser technique is significantly advantageous in terms of catheterization time and hospital stay. Trial sequential analysis provided firm evidence of shorter catheterization time and hospital stay associated with the treatment of HoLEP as compared to TURP.A relevant meta-analysis involving 4 RCTs by Tan et al Another earlier review/meta-analysis http://www.crd.york.ac.uk/PROSPERO/display_record.asp?ID=CRD42014007334). Second, our study included 8 RCTs and considered more outcomes, which can provide a more comprehensive view on the efficacy and safety. Third, our study followed the recommended Cochrane collaboration's tool for assessing risk of bias. The previous meta-analyses Compared with previous meta-analyses, our systematic review has several strengths. First, we based it on a published protocol with rigid inclusion criteria for randomized clinical trials , we were unable to provide any long-term evidence. Thirdly, data were sparse for sexual function. Fourthly, the included studies do not provide enough information as to prostate size and anti-coagulated patients for in-depth subgroup analysis. Lastly, the overall sample size was still small.Our meta-analysis may also have some implications for further researches and clinical practice. Future researches should clarify the effectiveness, safety, potential advantages and disadvantages of HoLEP compared with TURP in large, high-quality RCTs, which also evaluate long-term outcomes and sexual functions relevant outcomes and focus more on prostate size, anti-coagulated patients and so on. In clinical practice, surgeons should not be limited to only conventional TURP as a treatment option for BPH. Although conventional TURP is still regarded as “gold-standard” in clinical guidelines, our findings have illustrated several advantages of HoLEP including a more favorable procedural safety profile, shorter catheterization duration time and hospital stay. We would thus like to highlight to clinicians that HoLEP presents as a viable treatment option for BPH. It is potentially a better treatment strategy, especially for elderly patients, those with large volume of prostate or high risk patients.In summary, our study provided the strongest available evidence and showed that there were no clinically relevant differences in early and late postoperative complications between the two techniques. Although the operative time favored TURP, HoLEP was more preferable due to its more favorable profile, defined by the clinically relevant differences detected regarding curative effect and less blood transfusion. Additionally, catheterization time and hospital stay were significantly shorter in HoLEP. After TSA adjustment for sparse data and multiple updating in cumulative meta-analysis, it seems unsure that HoLEP provides overall clinical benefit for BPH patients. Considering our main limitations, data from large, well-conducted international/multicentre RCTs with long-term data (follow-up duration>12 months) are necessary; sexual function-analysis and cost-analysis are still needed, and the comparison between the two approaches remains open.Figure S1Trial sequential analysis of International Prostate Symptom Score (IPSS) at 12 months. The required information size for IPSS at 12 months was calculated based on a two side α = 5%, β = 20% (power 80%), a minimal relevant difference of 0.5, a standard deviation of 3.5, and D2 = 77% as estimated in a random effects model.(TIF)Click here for additional data file.Figure S2Trial sequential analysis of maximum flow rate (Qmax) at 3 months. The required information size for Qmax at 3 months was calculated based on a two side α = 5%, β = 20% (power 80%), a minimal relevant difference of 3.0 ml/s, a standard deviation of 13.8 ml/s, and D2 = 0% as estimated in a fixed effects model.(TIF)Click here for additional data file.Figure S3Forest plot for quality of life (QoL) at 3 months, 6 months, and 12 months based on a random effects model. WMD = weight mean difference; CI = confidence interval.(TIF)Click here for additional data file.Figure S4Trial sequential analysis of postvoid residual volume (PVR) at 6 months. The required information size for PVR at 6 months was calculated based on a two side α = 5%, β = 20% (power 80%), a minimal relevant difference of 5.0 ml, a standard deviation of 20.7 ml, and D2 = 73% as estimated in a random effects model.(TIF)Click here for additional data file.Figure S5Trial sequential analysis of postvoid residual volume (PVR) at 12 months. The required information size for PVR at 6 months was calculated based on a two side α = 5%, β = 20% (power 80%), a minimal relevant difference of 5.0 ml, a standard deviation of 36.7 ml, and D2 = 0% as estimated in a random effects model.(TIF)Click here for additional data file.Figure S6Trial sequential analysis of blood transfusion. A diversity adjusted information size of 5112 patients was calculated using a two side α = 5%, β = 20% (power 80%), D2 = 0%, an anticipated relative risk increase of 35% and an event proportion of 4% in the control arm. Trials with no events were included in the study with a constant continuity correction of 1. The blue cumulative Z-curve was constructed using a fixed effects model.(TIF)Click here for additional data file.Figure S7Trial sequential analysis of hemoglobin decrease. The required information size for operation time was calculated based on a two side α = 5%, β = 20% (power 80%), a minimal relevant difference of 0.5 g/dl, a standard deviation of 2.3 g/dl, and D2 = 79% as estimated in a random effects model.(TIF)Click here for additional data file.Appendix S1Search strategy protocols used for each electronic database.(DOC)Click here for additional data file.Checklist S1PRISMA checklist.(DOC)Click here for additional data file.


As a critical tumor suppressor, p53 is inactivated in human cancer cells by somatic gene mutation or disruption of pathways required for its activation. Therefore, it is critical to elucidate the mechanism underlying p53 activation after genotoxic and cellular stresses. Accumulating evidence has indicated the importance of posttranslational modifications such as acetylation in regulating p53 stability and activity. However, the physiological roles of the eight identified acetylation events in regulating p53 responses remain to be fully understood. By employing homologous recombination, we introduced various combinations of missense mutations (lysine to arginine) into eight acetylation sites of the endogenous p53 gene in human embryonic stem cells (hESCs). By determining the p53 responses to DNA damage in the p53 knock-in mutant hESCs and their derivatives, we demonstrate physiological importance of the acetylation events within the core domain (K120 and K164) and at the C-terminus (K370/372/373/381/382/386) in regulating human p53 responses to DNA damage. Tumor suppressor p53 is a transcription factor that directly activates the transcription of hundreds of genes, including p21, Mdm-2, noxa, and puma that are mediating p53-dependent functions  is required for activating p53-dependent transcription but dispensable for p53 stabilization  into the endogenous p53 gene of hESCs is the same as we described for the K120/164R (K2R) knock-in mutation  mutation is important for p53 stabilization and activation in hESCs after DNA damage  hESCs. In contrast to the findings that p53 protein levels in K8R hESCs were much lower than those in p53+/− control hESCs both before and after DNA damage, indicating that K6R mutation increases the p53 responses to DNA damage in hESCs , 10% plasmanate, 1% PenStrep, 1% Glutamine, 1% nonessential amino acids, 10 ng/mL bFGF, and 55 μmol/L β-mercaptoethanol as described , monoclonal antibody against Mdm2 , polyclonal antibody against p21 or β-actin (Santa Cruz Biotechnology). The membrane was subsequently probed with a horseradish peroxidase-conjugated secondary antibody and developed with ECL PLUS . For co-immunoprecipitation analysis, 1–2 mg of whole cell protein extracts was immunoprecipitated with anti-p53 antibody . The amount of p53 and Mdm2 in the immunoprecipitate was analyzed by Western blotting using monoclonal antibody against p53 , monoclonal antibody against Mdm2 .Real-time PCR was performed as previously described . hESCs were treated with doxorubicin (0.2 μmol/L), or irradiated with γ-irradiation (5 Gy), and harvested 8 and 16 h after treatment. Apoptotic cells were identified by staining with Annexin V as previously described , or MG132 (25 μmol/L) for 4 h, and then cells were lysed in modified RIPA buffer  with mild sonication. Cell lysates were immunoprecipitated with anti-p53 antibody  and analysed by Western blotting using anti-ubiquitin antibody  or anti-p53 antibody  . Cells were harvested at 20, 40, and 80 min later, subjected to Western blotting using anti-p53 antibody . The intensity of bands was measured by densitometry.For Karyotyping of the mutant hESC clones was carried out by Cell Line Genetics .


S. aureus is the leading cause of infective endocarditis in the developed world, affecting ~40,000 individuals each year in the United States, and the second leading cause of bacteremia . Even with current medical advances, S. aureus bloodstream infections and infective endocarditis carry mortality rates of 20 to 66% . S. aureus lineages associated with human disease worldwide include clonal complex 5 (CC5)/USA100, CC30/USA200, CC8/USA300, CC1/USA400, and CC45/USA600. The CC5/USA100, CC30/USA200, and CC45/USA600 lineages cause invasive disease yet remain poorly characterized. USA300 and cytotoxins are central to most S. aureus virulence studies, and yet, we find evidence that clonal groups are quite heterogeneous in parameters canonically used to measure virulence, including cytotoxicity, biofilm formation, and blood survival, and that the superantigen profile is an important parameter to consider when defining the virulence of S. aureus strains. Staphylococcus aureus diseases affect ~500,000 individuals per year in the United States. Worldwide, the USA100, USA200, USA400, and USA600 lineages cause many of the life-threatening S. aureus infections, such as bacteremia, infective endocarditis, pneumonia, toxic shock syndrome, and surgical site infections. However, the virulence mechanisms associated with these clonal lineages, in particular the USA100 and USA600 isolates, have been severely understudied. We investigated the virulence of these strains, in addition to strains in the USA200, USA300, and USA400 types, in well-established in vitro assays and in vivo in the rabbit model of infective endocarditis and sepsis. We show in the infective endocarditis and sepsis model that strains in the USA100 and USA600 lineages cause high lethality and are proficient in causing native valve infective endocarditis. Strains with high cytolytic activity or producing toxic shock syndrome toxin 1 (TSST-1) or staphylococcal enterotoxin C (SEC) caused lethal sepsis, even with low cytolytic activity. Strains in the USA100, USA200, USA400, and USA600 lineages consistently contained genes that encode for the enterotoxin gene cluster proteins, SEC, or TSST-1 and were proficient at causing infective endocarditis, while the USA300 strains lacked these toxins and were deficient in promoting vegetation growth. The USA100, USA200, and USA400 strains in our collection formed strong biofilms in vitro, whereas the USA200 and USA600 strains exhibited increased blood survival. Hence, infective endocarditis and lethal sepsis are multifactorial and not intrinsic to any one individual clonal group, further highlighting the importance of expanding our knowledge of S. aureus pathogenesis to clonal lineages causative of invasive disease.IMPORTANCES. aureus is the leading cause of infective endocarditis in the developed world, affecting ~40,000 individuals each year in the United States, and the second leading cause of bacteremia . Even with current medical advances, S. aureus bloodstream infections and infective endocarditis carry mortality rates of 20 to 66% . S. aureus lineages associated with human disease worldwide include clonal complex 5 (CC5)/USA100, CC30/USA200, CC8/USA300, CC1/USA400, and CC45/USA600. The CC5/USA100, CC30/USA200, and CC45/USA600 lineages cause invasive disease yet remain poorly characterized. USA300 and cytotoxins are central to most S. aureus virulence studies, and yet, we find evidence that clonal groups are quite heterogeneous in parameters canonically used to measure virulence, including cytotoxicity, biofilm formation, and blood survival, and that the superantigen profile is an important parameter to consider when defining the virulence of S. aureus strains. Staphylococcus aureus is a Gram-positive opportunistic pathogen that colonizes the mucosal surfaces and skin of approximately 30% of humans , TSS as a consequence of any infection, bloodstream infections, and infective endocarditis  contributes significantly to the burden of S. aureus invasive disease /USA100, CC30/USA200, CC8/USA300, CC1/USA400, and CC45/USA600 . This combination may be linked to a particular clonal group or be common to several lineages.Various studies have linked specific  disease . S. aureproduced . Hence, S. aureus strains, we characterized a subset of isolates from five United States types associated with human disease, many of which (such as USA100 and USA600) are clinically important yet inadequately characterized , SEl-N, SEl-O, and SEl-U genes, collectively known as the enterotoxin gene cluster (egc), in 100% of isolates from the USA100, USA200, and USA600 lineages. All USA200 strains encoded toxic shock syndrome toxin 1 (TSST-1), their hallmark SAg. SEC was not only highly prevalent in USA400 isolates but was also present in ~60% of USA600 strains and in ~10% of USA100 and USA200 strains. USA300 strains encoded the least number of SAgs and lacked those frequently associated with invasive disease. The majority of USA300 strains encoded SEl-K, SEl-Q, and SEl-X. These results demonstrate the high frequency of SAg genes in S. aureus backgrounds associated with invasive and life-threatening diseases.There is a strong association between select superantigens (SAgs) and invasive syndrome , 6, 27. syndrome ; Table 2l-X in the S. aureus isolates used for in vivo studies and found the strains to produce 5 to 50 µg/ml TSST-1, 50 µg/ml SEB, 58 to 81 µg/ml SEC, and 70 to 180 ng/ml SEl-X. The production of TSST-1, SEB, and SEC in liquid culture has been quantified previously in S. aureus isolates from atopic dermatitis, vaginal mucosa, and diabetic foot ulcers, where strains produced 3 to 39 µg/ml TSST-1, 25 to 120 µg/ml SEB, and 10 to 120 µg/ml SEC  and 244 ± 45 mm2, respectively, suggesting high alpha-toxin production  or very low hemolysis (below 100 mm2). Strains were also screened for the presence of hla, hlb, and pvl , MN8 (USA200), and IA1871 (USA600) caused infective endocarditis at a dose of 2 × 108 to 5 × 108 CFU, and strain Col caused it at 5 × 108 to 1 × 109 CFU. Strain RN450 and the USA300 strains LAC and PSLV were severely deficient in infective endocarditis development regardless of the infection dose  still led to lethality in rabbits infected with S. aureus strains IA116 , IA209 , MNPE , IA1471 , MW2 , and LAC   . Of impoS. aureus presents a significant clinical and public health problem, causing some of the most severe hospital- and community-associated illnesses and affecting approximately 500,000 individuals each year in the United States , such as SEethality , 43. TheS. aureus infective endocarditis isolates to those of skin/soft tissue infection isolates indicated a strong association between infective endocarditis and a select group of staphylococcal SAgs, where 18 to 25% of strains encoded SEC, 9 to 20% encoded TSST-1, and 58 to 90% contained the enterotoxin gene cluster , biofilm formation (as a measure of tissue adherence/colonization), and blood survival (as a measure of ability to resist immune attack). Interestingly, we found clonal group heterogeneity in each of the aforementioned parameters. For example, although we did find the USA300 isolates to be highly hemolytic, strain IA2980 was an obvious exception. Both the USA100 and the USA600 lineage had isolates that were high or low in hemolysis. Intraclonal variation in biofilm formation was also observed, but it was specific to the USA300 and USA600 isolates in our collection. The USA100, USA200, and USA400 isolates consistently produced strong biofilms. Furthermore, blood survival variability was also observed among all clonal groups and was independent of the isolation site. S. aureus strains isolated from blood exhibited as much heterogeneity as the rest. We found no correlation between hemolytic potential and survival in the blood. For example, strains IA116 and IA209 (USA100) and strains PSLV and IA1012 (USA300) are some of the most highly hemolytic strains in our collection, and yet these strains do not exhibit a commensurate increase in blood survival. Conversely, strains MN8 and IA1101 (USA200), MW2 (USA400), IA88 (USA600), and Col are low in hemolytic activity, and yet they exhibit high blood survival. Whether biofilm formation can compensate for reduced ability to survive in blood, allowing hematogenous spread by USA100 strains, is currently unknown. It is also unknown whether increased ability to survive in blood compensates for reduced ability to form biofilms in USA600 strains. Which combination of traits contributes to or is required for specific pathologies is also undetermined. These are areas of interest currently being pursued by our laboratory.To further characterize strains in the USA100 and USA600 lineage, we performed in vitro, is not an intrinsic property of all S. aureus isolates, as many isolates belonging to the USA300 and USA600 lineages form poor biofilms. Finally, the production of high-level SAgs, such as TSST-1, SEB, and SEC, is also not intrinsic only to USA200 and USA400 isolates, as many USA600 isolates and some USA100 isolates also produced SEC. Therefore, it is important to look at multiple parameters when determining strain virulence and to be cautious about classifying strains with low cytotoxicity as avirulent. Strains with low cytotoxicity can have increased virulence due to the production of a high-level SAg (exemplified by S. aureus strains MN8 and MW2) or cause serious invasive disease due to their proficiency in key functions like blood survival and/or biofilm formation (exemplified by S. aureus strains IA1871 and Col). The infective endocarditis and sepsis studies further highlight the importance of expanding our knowledge of S. aureus pathogenesis to include isolates belonging to clonal groups associated with invasive disease and high mortality worldwide, namely, isolates from the USA100/CC5, 200/CC30, and 600/CC45 lineages  broth  at 37°C.The Taq polymerase (Qiagen) or Phusion HF DNA polymerase  with superantigen-specific primers (hla was detected using primer set hlaBamHIfor (5′ TTACGGATCCATGAAAACACGTATAGTCAGCTCAGTAAC 3′) and hlaXhoIrev (5′ TTACCTCGAGATTTGTCATTTCTTCTTTTTCCCAATCG 3′). hlb was detected with hlbscreenF (5′ CATCAACTGTTGCTGAAGATGGC 3′) and hlbscreenR (5′ CGGTTTCTCAGTAACAACTTCATTGAC 3′), a primer set that detects the gene regardless of phage integration. pvl (luks) was detected using primer set pvluksfor (5′ GACTATTAGCTGCAACATTGTC 3′) and pvlluksrev (5′ CCAGTTCACTTCATATTTAACTG 3′).Genomic DNA was extracted from overnight cultures using the DNeasy blood and tissue kit . PCR amplification of superantigen genes was carried out using  primers , 47. Cytl-X were detected using standard Western blotting techniques. Briefly, organisms were cultured in TH broth  at 37°C with shaking overnight. The supernatants were concentrated by precipitating them with 4 volumes of 100% ethanol overnight at 4°C, centrifuging at 4,000 × g for 10 min, and resolubilizing in distilled water to 1/20 the original volume. Western immunoblotting with specific polyclonal rabbit antisera prepared against the designated SAgs was used to quantify the amounts of SAgs produced by each isolate. Purified SAgs were used to generate standard curves for quantification.SEB, SEC, TSST-1, and SE9 CFU/ml in phosphate-buffered saline (PBS) were spotted onto rabbit blood plates (RBP) in 5-µl volumes and incubated at 37°C with 5% CO2 for 18 to 24 h. The areas of hemolysis were calculated with ImageJ (Overnight cultures diluted to 1 × 10h ImageJ .5 CFU/ml bacteria in tryptic soy broth (TSB)  supplemented with 2% glucose and 2% sodium chloride growing in plates coated with 5% human plasma . The plates were incubated overnight at 37°C with 5% CO2. The absorbance of solubilized crystal violet was measured at an optical density at 562 nm (OD562) . We performed one-way analysis of variance (ANOVA), the Holm-Sidak multiple-comparison test, and individual pairwise comparisons derived from post hoc tests. This resulted in roughly four statistically significant groups, as follows: (i) strains producing biomass mean values (OD562) of less than 0.5; (ii) strains producing biomass mean values equal to or higher than 0.5 but less than 1.0; (iii) strains producing biomass mean values equal to or higher than 1.0 but less than 1.5; and (iv) strains producing biomass mean values equal to or higher than 1.5. Each group was comprised of strains with biofilm formation that was not statistically different than that of the other strains within the group but was statistically different than strains in the groups with higher or lower biomass values.The well-established microtiter biofilm assay was used , with 3 7 to 5 × 107 CFU/ml into 1 ml of freshly collected heparinized rabbit blood. Samples were incubated at 37°C with 360° rotation for a total of 120 min. Samples were collected at time points indicated above. Percent survival was calculated as the bacterial count recovered at a specific time point divided by the bacterial count at time zero.Blood survival assays were performed as previously described . BacteriThe native valve infective endocarditis and sepsis models were performed as previously described , 51. Yout test analyses derived from post hoc tests.Statistical significance in survival experiments was determined using the log-rank Mantel-Cox test (GraphPad Prism Software). Significance across means was carried out using one-way ANOVA and the Holm-Sidak multiple-comparison test (GraphPad Prism Software) and


Cupriavidus metallidurans NA1, NA4, and NE12 were isolated from space and spacecraft-associated environments. Here, we report their draft genome sequences with the aim of gaining insight into their potential to adapt to these environments. Cupriavidus metallidurans strains have previously been isolated from industrial sites linked to mining, metallurgic, and chemical industries  using the 454 GS-FLX sequencing platform. The sequencing data for strain NA4 and strain NE12 showed average read lengths of 307 nucleotides (nt) and 333 nt, average insert sizes of 2,972 nt and 2,777 nt, and total sequencing data of 258 Mbp and 409 Mbp, respectively. Both genomes were assembled using Newbler software (version 2.3), resulting in 109 and 87 contigs and N50 values of 226,033 nt and 197,312 nt for NA4 and NE12, respectively. Full-genome sequencing of C. metallidurans NA1 was performed by Baseclear  on the Illumina HiSeq 2000 platform using a combination of a paired-end sequencing library (101 nucleotides) with an average insert size of 240 nt and a mate-pair sequencing library (51 nucleotides) with an average insert size of 3,233 nt. Reads were filtered to remove low-quality reads, resulting in a total of 5,832,127 paired reads (594 Mbp) and 9,473,070 mate-pair reads . De novo genome assembly based on the paired-end reads was performed using Velvet , JFZE00000000 (version JFZE01000000), and JFZF00000000 (version JFZF01000000) for strains NA1, NA4 and NE12, respectively.These whole-genome shotgun projects have been deposited at DDBJ/EMBL/GenBank under the accession numbers


To investigate the performance of hematoma shape, hematoma size, Glasgow coma scale (GCS) score, and intracerebral hematoma (ICH) score in predicting the 30-day mortality for ICH patients. To examine the influence of the estimation error of hematoma size on the prediction of 30-day mortality.This retrospective study, approved by a local institutional review board with written informed consent waived, recruited 106 patients diagnosed as ICH by non-enhanced computed tomography study. The hemorrhagic shape, hematoma size measured by computer-assisted volumetric analysis (CAVA) and estimated by ABC/2 formula, ICH score and GCS score was examined. The predicting performance of 30-day mortality of the aforementioned variables was evaluated. Statistical analysis was performed using Kolmogorov-Smirnov tests, paired t test, nonparametric test, linear regression analysis, and binary logistic regression. The receiver operating characteristics curves were plotted and areas under curve (AUC) were calculated for 30-day mortality. A P value less than 0.05 was considered as statistically significant.P = 0.0018), 0.715 (P = 0.0008) (by ABC/2) to 0.738 (P = 0.0002) (by CAVA), 0.877 (P<0.0001) (by ABC/2) to 0.882 (P<0.0001) (by CAVA), and 0.912 (P<0.0001), respectively.The overall 30-day mortality rate was 15.1% of ICH patients. The hematoma shape, hematoma size, ICH score, and GCS score all significantly predict the 30-day mortality for ICH patients, with an AUC of 0.692 (Our study shows that hematoma shape, hematoma size, ICH scores and GCS score all significantly predict the 30-day mortality in an increasing order of AUC. The effect of overestimation of hematoma size by ABC/2 formula in predicting the 30-day mortality could be remedied by using ICH score. Hematoma shape has been recently reported to be related to hematoma size and hematoma growth of intracerebral hematomas (ICH). An irregular shape of hematoma has been linked to a larger hematoma size Hematoma size is an important prognostic factor predicting the mortality of ICH patients By weighting Glasgow coma scale (GCS) score, age, hematoma size, origin of ICH, and ventricular involvement, the ICH score has been raised as a simple method in predicting 30-day mortality In this study, we aimed to investigate the performance of hematoma shape, hematoma size, GCS score, and ICH score in predicting the 30-day mortality for ICH patients. In the meanwhile, we also examined the influence of the estimation error of hematoma size on the prediction of 30-day mortality.This retrospective study was approved by the institutional review board of Tri-Service General Hospital (1-103-05-014). The institutional review board waived the need of written informed consent from the patients in this study. All data underlying the findings described in this manuscript are in Brain computed tomography (CT) images of 137 patients, who were admitted to our hospital under the diagnosis of acute intracerebral hemorrhage during a period of 10 months, were initially reviewed. All patients received non-enhanced CT with an interval between the time of CT study and the onset of symptoms within 24 hours. Twenty-three patients were excluded based on the following exclusion criteria including concurrent subarachnoid hemorrhage (N = 4), subdural hematoma (N = 1), brain tumor (N = 2), acute ischemic infarction with hemorrhagic transformation (N = 3), old ischemic infarction (N = 5), old hemorrhage (N = 2), severe motion artifact (N = 2), beam hardening artifact (N = 1), and recent intracranial operations (N = 3). Eight patients who had a ventricular shunting catheter seen on CT images were also excluded. Finally, 106 patients  with intracerebral hematomas were recruited in this study. The clinical data , clinical history , and laboratory data  obtained on admission were recorded. To assess the 30-day mortality, the survival statuses of patients were confirmed based on the medical records not only on admission but also in outpatient follow up.Non-enhanced CT images were acquired using single or multi-row spiral CT scanners , which were compatible with the regulation of digital imaging and communications in medicine (DICOM). Imaging parameters included 140 kV, 170 mA, slice thickness of 5 mm, slice gap of 0 mm, field of view of 210 mm, matrix size of 512×512 and gantry angulation being parallel to the supraorbitomeatal line.The hematoma shape was independently evaluated by two neuroradiologists  using a binary scoring system, which was first introduced here in this study. A score of 0 represented a regular hematoma shape, including a roundish or ellipsoid hematoma with smooth margin . A scoreThe DICOM data were firstly stored in the picture archiving and communication system (PACS) immediately after acquisition. Maximal diameters of the hemorrhage in three orthogonal directions were measured on a PACS viewer  by a 10-year experienced neuroradiologist (C.J.J.) for calculating the hemorrhagic size using the ABC/2 method as described by Kothari et al. The ICH score was calculated based on the clinical information (age and GCS score) and the CT features  as developed by Hemphill et al Statistical analyses were performed using SPSS Version 16.0 software  and MedCalc Version 13.0 . The interobserver reliability was examined by linearly weighted kappa statistics. The normality of parameters was examined by using Kolmogorov-Smirnov tests. Cross-tabulations were analyzed using Fisher exact test. Paired student t test was used for 2-group comparisons regarding parameters with normal distribution, while nonparametric tests (Wilcoxon signed rank tests and McNemar tests) were used for 2-group comparisons regarding parameters without normal distribution. Linear regression analysis was used to assess the relationship between the hematoma size estimated by ABC/2 formula and the hematoma size measured by CAVA. Binary logistic regression was applied to examine the relationships between the independent variables  and dependent variable . The nonparametric receiver operating characteristics (ROC) curves were plotted and areas under curve (AUC) were calculated for 30-day mortality. A P value less than 0.05 was considered as statistically significant.P = 0.003), hematoma shape (P = 0.003), hematoma size (P = 0.009), presence of IVH (P<0.032), GCS score (P<0.001), and ICH score (P<0.001).Of 106 patients, 77 (73%) had a history of hypertension, 21 (20%) had type II diabetes mellitus, four (3.8%) had impaired coagulation function with international normalized ratio (INR) ≧ 1.5, and 47 (44.3%) had simultaneous IVH. The hematomas were located supratentorially in 93 (87.7%) patients, located infratentorially in 13 (12.2%) patients. The overall 30-day mortality was 15.1% (16 of 103). The demographic and clinical characteristics of our study were summarized in P = 0.391). Patients with pontine hematomas had a 30-day mortality as high as 33.3% (2 of 6), while patients with cerebellar hematomas had a 30-day mortality of 14.3% (1 of 7). Again, the difference was not statistically significant (P = 0.279).The 30-day mortality was 23.1% (3 of 13) for infratentorial hematomas and was 14.0% (13 of 93) for supratentorial hematomas. The difference between the aforementioned locations, however, was not statistically significant (Hematomas were judged as regular in 62 (58.5%) patients and irregular in 44 (41.5%) patients by observer 1, while regular in 61 (57.5%) patients and irregular in 45 (42.5%) patients by observer 2. Ten patients whose hematomas were judged as regular by observer 1 were considered as irregular by observer 2. On the other hand, 8 patients whose hematomas were judged as irregular by observer 1 were considered as regular by observer 2. Linear weighted Kappa statistics showed a weighted kappa value of 0.632 with a confidence level from 0.483 to 0.782, suggestive of substantial agreement.P = 1) and age (P = 0.92). Regarding the vital signs, the irregular group had a significantly higher diastolic pressure (P = 0.021) and higher pulse rate (P = 0.022) than the regular group. The laboratory data showed that the irregular group had a significantly higher WBC count (P = 0.003) than the regular group. With respect to the consciousness, the irregular group had a significantly lower GCS score than regular group (P<0.001). Taking the CT imaging features into consideration, the irregular group had a significantly larger hematoma size (P<0.001), significantly higher percentage of hematomas larger than 30 mL (P<0.001), and significantly higher ratio with intraventricular hemorrhage (P<0.001). The ICH score was significantly higher on irregular group than the regular group (P<0.001). The 30-day mortality was also significantly higher on irregular group than the regular group (P<0.006).The differences between regular hematomas and irregular hematomas were listed in 2 of 0.965 significantly (P<0.001). The size difference (y) between two methods was also positively correlated to the hematoma size (x) measured by CAVA characterized by the equation, y = 0.409x–0.434, with a R2 of 0.702 significantly (P<0.001)  was significantly higher than that measured by the CAVA (27.8±35.6 ml) (P<0.001). Linear regression analysis showed that the hematoma size (y) estimated by ABC/2 formula was positively correlated to the hematoma size (x) measured by CAVA characterized by the equation, y = 1.409x–0.431, with a RP<0.001) .The overall GCS score was 9.8±4.3. Nineteen patients (17.9%) had a GCS score of 3 or 4, 45 patients (42.4%) had a GCS score ranging 5 to 12, while 42 patients (39.6%) had a GCS score ranging from 13 to 15. Patients with a GCS score of 3 to 4 had a significantly higher 30-day mortality (68.4%) than those with a GCS score of 5 to 12 (6.7%) and a GCS score of 13 to 15 (0%).The relationship of 30-day mortality versus ICH score was shown on P = 0.0018), 0.715∼0.786 (P≤0.0008), 0.877∼0.882 (P<0.0001), and 0.912∼0.922 (P<0.0001), respectively. Pairwise comparisons of ROC curves, summarized in P = 0.022), predicted by GCS score and hematoma size (P = 0.0145∼0.0048), predicted by ICH score and hematoma size (P = 0.0336∼0.0076), predicted by GCS score and hematoma shape (P = 0.0009), and predicted by ICH score and hematoma shape (P = 0.0012∼0.0076). The difference between AUCs predicted by ICH score in which the hematoma size was estimated by ABC/2 formula and AUCs predicted by ICH score in which the hematoma size was measured by CAVA was not statistically significant (P = 0.11). Likewise, the difference between AUCs predicted by ICH score and predicted by GCS score was also not statistically significant (P = 0.14∼0.24).Comparison of ROC curves in predicting the 30-day mortality by GCS, ICH scores, ICH size, and ICH shape was graphically demonstrated . The preP<0.001) and higher ratio of hematoma size which is more than 30 mL (60% versus 4.9%) (P<0.001). In addition to hematoma size, the irregular hematoma is also associated with significantly higher ratio of IVH (69% versus 26%) (P<0.001), lower GCS score (7.2 versus 11.7) (P<0.001), and higher ICH score (2.8 versus 1.1) (P<0.001). The irregular hematoma has significantly higher 30-day mortality rate (26.7% versus 6.6%) than the regular hematoma (P<0.006). ROC analysis discloses that the hematoma shape significantly predicts the 30-day mortality with an AUC of 0.692 (P = 0.0018). These associations between hematoma shape and the hematoma size, IVH, GCS score, and ICH score may explain why the hematoma shape serves as an independent predictor of the 30-day mortality.ICH shape is getting increasing attention recently. Compared to the regular hematoma, the irregular hematoma has been related to a higher risk of hematoma growth P = 0.02).Although the hematoma size has been proposed as an independent predictor of mortality and used in ICH score for ICH in some researches P = 0.034 to 0.008). The AUC of ICH score with the hematoma size estimated by ABC/2 is not different to the AUC of ICH score with the hematoma size measured by CAVA.In predicting the 30-day mortality, the mean AUC of ICH scores in our study  are higher than Pengs' study (0.72) Our study has three limitations. First, we did not evaluate the functional outcome or quality of life in this study. Nevertheless, 30-day mortality remains an important outcome in ICH patients and easy to be examined by neuroradiologists. Second, we didn't take surgical intervention into account in this study. According to a random trial performed by Auer et al, the mortality rate of patients who receive endoscopic aspiration of lobar intracerebral hemorrhage was significantly lower than those received medical treatment only Our study shows that hematoma shape, hematoma size, ICH scores and GCS score all significantly predict the 30-day mortality in an increasing order of AUC. Although the ABC/2 formula significantly overestimates the hemorrhagic size than the CAVA method, the effect of overestimation of hematoma size by ABC/2 formula in predicting the 30-day mortality could be remedied by using ICH score and GCS score.Table S1Original data and ICH score of patients with intracerebral hematomas.(DOCX)Click here for additional data file.


These hydrogels, together with SaOS-2 cells, were bioprinted to computer-designed scaffolds. The results revealed that bioglass (nano)particles, with a size of 55 nm and a molar ratio of SiO2∶CaO∶P2O5 of 55∶40∶5, did not affect the growth of the encapsulated cells. If silica, biosilica, or polyP•Ca2+-complex is co-added to the cell-containing alginate/gelatin hydrogel the growth behavior of the cells is not changed. Addition of 5 mg/ml of bioglass particles to this hydrogel significantly enhanced the potency of the entrapped SaOS-2 cells to mineralize. If compared with the extent of the cells to form mineral deposits in the absence of bioglass, the cells exposed to bioglass together with 100 µmoles/L polyP•Ca2+-complex increased their mineralization activity from 2.1- to 3.9-fold, or with 50 µmoles/L silica from 1.8- to 2.9-fold, or with 50 µmoles/L biosilica from 2.7- to 4.8-fold or with the two components together (100 µmoles/L polyP•Ca2+-complex and 50 µmoles/L biosilica) from 4.1- to 6.8-fold. Element analysis by EDX spectrometry of the mineral nodules formed by SaOS-2 revealed an accumulation of O, P, Ca and C, indicating that the mineral deposits contain, besides Ca-phosphate also Ca-carbonate. The results show that bioglass added to alginate/gelatin hydrogel increases the proliferation and mineralization of bioprinted SaOS-2 cells. We conclude that the development of cell-containing scaffolds consisting of a bioprintable, solid and cell-compatible inner matrix surrounded by a printable hard and flexible outer matrix containing bioglass, provide a suitable strategy for the fabrication of morphogenetically active and biodegradable implants.We investigated the effect of bioglass (bioactive glass) on growth and mineralization of bone-related SaOS-2 cells, encapsulated into a printable and biodegradable alginate/gelatine hydrogel. The hydrogel was supplemented either with polyphosphate (polyP), administered as polyP•Ca It is the aim of bone tissue engineering to restore the function of diseased or damaged bone tissue by combining biological and biomechanical laws for the purpose of development of biodegradable scaffolds, allowing isolated functional cells to colonize. In recent years, growing emphasis has been put on three-dimensional (3D) bioprinting of cells to tissue-units due to the various advantages over existing patterning methods especially because it is anticipated that the bioprinting technique allows for the fabrication of personalized, customized free-form implants.Osseous tissue, bone, is formed by two different structures, cancellous bone and cortical bone. Cancellous bone represents the inner part of bone tissue, is spongy and shows a porosity of 50–90 vol%. In contrast, the dense outer bone layer, the cortical bone, comprises less than 10 vol% porosity. Both bone types undergo a continuous and dynamic remodeling, involving maturation and resorption, that is controlled by the tuned interaction of osteoblasts and osteoclasts, as well as by osteocytes  have been developed , tetraethyl orthosilicate (TEOS), and ammonium hydrogen phosphate were obtained from Sigma-Aldrich ; low-melting gelatin (bovine) from SERVA ; McCoy's medium and fetal calf serum [FCS] from Biochrom-Seromed ; and agarose from Biozym .2 in a stoichiometric ratio of 2∶1 (polyP∶CaCl2) as described 2+-complex”. Usually a concentration of 100 µM (14 µg/ml polyP•Ca2+-complex) was added to the assays.Sodium polyphosphate (Na-polyP of an average chain of 40 phosphate units) was obtained from Chemische Fabrik Budenheim . The chelating effect, caused by polyP, was compensated by mixing with CaCl5 cells/ml. Where indicated the alginate/gelatin gel was supplemented with 100 µmoles/L polyP•Ca2+-complex and 50 µmoles/L ortho-silicate (silica) or 50 µmoles/L biosilica.A sodium alginate solution (50 mg/ml), supplemented with 50 mg/ml of low-melting gelatin, was prepared in physiological saline. The gel was supplemented with 10 µl/ml of phenol red [0.5%] solution. SaOS-2 cells, growing at the end of the growing phase, were added to the gel at a concentration of 5×10Ortho-silicate (silica) was prepared from prehydrolyzed TEOS The particles were prepared as described 2+-complex, silica or biosilica, was filled into sterile 30 ml printing cartridges , connected with a 1.98×0.41 mm steel needles  and placed into the preheated (30°C) printing head of the 3D-Bioplotter . Using the following settings: 0.9 bar pressure, speed of 26 mm/sec, 30°C, the round gel cylinders were printed to a hydrogel scaffold . The hydrogel cylinders were injected into Petri dishes, filled with 0.4% CaCl2 as cross-linking solution The freshly prepared alginate/gelatin/SaOS-2 cell suspension, either as it is, or supplemented with polyP•CaWe used the human osteogenic sarcoma cells, SaOS-2 cells 2+-complex, silica or biosilica, were supplemented with 5 mg/ml of bioglass (nano)particles. For the determination of the effects on growth incubation lasted 3 d. In order to determine the effect of the different components on the mineralization capacity of SaOS-2 cells the cultures were incubated for 3 d in the absence of the osteogenic cocktail and subsequently for 5 d in the presence of the cocktail.Where indicated, the added 2.5 mL of medium/FCS around the bioprinted cylinders, formed of alginate/gelatin/SaOS-2 cells without or with polyP•CaThe scanning electron microscopic (SEM) images were performed with a HITACHI SU 8000  at low voltage  detector. The SEM mapping experiments  were performed with a XFlash FlatQUAD  using the following settings, 5.5 kV, 260 pA, 120 kcps, 640×480 pixels, 33 nm pixels, 62 min.Digital light microscopic studies were performed using a VHX-600 Digital Microscope  equipped with a VH-Z25 zoom lens.i) ortho-silicate, prepared from prehydrolyzed TEOS (“silica”), (ii) biosilica, prepared enzymatically from prehydrolyzed TEOS and silicatein (“biosilica”), (iii) silicatein alone without any silica precursor (“silicatein”), (iv) polyP•Ca2+-complex (“polyP”) and finally (v) polyP•Ca2+-complex together with biosilica (“polyP + biosilica”). The assays with these printed hydrogel samples had been performed in the absence (“minus bioglass”) or presence of bioglass, added to the medium/serum (“plus bioglass”).The polymers/hydrogels had been tested in the assays as follows: Control assays without any component (termed “none”). Additional components in the hydrogel samples that had been used for printing had been  or presence of bioglass (“plus bioglass”).Like described for the cell viability assays the respective assays of the hydrogels, composed for the mineralization studies have been termed “none”, hydrogel without any component; “silica”, addition of ortho-silicate, prepared from TEOS; “biosilica”, prepared from prehydrolyzed TEOS and silicatein; “silicatein”, addition of only the enzyme; “polyP”, addition of polyP•CaThe mineral deposits onto the cells were stained with Alizarin Red S. The cells were incubated for 3 d in the absence of the osteogenic cocktail and subsequently for 5 d in the presence of the cocktail. Then the hydrogel was gently dissolved by addition of 55 mmoles/L Na-citrate and the cells were allowed to adhere to the plastic bottom of the 24-well plates during a 24 h incubation period. After a further careful washing cycle with physiological saline (without sudden shaking) the reaction with Alizarin Red S staining reaction was performed. The color developed was quantitatively assessed, using the fluorochrome Alizarin Red S as an indicator by applying the spectrophotometric assay t-test The results were statistically evaluated using paired Student's Bioglass is a well established hard, porous basis material for the formation of bone-replacing scaffolds et al. 2∶CaO∶P2O5 was 55∶40∶5 2, 24 mol% Na2O, and 30 mol% CaO.As solid (nano)particles of bioglass we used the solid glass powder, prepared according to and characterized by Hong The SaOS-2 cells were encapsulated into a hydrogel with the components alginate and gelatin, as described under “2+-complex to the hydrogel, prior to the bioprinting process, revealed the appearance of crystal-like precipitates in the hydrogel cylinders  increase in cell density from 0.52±0.06 optical density units (controls without any of those polymers) to 1.38±0.21 units. Addition of 50 µmoles/L biosilica to the hydrogel resulted in an increase to 1.63±0.22 units; co-addition of 100 µmoles/L polyP•Ca2+-complex and 50 µmoles/L biosilica did not change the cell density significantly. This might imply that under these conditions the maximal potency of the cells to proliferate has been reached. During the 3 d incubation period the cells underwent 1.6 doublings (1.6-fold increase in cell density during the 3 d incubation). If 50 µmoles/L of chemically polycondensed silica, as ortho-silicate, is added to the hydrogel the cell growth increased to a smaller extent, but still significantly, to 0.87±0.11 units. In contrast, 10 µg/ml of silicatein added alone without ortho-silicate substrate to the hydrogel, had no significant effect on the cell density . Significantly higher values for the optical density units had been measured for those experimental sets which contained bioglass.A statistical analysis of the values for the cell growth in the absence and presence of bioglass had been performed by two-way analysis of variance (ANOVA). The analyses revealed that all experimental test series between the two groups, without and with bioglass, with the exception of the assays that contained no additional component particles to the cells embedded into alginate/gelatin hydrogel, containing in separate series of experiments, the polymers polyP•Cahe cells ; the ext2+-complex), to 0.61±0.10 nmoles/µg (50 µmoles/L silica), to 0.91±0.13 nmoles/µg (50 µmoles/L biosilica) and to 1.38±0.29 nmoles/µg (100 µmoles/L polyP•Ca2+-complex together with 50 µmoles/L biosilica); the values in the controls without any polymer and with 10 µg/ml silicatein vary around 0.35 nmoles/µg. Addition of 5 mg/ml of bioglass to the incubation assay increased the extent of mineralization significantly, for polyP•Ca2+-complex to 1.31±0.14 nmoles/µg, for silica to 0.98±0.14 nmoles/µg, for biosilica to 1.62±0.19 nmoles/µg and for the two components together (polyP•Ca2+-complex and biosilica) to 2.31±0.43 nmoles/µg (As outlined under “moles/µg . LikewisP<0.001) is seen between the samples lacking bioglass and those that had been treated with bioglass .To ascertain that only the minerals, synthesized by the cells had been quantified by the Alizarin Red S staining procedure, the scaffold had been printed without cells. After an incubation period of 5 d the hydrogel was dissolved by addition of 55 mmoles/L Na-citrate. After a washing step with saline and a subsequent keeping of the well plates in the culture chamber, the staining reaction was performed. In all assays, irrespectively if initially the hydrogel contained polyP, biosilica or silica the absorbance at 405 nm was determined. The values obtained had been lower than 0.8 nmoles dye bound per 1 cmIncubation period of 5 d of the cells in medium/FCS, supplemented with osteogenic cocktail, results in the formation of mineral deposits. Those 1 to 10 µm large deposits can be visualized by SEM  D.In order to make sure that the crystallites, formed onto the SaOS-2 cells indeed contain phosphate we analyzed the samples, after SEM analysis by energy-dispersive X-ray spectroscopy. In turn, the EDS analysis hat been performed at sub-micrometer spatial resolution; the iamages revealed an accumulation of the signals for the elements O, P and C ; the eleThe free-formed fabrication of bone implants requires the solution of two printable matrices. First a solid, bendable porous matrix and a more soft cell biocompatible hydrogel into which the embedded cells can proliferate and differentiate. In the present study it is shown that the bone-related SaOS-2 cells can be embedded into an alginate/gelatin matrix which, after supplementation with silica, but especially with polyP and biosilica, allows the cells to proliferate. Based on earlier findings that both polyP and biosilica display the potency to enhance the induction of morphogenetically active cytokines, e.g. BMP-2


We aimed to compare patient-controlled thoracic or lumbar epidural analgesia methods after thoracotomy operations.One hundred and twenty patients were prospectively randomized to receive either thoracic epidural analgesia (TEA group) or lumbar epidural analgesia (LEA group). In both groups, epidural catheters were administered. Hemodynamic measurements, visual analog scale scores at rest (VAS-R) and after coughing (VAS-C), analgesic consumption, and side effects were compared at 0, 2, 4, 8, 16, and 24 hours postoperatively.P = 0.001, P = 0.01, P = 0.008, and P = 0.029, respectively; and for VAS-C, P = 0.035, P = 0.023, P = 0.002, and P = 0.037, respectively). Total 24-hour analgesic consumption was different between groups . The comparison of postoperative complications revealed that the incidence of hypotension , bradycardia , atelectasis , and the need for intensive care unit (ICU) treatment  were lower in the TEA group in comparison to the LEA group.The VAS-R and VAS-C values were lower in the TEA group in comparison to the LEA group at 2, 4, 8, and 16 hours after surgery (for VAS-R, TEA has beneficial hemostatic effects in comparison to LEA after thoracotomies along with more satisfactory pain relief profile. The traumatic process of a thoracotomy operation involves cutting of the muscles, separation of the ribs along with the use of retractors during the procedure, and acute pain is expected to occur after surgery due to damage to the intercostal nerves. For postoperative pain management, either lumbar or thoracic epidural analgesia is required to diminish the incidence rate of possible pulmonary and cardiac complications ,2. HowevThe lumbar epidural analgesia (LEA) method is an alternative method for thoracic epidural analgesia (TEA) for the treatment of post-thoracotomy pain ,5,6. TheOur goal was to compare the hemodynamic and analgesic effects of patient-controlled thoracic or lumbar epidural analgesia methods in a prospective randomized study design after thoracotomy operations.Institutional Review Board of Sureyyapasa Chest Disease and Thoracic Surgery Hospital, clearance and written informed patient consent were obtained. One hundred and twenty adult patients with a diagnosis of lung cancer underwent a posterolateral thoracotomy procedure by one surgeon. Most incisions were performed with posterolateral thoracotomy because of an advanced stage of lung cancer and a huge mass. Muscle-sparing thoracotomy was applied when possible. Patients in an age range of 46 to 86 years and of American Society of Anesthesiologists (ASA) physical status I, II or III were included in a prospective, randomized, double-blind controlled study. The allocation of patients into groups was performed according to a random numbers list. Patients were randomized to either the thoracic epidural  group or lumbar epidural  group for a 24-hour postoperative period after surgery. From 147 patients, 13 were excluded as 7 of them did not meet the inclusion criteria and 6 of them declined to participate. A total of 134 patients were randomized into two groups. Two patients did not receive allocated intervention, and intervention was discontinued in 12 patients. The Consort diagram is presented in Figure 1) and forced vital capacity (FVC). FEV1 (% predicted) and FVC (% predicted) values were determined. In addition, preoperatively, intraoperatively, and at 2, 12 and 24 hours after surgery, arterial blood gas values including pH, PaO2  and PCO2  were collected. Also, the Charlson comorbidity index was used in this study, and it is classified into four groups to define and grade any existence of comorbidities. There are nineteen conditions that are related to increased risk for mortality and a weighted score of 1 to 6 was provided depending on the relative mortality risk.Demographic data, age, height, weight, body mass index (BMI), cigarette smoking, pulmonary function parameters, histological type of cancer, surgical procedures and duration of operation were recorded. Preoperative pulmonary functions measured included forced expiratory volume in one second  monitoring. An ST segment increase or decrease of ≥ 0.2 mV in men or ≥ 0.15 mV in women was monitored to determine signs of myocardial ischemia.4-6 or L2-3 interspaces while patients were in a sitting position. The epidural space was identified by using the loss of resistance to saline technique, which is required to demonstrate a negative aspiration of cerebrospinal fluid and blood. Afterwards, a test dose of 20 mg of 2% lidocaine  with epinephrine  was administered. The epidural catheter  was advanced in a cephalad direction and 5 mL was administered to both groups from the epidural catheter before general anesthesia was administered from the solution prepared before catheterization. A basal infusion at a dose of 2 mL/h was started intraoperatively after the anesthesia induction and just before surgery. At the end of the surgery, a patient-controlled epidural analgesia device  was connected to the epidural catheter. The infusion rate of PCEA was 0.1 mL/kg/h, with 2 mL on demand, and a lock-out interval of 30 min in the 24 hours postoperative period.The epidural catheters were applied at the operating table prior to the induction of anesthesia. An analgesic solution that contains 0.125% bupivacaine with sufentanyl  (0.6 μg/mL) was prepared by an anesthesia technician. All epidural punctures were inserted with an 18 gauge Touhy needle  through T2). A left-sided double-lumen endobronchial tube was inserted. Correct tube position was confirmed with the help of a stethoscope and confirmed by flexible fiberoptic bronchoscopy in both the supine and lateral decubitus positions. For intraoperative monitoring, when mean arterial blood pressure (MAP) or HR was 25% higher than the baseline value, it was treated with 0.3 μg/kg of fentanyl. On completion of surgery, the patients were reversed with intravenous neostigmine and atropine at a dose of 0.04 mg/kg and 0.02 mg/kg, respectively.General anesthesia was induced with 1 μg/kg of fentanyl  and 5 mg/kg of thiopental  intravenously. Tracheal intubation was facilitated with 0.15 mg/kg vecuronium , and repeated intraoperatively if necessary. Anesthesia was maintained with desflurane  in 70% oxygen and 30% air. The oxygen concentration was adjusted according to arterial blood gas values of partial arterial oxygen concentration value  bolus was administered and repeated if necessary. Bradycardia was defined as heart rate below 50 beats/min and was treated with bolus doses of 0.4 mg atropine sulfate .During the first 24-hour period in the intensive care unit (ICU), MAP, HR and SpO2 ≥ 60 mmHg, 2) FiO2 ≤ 0.40, 3) peak end expiratory pressure (PEEP) ≤ 5 cm H2O, 4) respiratory rate (RR) < 30, 5) minute ventilation of < 12 L to maintain partial arterial carbondioxide tension (PaCO2) between 35 and 45 mmHg, 6) appropriate level of consciousness, 7) intact cough and gag reflex, 8) vital capacity > 10 mL/kg, and 9) minimum inspiratory pressure < -30 cm H2O.At the end of surgery, patients were weaned from mechanical ventilation. Reintubation was necessary in some patients in the ICU. Criteria for weaning from mechanical ventilation include the following: 1) PaOPostoperatively, the incidence of nausea or vomiting was noted. Nausea greater than 2/10 ) or vomiting were treated with 10 mg intravenous metoclopramide . Pruritus was treated by intravenous difenhydramine  10 mg. The doses were repeated if necessary.We administered intravenous 1,000 mg paracetamol  every 8 h to all patients during the ICU treatment. At a VAS score > 3, breakthrough pain relief was provided through bolus doses of epidural analgesic agent at a dose of 0.1 mL/kg in addition to PCA. Additional pain relief was provided with intravenous morphine at a dose of 2 mg  in both groups for a VAS score > 3 at rest despite four consecutive PCA boluses.After extubation, patients were assessed by a physician in ICU to evaluate their pain at a scale from 0 (no pain) to 10 (disabling pain) by the use of VAS. Postoperative pain scores at rest (VAS-R) and after a strong cough (VAS-C) were evaluated. Sedation scores were judged by the observer . In each case, pain and sedation scores were evaluated at 0, 2, 4, 8, 16, and 24 h by a blinded resident to the study protocol on call in the ICU.2 > 50 mmHg) treated with 100% oxygen supplementation via a face mask. Patients were discharged from the ICU when the following criteria were met: SpO2 ≥ 90% at FiO2 ≤ 0.5 by face mask; hemodynamic parameters including MAP, HR and RR within normal adult limits; chest tube drainage < 50 mL/h; urine output > 0.5 mL/kg/h; and no intravenous inotropic or vasopressor therapy.Excessive sedation was defined as a score higher than 3 or 4 and either respiratory depression (defined as a ventilatory frequency below 8 breaths/min) or hypercarbia (PaCO2) values that were recorded at 0, 2, 4, 8, 16, and 24 hours; 2) visual analog scale (VAS) score at rest (VAS-R) and after coughing (VAS-C); 3) sedation scores; 4) the total amount of analgesic (PCEA) and morphine consumption; 5) the determination of postoperative complications ; and 6) the record of analgesic- and morphine-related side effects such as nausea and vomiting, pruritus, sedation or other adverse events.The TEA and LEA groups were compared in the first 24-hour ICU stay for 1) hemodynamic data including MAP, HR and arterial oxygen saturation  for repeated measures. Results are expressed as mean ± standard deviation (SD) where appropriate. For all tests, P < 0.05 was considered significant.The Statistics Package for Social Sciences  was used for all calculations. During sample size evaluation, the aim was to detect a clinically relevant reduction in the VAS score by 30 mm. To be able to detect a difference of 20 cm/h in the area under the curve of the VAS score after coughing with an expected standard deviation of 50 cm/h, and α and β errors of 0.05 (two-sided hypothesis) at a power of 0.8, the calculated sample size was 60 patients. To compensate for unforeseen dropouts and a possibly higher variability than expected, we planned to study 80 patients. The compliance of the data of the continuous measurements to a normal distribution was examined by the Kolmogorov-Smirnov test. Group differences for categorical variables were examined by Chi-square or Fisher’s exact test, as appropriate. Whereas in cases of normal distribution, group differences for continuous data were examined by independent samples 1 (79.56 ± 13.92 versus 83.02 ± 13.75) and FVC (81.58 ± 16.86 versus 84.92 ± 15.33) values were not different between the TEA and LEA groups.The comparison of the baseline characteristics between groups showed no significant difference Table . The dis2 and PCO2, serum CK-MB and troponin values preoperatively, and postoperatively at 6 and 24 hours, did not show statistically significant differences between groups   in the LEA group had ST segment depression during and after surgery. In contrast, none of the patients in the TEA group had signs of myocardial ischemia during or after surgery. Another patient in the LEA group developed a severe depression in the ST segment (-0.2 mV) six hours after operation in the ICU. A comparison of creatinine kinase MB fraction (CK-MB) and troponin values at different time points is shown in Table The postoperative hemodynamic data for MAP, HR, RR, and SpOP = 0.001, P = 0.01, P = 0.008, and P = 0.037 and for VAS-C, P = 0.035, P = 0.023, P = 0.002, and P = 0.029; respectively). However, at 0 and 24-hour time points, the observed differences were not statistically significant.A comparison of VAS-R and VAS-C scores is presented in Tables P < 0.0001), 4 hours , 8 hours , 16 hours , and 24 hours  for the same group. VAS-R scores at 0 hours (5.36 ± 1.85) in the LEA group were higher than VAS-R scores at 2 hours , 4 hours , 8 hours , 16 hours , and 24 hours  of the same group.The comparisons within groups for VAS-R scores revealed that the scores at 0 hours (5.43 ± 1.8) in the TEA group were higher than the VAS-R scores at 2 hours , 4 hours , 8 hours , 16 hours , and 24 hours  in the same group. VAS-C scores at 0 hours (7.32 ± 1.52) in the LEA group were higher than VAS-C scores at 2 hours , 4 hours , 8 hours , 16 hours , and 24 hours  of the same group.Within group comparisons revealed that the VAS-C scores at 0 hours (7.75 ± 1.48) in the TEA group were higher than the VAS-C scores at 2 hours , morphine consumption was similar (8.2 ± 11.3 mg versus 10.3 ± 11 mg). There was no difference in the use of ephedrine (19.29 ± 9.97 mg versus 12 ± 4.47 mg) and atropine (0.61 ± 0.22 mg versus 0.5 ± 0.00) between groups. Sedation scores were similar at 0, 2, 4, 8, 16 and 24 hours postoperatively. Two patients (3.2%) in the LEA group versus none in the TEA group had a sedation score ≥ 3 for 24 hours.While total 24-hour analgesic consumption was different between groups . The patients in the TEA group were discharged home in a comparable time period to the LEA group (8.74 ± 5.62 days versus 9.17 ± 6.83 days).In the early postoperative period, the use of a PCEA through either a lumber or thoracic epidural catheter has been shown to decrease the risk of myocardial ischemia, improve lung ventilation and decrease incidences of atelectasis and pneumonia ,3,7. In 4 dermatome level, and 3) a decrease in heart rate secondary to either sympathectomy or increased vagal tone [Previous studies investigated the effects of TEA on left ventricular function, and in these studies, major concerns of TEA were 1) a decrease in preload related to venodilation, 2) impairment in cardiac contractility secondary to sympathectomy at Tgal tone ,14. Thergal tone . Other sgal tone . Recentlgal tone ,18.P = 0.001) [In our study, the incidences of hypotension and bradycardia were significantly higher in the LEA group in comparison to the TEA group. In a recent study, a significantly higher incidence of hypotension with lumbar PCEA (7.7%) was observed compared with thoracic PCEA (4.1%) (= 0.001) . We also= 0.001) -21. Also= 0.001) . A previThe other most prominent action of TEA is on pulmonary functions. TEA has been associated with earlier mobilization, reduced opioid consumption, and improved cough, and thus, it has beneficial effects on lung functions causing diminished incidences of atelectasis or pneumonia ,24. In aThe comparison of TEA and LEA for pain relief after thoracotomies in the early postoperative period revealed that both techniques provide efficient analgesia; however, TEA has superior pain relief profile. These findings are comparable with previous studies and meta-analyses ,15,19,21Although better cardioprotective effects on serum cardiac enzymes and ST segment analysis have not been shown, TEA has beneficial hemodynamic effects in comparison to LEA after thoracotomies along with a more satisfactory pain relief profile in the 24-hour postoperative period.2: arterial partial pressure of oxygen; PCEA: patient-controlled epidural analgesia device; PCO2: arterial partial pressure of carbon dioxide; RR: respiratory rate; TEA: thoracic epidural analgesia; VAS: visual analog score; VAS-C: visual analog score after coughing; VAS-R: visual analog score at rest.BMI: body mass index; CABG: coronary artery bypass graft; CK-MB: creatinine kinase MB fraction; FEV1: forced expiratory volume in one second; FVC: forced vital capacity; HR: heart rate; ICU: intensive care unit; LEA: lumbar epidural analgesia; MAP: mean arterial blood pressure; PaOThe authors declare that they have no competing interests.All authors read and approved the final manuscript.