The objective of this study was to evaluate the validity of self-reported smoking prevalence in Korean adolescents by using an improved gold standard by a combined method. Using a stratified sampling method, we selected 13 schools from among 397 high schools that participated in the 2015 Korean Youth Health Risk Behavior Web-Based Survey (KYRBS). A second survey (repeated self-reporting questionnaire and urinary cotinine test) was conducted on 1058 students who completed the KYRBS. The gold standard of current smoker was defined as those either self-reporting as a smoker in the second survey or having a urinary cotinine concentration ≥50 ng/mL. The current smoking prevalence in the first survey (KYRBS) was 7.9% (boys 16.5% and girls 1.8%), which was lower than the results based on gold standard . The sensitivity and specificity of self-reported smoking status was 62.5% and 99.0%, respectively. In particular, the sensitivity of girls (43.5%) was lower than that of boys (67.0%). The self-reported smoking prevalence in Korean adolescents was underestimated, particularly among girls. Careful attention should be paid to interpreting adolescents’ smoking prevalence, and supplementary surveys or periodic validity tests need to be considered in Asian countries. Generally, self-reported questionnaires, which are generally used for computing smoking statistics, provide underestimated outcomes compared to the actual smoking prevalence . This isThe gold standard used by previous studies for assessing the validity of smoking surveys were mostly biochemical tests using urine, saliva, serum, and hair specimens. However, when only a biochemical test is used as the gold standard to assess the validity of self-reported smoking surveys, a misclassification error may arise, where passive smokers are classified as smokers while occasional smokers are classified as nonsmokers ,7. VarioSchool-based self-reported smoking surveys have employed the bogus pipeline method as a psychological approach to curb social desirability biases ,12,13,14Therefore, we attempted to assess the validity of anonymous self-reported smoking surveys by applying a new gold standard utilizing the combined method (repeated self-reporting questionnaire and urinary cotinine test) on some respondents to the Korean Youth Health Risk Behavior Web-Based Survey (KYRBS), Korea’s nationally representative survey. Additionally, we attempted to identify characteristics of smoking adolescents who provide false responses in smoking surveys.We used a subgroup of participants in the 11th Korea Youth Risk Behavior Web-Based Survey (KYRBS-XI) conducted in 2015 by the Korea Centers for Disease Control and Prevention. The KYRBS uses a stratified multistage probability sampling design to produce nationally representative statistics on health behaviors in Korean adolescents. A total of 68,043 students from 797 schools (400 middle schools and 397 high schools) completed the KYRBS-XI (response rate = 96.7%) [Among the high schools participating in KYRBS-XI, we selected 13 high schools using a stratified sampling method considering schools’ location (seven metropolitan city schools and six city schools), characteristics , and sex . Of the 1214 students in the 13 high schools participating in the KYRBS-XI, 12th grade students (79 students) from two schools that refused to participate in the repeated survey due to academic constraints were excluded. Of the 1135 students in targeted populations, 1111 students participated in a repeated survey, and the final study population who completed the urinary cotinine test was 1059 students (participation rate 93.3%) after excluding students who refused the urinary cotinine test (52 students).The KYRBS-XI (hereafter first survey) and second survey were conducted in June 2016, and June or July 2016, respectively, and the median time between the two surveys was 15 days (interquartile range 12–28 days). There was no specific school schedule including school trips or vacations that influenced students’ smoking habits. The first survey was carried out in the same way as the usual KYRBS-XI (web-based anonymous self-reported questionnaires under supervision of school staff). Those who participated in the first survey were not informed about the second survey for validation of self-reported smoking status until after they completed the first survey. Upon completing the first survey, they were told that resurvey for validation of self-reported smoking status would proceed; they were advised to turn in an enclosed identification number to the investigator for matching individuals between surveys. The second survey was composed of an anonymous self-reported questionnaire and a urinary cotinine test. Before the second survey, the participants were told about the urinary cotinine test for their smoking validation by well-trained surveyors, and they assured the students of the anonymity and confidentiality of their survey results. The results of the first and second survey were matched individually by identification number. This study design was approved by the Institutional Review Board of the Daegu Catholic University Medical Center (CR-15-049).In both the first and second survey, a self-reported current smoker was defined as a person who had smoked one or more days in the most recent 30 days using the following question: “During the past 30 days, on how many days did you smoke cigarettes?”During the second survey, midstream urinary samples among students who completed the questionnaire were collected. The samples were analyzed by a liquid chromatography–tandem mass spectrometry (LC-MS-MS) system on an ABSciex API 4000 using the TurboIonSpray interface and multiple reaction monitoring  . StudentN = 1163). Since sex-specific smoking prevalence in co-educational classes cannot be calculated due to not asking the number of smokers by sex in each class, students in co-educational classes (six classes in two schools) were excluded from the calculation to estimate sex-specific smoking prevalence.In addition to this individual classification of current smokers, estimated smoking prevalence was suggested as a subsidiary method to assure the confidentiality and anonymity using the representative value rather than the individual response to smoking questionnaire questions. Estimated smoking prevalence based on the mode value of the number of smoking classmates was calculated by asking the following question: “How many students smoke in your classroom?” In cases where the mode value was 0 or extreme (ex. 13 in 10th grade girls), we selected the second most frequent value. When there were two mode values in a class, we applied the mean of the two mode values. Estimated smoking prevalence was calculated by dividing the sum of the estimated number of smoking classmates by the total number of the students in the selected class  results could leak out to teachers or parents?” Closest friends’ cigarette smoking was assessed using responses to the following question: “Do any of your closest friends smoke tobacco?” Participants were provided with three possible answers: (1) none of them; (2) some of them; and (3) most/all of them.Current smokers were assessed using the following four methods: (1) self-reported in first survey; (2) self-reported in second survey; (3) urinary cotinine concentration ≥50 ng/mL in second survey; (4) combined method in second survey (either by self-reported questionnaires and/or urinary cotinine concentration). The combined method was used as a gold standard for the classification of current smokers.Validation of anonymous self-reported smoking status was evaluated in two ways using three indicators . First, results between self-reported questionnaires and urinary cotinine concentration within the second survey were compared. Since students knew about the accompanying biochemical test before filling out the questionnaires, this method evaluated the validity of the questionnaire survey conducted by the bogus pipeline method. Second, the results of self-reported questionnaires in the first survey were compared with those of the second survey with three different criteria: (1) self-reported questionnaires; (2) urinary cotinine concentration; and (3) the combined method (gold standard). This comparison was newly designed to assess the validity of anonymous self-reported smoking status by applying improved criteria.Among current smokers based on gold standard criteria , multivariate logistic regression was performed to investigate the characteristics of respondents providing false information. Sex, grade, confidentiality of survey environment, friends’ cigarette smoking, and cotinine level were included in statistical model; cotinine levels were classified as tertiles.t-test for equivalence  was used to evaluate validity of self-reported height. All analyses were performed using SPSS version 19.0 , and a p-value of <0.05 was considered significant.For comparison with the validity of anonymous self-reported smoking status, the validity of both sensitive items (self-reported current drinking status) and non-sensitive items (self-reported height) were also evaluated between the first and second surveys. The validity of self-reported drinking status was evaluated in the same way as the smoking status, using three indicators . Paired p < 0.001). The proportion of smoking closest friends in girls was significantly lower than that in boys (p < 0.001). There was no statistically significant difference in perceived threats to the confidentiality of the survey response between boys and girls , which was lower than those of 9.0–11.3% in the second survey using the self-reported questionnaire, urinary cotinine concentration of ≥50 ng/mL, or the estimated prevalence based on the mode value of the number of smoking classmates. Of all of the methods, the overall prevalence of current cigarette smoking in the study population was the highest when smoking status was assessed using the combined method  in the second survey; 3.4 percentage point  higher than the results from the first survey .When the validity of the self-reported current smoking status in the first survey was evaluated through comparison with the second survey results with three criteria. The sensitivity based on the combined method was the lowest at 62.5% , and there was a difference of about 5% as compared with other methods. The results of stratification by sex also showed the lowest sensitivity in the combined method. According to this combined method, 33.0% of boy smokers and 56.5% of girl smokers reported that they did not smoke cigarettes in the first survey. Although there was no statistical significant difference between sexes, sensitivity in girls was lower than that in boys by 23.5 percentage points. When sensitivity was evaluated by a single method, the difference in sensitivity between sexes was also about 20 percentage points. Overall, the specificity of self-reported smoking was more than 95.0% regardless of sex or evaluation method .Compared with the second self-reported questionnaire, the sensitivity for self-reported current drinking status in the first survey was similar to that of smoking (drinking 69.9% vs. smoking 67.7%), but the specificity of the alcohol questionnaire was lower than that of smoking (drinking 91.0% vs. smoking 99.1%). As in the result of self-reported smoking status, the sensitivity for self-reported drinking status in girls was lower than that in boys. However, the sex difference in sensitivity was larger in the questionnaire for questions about smoking than for questions about alcohol (smoking 17.3 percentage points vs drinking 7.2 percentage points) see . Unlike Of 120 current smokers based on the combined method, girls were 2.64 times more likely to hide their smoking status in self-reported surveys than boys. However, this significant result became non-significant after multivariate analysis. There was no statistically significant association between hiding smoking status and perceived threat to the confidentiality of the survey responses. The risk of smokers hiding their smoking status significantly increased from 1.58  to 58.21  when the number of closest friends who smoked decreased. Students with low urinary cotinine concentrations were significantly more likely to hide their smoking status .This study assessed the validity of self-reported smoking surveys in adolescents against a gold standard based on a combined method (repeated survey and urinary cotinine test). About 37.5% of smoking adolescents lied about their smoking status, and the proportion of false responses was higher for girls (56.5%) than for boys (33.0%). In contrast, the specificity of the survey was 99%, suggesting that nonsmokers rarely claimed to be smokers. Although the validity of surveys varies across studies, adolescents’ self-reported smoking prevalence was underestimated compared to the actual smoking rate ,18,19,20In previous studies assessing the validity using a biochemical test alone as a gold standard on Korean adolescents, the sensitivity of the survey was high at about 90%, confirming the validity of self-reported smoking surveys ,26. HoweIn the present study, adolescents whose friends do not smoke or who had low urinary cotinine concentrations were more likely to lie about their smoking status. Because smoking is perceived as a socially undesirable behavior, occasional smokers who have only recently begun to smoke or those who do not have smoking friends are thought to have attempted to hide their smoking status from others and thus lied about it in the surveys. The NHSDA in the US also showed that adolescents who had more smoking friends were less likely to give false responses about their smoking status . Social This study has a few limitations. First, the second survey was conducted about 15 days (median) after the first survey, and the students’ smoking status may have changed within that period. However, we administered the survey during the semester to avoid special circumstances that may affect students’ smoking status, such as school trips or summer vacation. We performed the surveys before the summer break in Korea, considering that smoking rate among adolescents tends to rise during school breaks when adult monitoring is difficult . The NatSecond, it may be difficult to generalize the findings of our study because we did not include all of the participants of the KYRBS-XI, a nationally representative survey of Korean adolescents, in our investigation. Because it is practically difficult to investigate all the KYRBS-XI respondents , we selected the schools via a stratified sampling method considering the size of the school’s location, characteristics, and co-educational status to ensure the representativeness of the sample. Nonetheless, the results of this study cannot be generalized to non-Korean adolescents. However, our study can provide meaningful data to Asian countries because, to the best of our knowledge, there has been no validity study using a biochemical test on more than 1000 adolescents in an Asian country.There have been differences in validity results across countries  ,20,21,28In Asian countries, investigators should reconsider conducting their validity studies during periods of rapid decline of smoking prevalence or periods of changes in social norms concerning smoking. Administering biochemical tests may be practically challenging due to high costs or ethical reasons, so it may be useful to regularly perform surveys to estimate smoking prevalence using additional methods. In China, the adolescent smoking rate has been estimated using the capture-recapture method based on an additional survey that asked parents about their child’s smoking habits . In our Self-reported smoking prevalence in adolescents considerably underestimates the actual smoking prevalence in adolescents, where about one-third of boys and one-half of girls who smoke lie about their smoking status in smoking surveys. Thus, investigators should be careful when interpreting adolescent smoking prevalence, and supplementary surveys or regular validity tests should be performed to compensate for the limitations of self-reported smoking surveys.


According to data presented at the 2017 American Society of Oncology (ASCO) Annual Meeting, with more than 4 years of follow-up, ibrutinib continues to provide clinical utility in chronic lymphocytic leukemia (CLL). However, treatment of CLL patients with high-risk cytogenetics features remains a challenge and the outcome of these hard-to-treat patients is dismal. At the 2017 ASCO Meeting, results of the GENUINE phase III trial showed that, by adding ublituximab, a glycoengineered, anti-CD20 type 1 monoclonal antibody, to ibrutinib, the overall response rate (ORR), complete response rate (CRR), and minimal residual disease (MRD) negativity may be improved in high-risk CLL patients. A further way to improve the results obtained with Bruton’s tyrosine kinase (BTK) inhibitors is the parallel use of ibrutinib with chimeric antigen receptor (CAR) T-cell therapy. Through this investigational approach, the rate of MRD negativity was shown to be higher, implying potential eradication of CLL. These novel data indicate that ibrutinib continues to have a positive effect in CLL. With the regulatory approval of ibrutinib, idelalisib, and venetoclax, as well as of other therapeutic small molecules likely to become widely available in the coming years, the treatment of chronic lymphocytic leukemia (CLL) has changed dramatically. However, complete remissions (CRs) are rare in CLL and treatment options for patients relapsing after treatment with ibrutinib remain limited . The potAt the 2017 American Society of Oncology (ASCO) Annual Meeting, investigators presented mature results from key ibrutinib clinical trials and emerging data on novel associations with ibrutinib, demonstrating activity against highly resistant, harder-to-treat CLL. Between the new drugs  and immunotherapeutic approaches, such as chimeric antigen receptor T-cell (CAR T-cell) therapy, there is great hope for the future treatment of CLL patients.P < 0.0001; 3-year PFS 59% versus 3%). The significant benefit was observed across patient subgroups with genomic abnormalities usually considered at higher risk of progression. Patients with deletion 11q tended to have the most favorable outcome; furthermore, PFS was not statistically different for patients with deletion 17p or deletion 11q or without these FISH abnormalities. The safety profile of ibrutinib was consistent with previous reports [Pivotal RESONATE data on ibrutinib, a first-in-class Bruton’s tyrosine kinase (BTK) inhibitor, have significantly transformed the treatment landscape for patients with relapsed or refractory CLL . An updaAlthough results of the RESONATE trial highlight the value of ibrutinib in the treatment of CLL, to date, hematologists have little guidance on which kinase inhibitor (KI)  should be used first . A recenIbrutinib has been associated with an anti-CD20 monoclonal antibody under the assumption that the latter would help reduce the lymphocytosis often observed with ibrutinib single agent . RituximUblituximab is a glycoengineered, anti-CD20 type 1 monoclonal antibody that maintains complement-dependent cytotoxicity and enhances antibody-dependent cell-mediated cytotoxicity . At the P < 0.001). PFS showed a trend toward improvement in the patients treated with the combination, with a HR of 0.559, albeit not statistically significant at the time of analysis. The investigators concluded that the study met its primary endpoint, with a superior and deeper response rate of association in comparison to ibrutinib single agent; however, the results should be interpreted with caution due to the absence of a PFS improvement [In the combination arm, the best overall response rate was 78%, with 7% achieving CR, compared with 45% in the monotherapy arm with no CRs (A challenging issue is that of introducing immunotherapy with anti-CD19 CAR T-cell therapy into conventional CLL treatment . At the In conclusion, the ASCO 2017 Annual Meeting showcased a number of important studies expected to influence the scenario of CLL treatment in the near future. In particular, strategies of ibrutinib association are likely to contribute to the complete eradication of disease from the bone marrow and to potentially cure CLL.


Exposure to high doses of radiation poses a significant threat to human life. The current methods of accurately predicting the biological effect of radiation are still dependent on the manifestation of symptoms or rely on technically complex and time-consuming tests such as the dicentric chromosome assay . One of MicroRNAs (miRNAs) are non-coding RNAs of 19-22 nucleotides that regulate diverse cellular processes, and serum circulating miRNAs have recently emerged as promising biomarkers for diagnosing various malignancies and predicting treatment responses , 4. The In 2015 our team identified a panel of serum miRNAs that were differentially expressed in response to total body irradiation (TBI) in C57BL/6J mice 24 hours after exposure [Macaca mulatta, Chinese substrain) subjected to total doses of radiation ranging from 5.8Gy (LD30/60) to 7.2Gy (LD70/60) during a drug trial on radiation mitigating agents. We identified seven miRNAs  that were altered by irradiation in both mice and NHPs, proving that this pattern is evolutionarily conserved, most likely due to conserved transcription factor binding sites within promoter regions of genomic miRNA-coding sequences. Interestingly, bioinformatic analyses revealed seven transcription factors that are predicted to bind to the promoter sequences of all seven conserved radiation-responsive miRNAs in mice, macaques and humans. It was demonstrated that serum concentrations of the evolutionarily-conserved miRNAs miR-30a, miR-126, and miR-375 correlated with the efficacy of a radioprotective agent, γ-tocotrienol (GT3). However, the concentrations of remaining four conserved miRNAs showed a greater changed in expression change to radiation than that observed in controls. This suggests that the processes regulated by GT3 differ from those modulated by amifostine, however, this issue requires in-depth mechanistic studies. In addition, we developed a classification tool trimmed down to only three miRNAs  which accurately distinguished NHPs exposed to radiation from those who were not. We also found striking evidence of miRNAs sex-dependent changes in response to radiation: miR-16-2 was associated with survival and its decreased expression was predictive of death both in males and females, however, the males showed half the pre- and post-exposure expression than females. Therefore, the design of an effective prognostic model required us to account for the sex of the animals, and by doing so we showed that we can reproducibly predict radiation-induced mortality in NHPs using a model consisting of miR-30a and miR-126. Further studies are required to determine whether radiation-induced serum miRNA signatures are tissue-specific or related predominantly to damage of the hematopoietic system.The results obtained in a mouse model are not however easy to translate into clinical practice. Given the impossibility to test the efficacy of our biomarkers in humans directly, we sought to develop an experimental model mimicking the high-dose irradiation scenario as accurately as possible. Thus, we decided to test our miRNA-based diagnostic tool for lethal irradiation in nonhuman primates (NHPs) . We studUnresolved before miRNA-based tests or drugs may be put into clinical practice. The biological mechanism of secreting the miRNAs into the serum remains unknown, as are the regulatory mechanisms leading to differential expression of radiation-inducible miRNAs depending on sex and their complex interactions with radiation mitigating or radioprotective agents. Moreover, if the observed miRNAs are exosome-bound, they could actively participate in cell-to-cell transfer and play an active role in mitigating radiation-induced damage, rather than acting only as biomarkers. However, studies suggest that novel biodosimetric miRNA-based tools and even RNA-based drugs will be within our reach in the near future.Furthermore, the utility of miRNAs in detecting and quantifying radiation exposure may be potentially translated to the field of radiotherapy. Our clinical paper showed that circulating serum miRNA profile changes in response to the delivered dose of radiation of radiation in patients treated for lung cancer , while a


Background. Fracture load of implant-supported restorations is an important factor in clinical success. This study evaluated the effect of two techniques for screw access hole preparation on the fracture load of cement-screw-retained implant-supported zirconia-based crowns.Methods. Thirty similar cement-screw-retained implant-supported zirconia-based maxillary central incisor crowns were evaluated in three groups of 10. Group NH: with no screw access holes for the control; Group HBS: with screw access holes prepared with a machine before zirconia sintering; Group HAS: with screw access holes prepared manually after zirconia sintering. In group HBS, the access holes were virtually designed and prepared by a computer-assisted design/computer-assisted manufacturing system. In group HAS, the access holes were manually prepared after zirconia sintering using a diamond bur. The dimensions of the screw access holes were equal in both groups. The crowns were cemented onto same-size abutments and were then subjected to thermocycling. The fracture load values of the crowns were measured using a universal testing machine. Data were analyzed with ANOVA and Tukey test (P < 0.05).Results. The mean fracture load value for the group NH was 888.37 ± 228.92 N, which was the highest among the groups, with a significant difference (P < 0.0001). The fracture load values were 610.48 ± 125.02 N and 496.74 ± 104.10 Nin the HBS and HAS groups, respectively, with no significant differences (P = 0.44).Conclusion. Both techniques used for preparation of screw access holes in implant-supported zirconia-based crowns decreased the fracture load. However, they have some limitations, including early loosening, difficult or impossible retrieval, and impossible cleaning of excess cement in deeply placed implants.3Implant-supported restorations are considered a choice for replacing missing teeth. Selection of the type of attachment among different implant prostheses and implants plays a pivotal role in the clinical service and long-term success of an implant-supported restoration. Retention of implant-supported crowns may be provided by the use of a screw or cement. Screw-retained and cement-retained restorations have benefits as well as shortcomings. For instance, crown retrieval is easier in screw-retained restorations, but the crown may not be passively placed over the abutment, and the screw may become loose or broken.4 However, because of the increased patient demand for implant treatment, the prevalence of screw loosening and its associated biological and technical complications is on the rise.5According to a systematic review conducted by Jung et al,6 problems of the implant/abutment joint, such as screw loosening and fracture, are among the most common complications compromising the survival of implant-supported, screw-retained fixed partial dentures.6Currently, due to the improved design and a better understanding of the mechanics of screw, screw loosening or fractures are rare in dental implants.7 introduced a technique using a screw locked in the implant restoration to retrieve the cemented restoration. Okamoto et al8 suggested preparing cylindrical holes in the lingual surface for this purpose. McGlumphy et al9 suggested providing retention by both cement and screw, to facilitate accessing the screw hole of restorations and to enable cleaning of the subgingival cement. This technique was referred to as the “combination implant crown”.10This method has been recommended for use in implant-supported metal ceramic restorations; it also allows for permanent cementation of crowns instead of using a temporary cement.10Baige et al11 reported a technique to retrieve implant-supported cemented restorations that benefits from the advantages of both cement-retained and screw-retained fixations. A case report by Rojas-Vizcaya12 described restoration of a maxillary lateral incisor via a cement-screw-retained implant-supported zirconia-based crown. In this method, a screw-retained metallic substructure was designed and a cement-retained crown was fabricated over it. It seems that cement-screw-retained implant-supported restorations have been well developed in prosthodontics.Several methods have been proposed for the retrieval of cement-retained implant-supported crowns. Chee et al13 showed that treatment with implant-supported zirconia-based fixed prostheses yielded favorable outcomes and that screw retention was feasible at the implant level. However, these prostheses have to be screwed and suffer from the same limitations as metal-ceramic cemented crowns.3 Use of cement-screw-retained implant-supported zirconia-based restorations can be a solution to this problem. In this approach, a zirconia-base restoration with a screw access hole is fabricated, cemented on a titanium abutment in the lab, and finally screwed onto a dental implant in the oral cavity.On the other hand, implant-supported zirconia-based crowns, fabricated by computer-assisted design/computer-assisted manufacturing (CAD/CAM) systems, have acceptable esthetics, optimal biocompatibility, high fracture strength, resistance against crack propagation and high adaptation, which explains their popularity. Worni et al14the effect of screw hole preparation on the crown fracture load has yet to be clearly understood. Some previous studies on the fracture load of cement-retained and screw-retained implant-supported metal-ceramic restorations with and without screw access holes have shown that crowns with screw access holes had lower fracture load.16 In a clinical setting, the mechanical resistance of implant-supported restorations plays a pivotal role in the clinical service and long-term success of dental implants.17 The fracture load is defined as the maximum force that a material can tolerate before fracture. Several factors influence the fracture load of zirconia ceramics, including loading conditions, type of cement, and restoration design, the density of pre-sintered zirconia blocks, zirconia sinterability, mechanically created cracks, residual compressive stresses during preparation, yttria contents of zirconia, external stresses due to sandblasting, wear, cyclic loading and thermal or chemical aging .18Considering the structure of zirconia-based crowns and the mechanism of crack propagation and its control,24 the effect of screw access hole preparation on the fracture load of zirconia-based crowns has yet to be fully evaluated. Hence, the aim of this study was to assess the effect of two techniques for screw access hole preparation on the fracture load of implant-supported zirconia-based crowns. The null hypothesis was that screw access hole preparation would not affect the fracture load of implant-supported zirconia-based crowns.Despite some previous studies related to the fracture load of zirconia ceramics,This was an in vitro study which did not involve the use of any animals or human data or tissues, and thus an ethics approval was not required.22 Since all the abutments had equal dimensions, a wax pattern of an anterior crown without a screw access hole was made on the abutment for laser scanning using the Sirona inEos machine .The wax pattern was scanned and the scanned image was transferred to the Sirona inEos CAD/CAM machine .Then a screw access hole with predetermined dimensions  was created on the same wax pattern and scanning process was performed again. The scanned images were used to fabricate 30 zirconia cores with the thickness of 0.6 mm from zirconia blanks  (20 cores without screw access hole and 10 cores with screw access hole) , measuring 4.3mm in diameter and 15mm in height, were used in this experimental study. Three 15° esthetic abutments , measuring 0.5 in mm collar height and 7.5 mm in height, were selected. The abutments were fixed in the implant analogs by applying a torque of 35 N.cm based on the manufacturer’s instructions. A two-piece custom metal block was fabricated and the implant analogs were then embedded in the metal block at a 35° angle relative to the vertical axis. Taking into account the 15° angulation of the abutment relative to the longitudinal axis of implant analog, the final angulation of abutment relative to the vertical axis was 50°.ss hole) . The zirss hole) with a siss hole)  and diviGroup NH: This group included 10 zirconia-based crowns with no screw access holes.Group HBS: This group included 10 zirconia-based crowns with screw access holes which were prepared before zirconia core sintering. The holes were designed by the CAD/CAM system according to the scanned image, with predetermined dimensions. The zirconia cores were sintered and veneered.Group HAS: This group included 10 zirconia-based crowns with screw access holes which were prepared after zirconia core sintering. The holes were manually prepared in the palatal surface of zirconia cores using the mentioned bur, and matched dimensionally to group HBS.23The excess cement was removed. For specimens in groups HBS and HAS, after cementation, the screw access holes were completely filled with the same composite resin and the composite resin was light-polymerized. All the specimens were subjected to 500 thermal cycles in a thermocycler between 5 and 55°C, with a dwell time of 60 seconds for each specimen and 12 seconds of intermediate pause.The abutments were cleaned with pumice powder and water. Cotton pellets were placed over the abutment screws. For specimens in group NH, the abutment screw holes were filled with composite resin . Next, the composite resin was light-polymerized prior to cementation. All the thirty crowns were cemented over the abutments using resin cement  according to manufacturer’s instructions and pressed with finger pressure for 2 minutes.24 The load was applied 3 mm below the incisal edge23 out of the hole area. The load applicator of the universal testing machine was similar to the incisal edge of a mandibular incisor (dimension of 5.5×1 mm), to simulate clinical settings.25 The magnitude of the load, which was applied by the universal testing machine, increased until the zirconia core fractured. The fracture load was recorded. In case of ceramic veneer fracture without core fracture, the specimen was eliminated from the study. After fracture occurred, the specimens were heated to decrease the retention of cement on the abutment, and the crown was removed. After removal of the crown, the cement remaining on the abutment was thoroughly removed by an explorer. The abutment surface was cleaned with an acetone solution and water. Then the abutment was dried, tightened using a torque of 35 N.cm, and prepared to cement the next crown. This process was repeated for all the specimens. The fracture load was measured for each specimen in Newton (N). Data were analyzed with ANOVA and Tukey test using SPSS 20 . P < 0.05 was considered statistically significant.To determine the fracture load, the specimens were subjected to compressive load testing in a universal testing machine . Compressive loads were applied vertically and at a 50° angulation relative to the abutments at a crosshead speed of 2 mm/min .22,24 ThThe means and standard deviations for the fracture load values of the specimens in the three groups were 888.3 ± 228.9, 610.4 ± 125, and 496.7 ± 104.1 Nin the NH, HBS and HAS groups, respectively . After eBased on the results of the current study, preparation of screw access holes in cement-screw-retained implant-supported zirconia-based crowns before and after sintering  resulted in a significant reduction in the fracture load values. Moreover, considering the technique of the preparation, the manual and machine techniques gave rise to no differences in this respect. Thus, the null hypothesis of the study was refuted.26Therefore modifications on the substructures may affect the fracture of restorations. One of these modifications is to prepare screw access holes. Most relevant previous studies have evaluated the effect of access hole preparation on the fracture load of posterior metal-ceramic restorations and to the best of authors’ knowledge, no similar study has been conducted on cement-screw-retained implant-supported zirconia-base crowns.Cement systems, bonding procedures, occlusion and metal copings all play a role in the success of fixed prostheses and all these factors must be taken into account in order to be able to make a comparison among novel ceramic and metal substructures.27 Some researchers believe that the preparation of screw access holes in screw-retained restorations can undermine the ceramic around the hole and also the ceramic at the cusp tip, leading to ceramic fracture; however, this problem does not exist in cement-retained restorations.28Evidence has shown that cement-retained restorations with intact occlusal surfaces have higher porcelain fracture load than screw-retained restorations.30 Similar results were obtained in the current study since the preparation of screw access holes before and after sintering significantly decreased the fracture load of zirconia crowns. Wood et al31evaluated the effect of access cavity preparation for root canal therapy on the fracture load of zirconia and alumina restorations. All the specimens showed porcelain chipping around the access cavity and the decrease in the fracture load of the zirconia restorations was greater than that of the alumina restorations.31 However, it should be noted that in the study by Wood et al,31the access cavity prepared for root canal therapy was much larger than the screw access hole prepared in implant-supported restorations. This difference may have been responsible for the inconsistent comparisons. Screw access hole preparation in screw-retained restorations disrupts the structural integrity of the zirconia ceramic and results in subsequent displacement of the center of ceramic mass towards ceramic gaps during the sintering process. Such a displacement changes the behavior of ceramic in these restorations compared with cement-retained restorations.30 Nonetheless, it should be noted that due to the variability in the use of different methods, implant systems, loading conditions, cement materials, restoration design, type of abutment and type of alloy, a direct comparison of the results across different studies may not be feasible.Several studies have demonstrated that preparation of screw access holes in screw-retained metal-ceramic restorations decreased the fracture load of porcelain.19The strength of zirconia directly relates to the size, number, location and distribution of defects, cracks and superficial gaps/porosities.32 However, mass cracks possess a covering protection and are supported by the surrounding material. Superficial cracks directly serve as stress accumulation areas and intensify the applied stresses, depending on their extent.32This is due to the size of superficial cracks in all-ceramic restorations. Sharp, deep defects result in an increased concentration of stress at the tip of the cracks and likely serve as sites of crack initiation.33 Although the superficial defects created in the process of CAD/CAM preparation may have insignificant effects on the strength of specimens, they may have a significant impact on the fatigue behavior of zirconia restorations.34 Small defects propagate due to cyclic loading of mastication and eventually cause sudden fractures in ceramic.34 Consequently, despite their small size, superficial defects can serve as stress concentration sites and can lead to restoration fracture.34Fully sintered zirconia is very tough, making its preparation extremely difficult. Moreover, the preparation of zirconia ceramic has adverse effects on its mechanical properties.35 reported that many factors can affect the mechanical properties of zirconia ceramics such as hardness and strength. These factors include the density of pre-sintered zirconia blocks , sinterability of the powder (due to its correlation with the size of primary particles), mechanically created cracks, residual compressive stresses during preparation of specimens and high yttrium content .36Consequently, the screw access hole preparation may mechanically create cracks. This may be the reason for a decrease in fracture load following screw access hole preparation in the present study.Roebben et al36 Denry et al37suggestedthat the minimum thickness of zirconia core should be 0.5 mm to avoid fractures; accordingly, a durable zirconia core thickness was employed in the present investigation. Haraldson et al38reported a mean load of 206 N applied to the anterior region of dental arch during masticatory function. Maximum bite forces in the anterior incisor region correspond to approximately 150 Nin adults.39 Gay et al40 estimated human incisal bite forces ranging from a minimum of 25 N to a maximum of 400 N. Although the results of the present study cannot be directly generalized to a clinical setting, all the study groups showed higher fracture loads than the anterior biting force. Based on the results of this study and also the range of occlusal loads applied to the anterior teeth, the fracture load values of the study groups were within an optimal range of functional loads. Similarly, Worni et al13 demonstrated that screw-retained implant-supported zirconia crowns had optimal survival rates in the oral environments.In the current study, the thickness of the zirconia core was adjusted at 0.6mm according to a study by Potiket et al.41 Obviously, application of a single, compressive load cannot simulate all the clinical loads that a restoration is exposed to.42 In fact, one may not be able to exactly relate a fracture load test, performed with the application of single compressive load until fracture in a dry environment, to the dynamics of numerous fractures that occur in the wet oral environment due to cyclic loading.43 Therefore, generalization of the results would have been possible if the specimens had been tested under fatigue loading. Moreover, quick load application to a single point is different from normal mastication in the oral environment because the clinical pattern of loading decreases the loads applied to the restorations.44 Furthermore, a clinical pattern of load application can cause fatigue fractures in small amounts compared to in vitro loading conditions. Therefore, further studies are necessary under the conditions noted above. If the results of the current study can be supported by further successful clinical researches, screw access hole preparation will be deemed a useful method to fabricate cement-screw-retained implant-supported zirconia-based restorations.The current study had some limitations; the crowns studied were only subjected to one cycle of compressive load until fracture; whereas, in a clinical setting, the crowns may undergo fracture following fatigue processes and the formation of small cracks.Based on the results of the present study in relation to a decreased fracture load in cement-screw-retained implant-supported zirconia-based crowns, reinforcement of the zirconia core is recommended in these cases. Also screw access hole preparation techniques need innovations and more investigations.Within the limitation of the study it was concluded that:The preparation of screw access hole in cement-screw-retained implant-supported zirconia-based crowns before sintering (machined technique) and after sintering  decreased the fracture load.In this respect, there was no significant difference between the two techniques of access hole preparation .The authors do not have any individual or organization to acknowledge with regards to this paper.HM, REA, and MM contributed to the concept and design of the work. The acquisition, analysis, and interpretation of data were accomplished by MM and FT. HM, MM, FT, and MA drafted and revised it critically for intellectual content. All the authors read and approved the final manuscript.This project was supported and funded by the authors.The authors declare no competing interests with regards to the authorship and/or publication of this article.Not applicable.


A new study uncovers a potential mechanism that may allow zinc-finger domains in metazoans to recognize and bind virtually any DNA sequence.The human genome encodes about 700 proteins with Cys2-His2 zinc finger (C2H2-ZF) domains, the majority of which are likely to bind to DNA and regulate transcription. This makes C2H2-ZF the largest transcription factor (TF) family in our species , and theA combination of evolutionary processes has resulted in a diverse range of C2H2-ZF proteins that show affinity for different DNA targets in metazoans . This imGenome Biology, Najafabadi and colleagues [In a recent study published in lleagues  revealedZinc-finger domains are rarely found alone; instead, they tend to form tandem arrays and sometimes they combine with different domains. This increases the length of the interaction surface and thus the number of different potential targets. The amount of redundancy in the binding sites is astonishing and it has been estimated that hundreds or even thousands of different C2H2-ZF sequences can recognize the same DNA triplet .In general, transcription networks evolve through modifications of cis-regulatory sequences rather than through changes in the TFs. Mutations in the latter are often deleterious because they change the regulation of several genes at the same time. As a result, many developmental TFs are highly conserved across species. Zinc-finger containing proteins, however, do not appear to follow this general rule and have instead undergone very rapid diversification. At least some of these changes appear to have been adaptive , suggestAnother interesting property of zinc-finger proteins is that, unlike other abundant TF families, they have undergone bursts of gene duplication at different evolutionary time points, such as at the base of the vertebrates or in the primate branch , 7. AlthA longstanding question is whether a zinc-finger–DNA recognition code exists. In other words, given the sequence of a zinc-finger domain, can we predict its DNA target? Deciphering such a code would be useful to identify the actual targets in the genome and to better understand the contribution of different amino acids to the binding mechanism.Genome Biology [To attempt to decipher the code, two studies , 5 used  Biology  that theHow does the relative abundance of the C2H2-ZF domain in metazoans compared to other eukaryotes affect the diversity of DNA targets? Najafabadi et al.  describeThe researchers discovered that there were other fundamental differences between the two groups of organisms. The recognition code in non-metazoans was mostly determined by the base-contacting residues, with little influence from other amino acids in the sequence. However, in metazoans, both base-contacting and non-base-contacting residues were required for binding.To unravel the complexity of zinc-fingers in metazoans, Najafabadi et al.  developeNajafabadi et al.  also anaThe metazoan zinc-finger domain provides a fascinating example of exhaustive exploration of the sequence space, with only four residues being completely conserved and the rest being highly variable. As shown in the study by Najafabadi and co-workers , the binThe large-scale studies performed to date have focused on single domains; therefore, much remains to be learnt about the combinatorial effects of several C2H2-ZFs. New experiments will be required to validate the model and to understand how proteins with multiple domains behave. Another open question is why the fast evolution of zinc-fingers is not harmful, considering the potential of these domains to impact gene expression. Many C2H2-ZF proteins contain other types of domains, such as the Krüppel-associated box (KRAB), which represses the expression of endogenous retroviral elements. This may impose limits to the functionality of C2H2-ZFs, effectively taming their power.


Lung cancer is the top cancer killer worldwide with high mortality rate. Majority belong to non-small cell lung cancers (NSCLCs). The epidermal growth factor receptor (EGFR) has been broadly explored as a drug target for therapy. However, the drug responses are not durable due to the acquired resistance. MicroRNAs (miRNAs) are small non-coding and endogenous molecules that can inhibit mRNA translation initiation and degrade mRNAs. We wonder if some downstream molecules shared by EGFR and the other tyrosine kinase receptors (TKRs) further transduce the signals alternatively, and some miRNAs play the key roles in affecting the expression of these downstream molecules. In this study, we investigated the mRNA:miRNA associations for the direct EGFR downstream molecules in the EGFR signaling pathway shared with the other TKRs, including c-MET (hepatocyte growth factor receptor), Ron (a protein tyrosine kinase related to c-MET), PDGFR (platelet-derived growth factor receptor), and IGF-1R (insulin-like growth factor receptor-1). The multiple linear regression and support vector regression (SVR) models were used to discover the statistically significant and the best weighted miRNAs regulating the mRNAs of these downstream molecules. These two models revealed the similar mRNA:miRNA associations. It was found that the miRNAs significantly affecting the mRNA expressions in the multiple regression model were also those with the largest weights in the SVR model. To conclude, we effectively identified a list of meaningful mRNA:miRNA associations: phospholipase C, gamma 1 (PLCG1) with miR-34a, phosphoinositide-3-kinase, regulatory subunit 2 (PIK3R2) with miR-30a-5p, growth factor receptor-bound protein 2 (GRB2) with miR-27a, and Janus kinase 1 (JAK1) with miR-302b and miR-520e. These associations could make great contributions to explore new mechanism in NSCLCs. These candidate miRNAs may be regarded as the potential drug targets for treating NSCLCs with acquired drug resistance. Lung cancer is the top cancer killer worldwide with high mortality rate  are small non-coding and endogenous molecules with 19–22 nucleotides, which inhibit mRNA translation initiation and degrade mRNAs , and to further explore the candidate miRNAs affecting the mRNA expressions of these proteins. We hypothesize that there are some important downstream proteins shared by EGFR and the other TKRs, possibly leading to the resistance of EGFR TKIs, and some key miRNAs targeting these proteins are dysregulated in NSCLCs. In order to support our hypotheses, we proposed a novel mRNA:miRNA association identification approach that consists of two steps. The first step involved the identification of putative miRNA and mRNA pairs from the prediction databases. The second step is to identify the statistically significant and the best weighted miRNAs for the mRNAs of the shared downstream molecules using multiple linear regression and support vector regression models based on the microarray expression profiles. Our approach could reduce the intricate interactions between miRNAs and mRNAs to a few meaningful mRNA:miRNA associations, which facilitates the subsequent biological analysis and more efficient experimental validation. These candidate miRNAs may be served as potential drug targets for the treatment of NSCLCs with drug resistance.In this study, the microarray datasets GSE51852 and GSE51853 for mRNAs and miRNAs were collected from Gene Expression Omnibus (GEO) repository database  were also considered to select the shared downstream molecules. The miRNAs regulating the mRNAs of the shared downstream molecules were selected using the representative resources: MicroCosm-Targets  machine : miR-34a was negatively significantly associated with PLCG1, miR-30a-5p was negatively significantly associated with PIK3R2, miR-27a was negatively significantly associated with GRB2, miR-302b was positively significantly associated with JAK1 and miR-520e was negatively significantly associated with JAK1, as well as miR-155 was positively significantly associated with JAK2 . In this method, the first step is to select the putative miRNA-mRNA pairs using the prediction databases. And then, the GST is performed to get the significant mRNAs for a given miRNA based on the microarray data  and wester-blot. Furthermore, knock-down of the other TKRs and the shared downstream molecules, as well as miRNA mimics/inhibitors experiments will be performed to support the hypothesis and the current findings.FW and LC initiated the project and participated in its design. FW, FM, and LC performed the multiple linear regression analyses and participated in the interpretation of data. FW and LW constructed the support vector regression (SVR) model. FM, LW, SW, and WC participated in the design and coordination of the study. FW was responsible for writing the manuscript. All the authors participated in the discussion and editing of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Umbilical hernias are common in patients with end-stage liver disease undergoing liver transplantation. Management of those persisting at the time of liver transplantation is important to define. To evaluate the long-term results of patients undergoing simultaneous primary umbilical hernia repair (UHR) at the time of liver transplantation at a single institution. Retrospective chart review was performed on patients undergoing simultaneous UHR and liver transplantation from 2010 through 2016. 30-day morbidity and mortality outcomes and long-term hernia recurrence were investigated.59 patients had primary UHR at the time of liver transplantation. All hernias were reducible with no overlying skin breakdown or leakage of ascites. 30-day morbidity and mortality included 5 (8%) superficial surgical site infections, 1 (2%) deep surgical site infection, and 7 (12%) organ space infections. Unrelated to the UHR, 10 (17%) patients had an unplanned return to the operating room, 16 (27%) were readmitted within 30 days of their index operation, and 1 (2%) patient died. With a mean follow-up of 21.8 months, 7 (18%) patients experienced an umbilical hernia recurrence. Despite the high perioperative morbidity associated with the transplant procedure, concurrent primary UHR resulted in an acceptable long-term recurrence rate with minimal associated morbidity.  Chronic liver disease (CLD) affects 4.5% to 9.5% of the general population worldwide . Those pApproximately 7000 liver transplantations were performed in the USA in 2015 [The management of umbilical hernias in patients on the liver transplant list is controversial, with little published data to guide transplant and general surgeons. Our institution is a high-volume transplantation center with significant experience with this patient population. Our group prefers to perform a primary tissue repair of the umbilical hernia defect when present at the time of liver transplantation. The objective of this study was to evaluate 30-day morbidity and mortality outcomes and long-term hernia recurrence rates in those patients undergoing concurrent primary umbilical hernia repair (UHR) at the time of orthotopic liver transplantation at a single institution.Consecutive adult patients undergoing primary UHR at the time of liver transplantation from January 2010 through June 2016 at the Cleveland Clinic Foundation were identified within a prospectively-maintained, institutional liver transplantation database. All umbilical hernias were repaired primarily via exposure from the Chevron incision used for the liver transplantation with the use of simple interrupted or figure-of-eight permanent sutures by the transplant surgeon without an additional incision over the site of the umbilical hernia. Patients undergoing only liver transplantation and those patients undergoing repair of abdominal wall hernias other than umbilical hernias, including ventral/incisional hernia repair or inguinal hernia repair, were excluded from this analysis. After obtaining Institutional Review Board approval, retrospective chart review was performed on all identified patients. Patient demographic information, operative details, 30-day post-operative outcomes, and long-term hernia recurrence outcomes were collected. At our institution, all patients undergoing liver transplantation are followed at the transplant clinic at one week, one month, three months, six months, nine months, and one year, post-operatively and then annually, thereafter. ver 9.4, Cary, NC, USA).Disease severity was assessed by the Charlson Comorbidity Index, the Model for End-Stage Liver Disease (MELD) score, and the Sodium-Adjusted MELD Score -8. TheseApproximately 135 patients are transplanted at our institution annually. From January 2010 through June 2016, 59 patients underwent primary UHR at the time of liver transplantation at our institution . The majIn terms of 30-day morbidity and mortality outcomes, five (9%) patients experienced a superficial SSI, one (2%) patient experienced a deep SSI, and seven (12%) patients experienced an organ space infection . Of the Ten (17%) patients experienced an unplanned return to the OR; eight (80%) patients returned to the OR for coagulopathy-related intra-abdominal hemorrhage, one (10%) patient returned to the OR for repair of a biliary duct anastomotic leak, and one (10%) patient returned to the OR for emergent repair of an incarcerated inguinal hernia. Sixteen (27%) patients had an unplanned readmission to the hospital, all of which were unrelated to the UHR, and one (2%) patient died within 30 days of their index procedure due to multiorgan system failure following a biliary anastomotic leak. With respect to long-term outcomes, patients were followed for a mean of 21.8 months. At the time of chart review, there were data available for 39 (66%) patients. Of the original 59 patients, there were nine (15%) patient deaths and 11 (28%) patients who were lost to follow-up. Seven (18%) patients had an umbilical hernia recurrence diagnosed by either physical examination (n=5) or radiographic imaging (n=2). The mean±SD time to recurrence was 4.3±2.5 months. The aforementioned UHR recurrence after superficial SSI was the only one of the seven recurrences to have developed a post-operative infection of any kind. Of the seven umbilical hernia recurrences, three patients underwent elective recurrent hernia repair and one had urgent primary repair for incarceration. Two patients had primary suture repair of their recurrent hernia, one was repaired using an open retrorectus placement of synthetic mesh, and another had an onlay repair of their umbilical hernia with Permacol mesh concurrently during primary repair of bilateral subcostal incisional hernias and a roux-en-y hepaticojejunostomy for biliary stricture.Due to the inherent death and loss to follow-up over time, a Kaplan-Meier analysis was performed to determine the rate of hernia recurrence-free survival over time. Umbilical hernia recurrence was noted to occur early (within six months) following primary UHR at the time of transplantation .The timing of UHR in patients with end-stage liver disease gives pause to many surgeons due to the inherent risk and comorbidities of the patient. Some surgeons choose to undertake an elective repair prior to transplantation; others choose a conservative “watch-and-see” approach with repair at time of symptom development, whether it is before or after transplantation, while others choose elective repair at the time of liver transplantation. In a retrospective review of 34 patients, Marsmen, et al, compared the outcomes of umbilical hernia management in patients with liver cirrhosis and ascites. Due to the high incidence of morbidity and mortality noted with conservative “watch-and-see” treatment, they advocated that all umbilical hernias be repaired electively prior to transplantation, if possible . DespiteFor persistent umbilical hernias, the risk of incarceration and strangulation has been observed to be the highest following liver transplantation due to the resolution of ascites and associated fluid shifts, necessitating emergency surgery shortly after liver transplantation . FurtherOur results demonstrated that repair of the umbilical hernia from within the abdomen using the same transplantation incision added very little to the operation time or the overall morbidity of the liver transplant surgery. Furthermore, we found that the long-term hernia recurrence rate in our series was 18%—a much more acceptable rate than that previously described by de Goede . In thisCompared to the long-term umbilical hernia recurrence rates of 8%–54% mentioned for the general population, our recurrence rate of 18% was acceptable . Our recDespite our results, our study did have limitations that are worth mentioning. First, this was a retrospective study with all of its inherent weaknesses. Second, this study was performed at a quaternary referral center. While this allowed for significant long-term follow-up, these results may not be reproducible amongst all centers performing liver transplantation. Finally, hernia-specific data including hernia size were not available for analysis. In the absence of these data, we were unable to comment specifically on the management of umbilical hernias with regard to the defect size.In conclusion, we found that despite the high perioperative morbidity associated with the index transplant procedure, the long-term results of primary UHR from within the abdominal cavity without the use of a separate incision resulted in acceptable wound and hernia-related outcomes at our institution. These findings suggested that primary repair of asymptomatic umbilical hernias at the time of liver transplantation would be a safe and effective approach. Further hernia-specific data are required to determine patient populations that may benefit from alternative management strategies.


To modify the CAD surface model using different parameters , varying fit was created. Five light-body silicone replicas representing the gap between the canine and the coping were made for each coping and for each measurement method: (1) light microscopy measurements (LMMs); and (2) computer-assisted measurements (CASMs) using an optical digitizing system. Two investigators independently measured the marginal and internal fit using both methods. The inter-rater reliability [intraclass correlation coefficient (ICC)] and agreement [Bland-Altman (bias) analyses]: mean of the differences (bias) between two measurements  were calculated for several measurement points . For the LMM technique, one investigator repeated the measurements to determine repeatability (intra-rater reliability and agreement).For inter-rater reliability, the ICC was 0.848-0.998 for LMMs and 0.945-0.999 for CASMs, depending on the measurement point. Bland-Altman bias was −15.7 to 3.5 μm for LMMs and −3.0 to 1.9 μm for CASMs. For LMMs, the marginal-distal and marginal-buccal measurement points showed the lowest ICC (0.848/0.978) and the highest bias (-15.7 μm/-7.6 μm). With the intra-rater reliability and agreement (repeatability) for LMMs, the ICC was 0.970-0.998 and bias was −1.3 to 2.3 μm.LMMs showed lower interrater reliability and agreement at the marginal measurement points than CASMs, which indicates a more subjective influence with LMMs at these measurement points. The values, however, were still clinically acceptable. LMMs showed very high intra-rater reliability and agreement for all measurement points, indicating high repeatability. A poor marginal fit is associated with secondary caries,-,,,,Many measurement methods have been established to investigate the fit of dental restorations. To confirm a method's validity, however, two methods should be studied and compared,Another possibility for determining the replica's thickness is a computer-assisted technique that measures the optically captured replicas digitallyThis study focused on analyzing the reliability and repeatability of the conventional light microscopy replica technique by determining both inter-rater and intra-rater reliability and agreement for specific measurement point locations. As a second step, the study aimed to compare the conventional light microscopy replica technique to the more recent computer-assisted replica technique by means of the respective inter-rater reliability and agreement for specific measurement point locations.in vitro study were that: (1) the conventional light microscopy replica technique for the analysis of dental coping fit shows high intrarater reliability and agreement; (2) the conventional light microscopy method and the computer-assisted replica technique for the analysis of dental coping fit show high inter-rater reliability and agreement; and (3) the intra-rater and inter-rater reliability and agreement are independent of the specific measurement point location for the conventional light microscopy technique (intra-rater and inter-rater) and the computer-assisted replica technique (inter-rater) for analyzing dental coping fit.The hypotheses for this ®V. 10.0; Imageware Inc., Ann Arbor, MI, USA), the parameters were modified to create eight different CAD models resulting in varying fit. Therefore, the following parameters were modified: four CAD models showed high data density  and four CAD models showed low data density . Both data density groups were modified with regard to the fit by enlarging the originally sized data (1st CAD model) in z-direction , in x-/y- direction  and x-/y-/z-direction . Two titanium copings were manufactured for each of the eight different CAD models, resulting in 16 copings. For each titanium coping, five silicone replicas were produced for the light microscopy measurements (LMMs) and for the computer-assisted measurements (CASMs), respectively. This resulted in 80 (16×5) silicone replicas for the LMMs and 80 (16×5) silicone replicas for the CASMs, respectively (A prepared (chamfer) stainless steel maxillary canine (FDI 13) and its computer-aided design (CAD) surface model served as the master die . Using the CAD software  on the master die with a force of 20 N using a scale with a digital display . An individual marker ring with grooves fabricated with training alloy  was used for subsequent reproducible segmentation. After 10 min, using pliers, the restoration was removed from the master die in the axial direction. The master die replicas were reinforced by coating them with a heavy-body addition-curing silicone (clear color contrast) before cutting.For the LMMs, the restoration inside was isolated by silicone oil , which guaranteed that the replica would stay on the master die. The restoration was "cemented" with light-body addition-curing silicone  were manufactured as described above for the LMMs. The light-body silicone replica was not coated with heavy-body silicone for stabilization because no cutting was involved.The silicone replicas for the CASMs could not be produced on the master die due to its highly light- reflecting metallic surface, which was inappropriate for digitizing by the optical three-coordinate measurement system used . Therefore, 80 plaster dies — one for each replica — were fabricated using the double mix impression technique. Using the plaster dies, replicas . Calibration was performed each time the program was initiated and after every 10 measurements. While topical digitalization devices usually perform an automatic calibration before starting a measurement, older systems such as the ODKM 97 needed to be calibrated using standard geometries and a calibration software. The point clouds were processed using software tools  and aligned  with the corresponding CAD master model, as described by Luthardt, et al.For the CASMs, the digitizing system ODKM 97 was used The accordance between the two measurements at both sides of each sectional cut (LMMs) was checked. For this purpose, the mean, standard deviation, and minimum and maximum values of the difference  between those measurements were calculated exemplarily for the first measurement of the first investigator [Investigation 1.1 (I1.1)] .,,The first investigator repeated the examination of all existing 80 silicone replicas for the LMMs with the frequently used interval of one week in intra-rater reliability studiesThe measurements were performed by two investigators (postgraduates) with the same professional status (Dr. med. dent. candidates). The training time for the LMMs and ODKMs before starting the measurements was the same for each investigator. The first investigator performed both LMMs  and the CASMs . For calculation of inter-rater reliability and agreement, only one measurement of the first investigator (LMM) was randomly chosen (I1.2). The second investigator repeated the measurements independently from the first investigator, once for both measurement systems (LMM: I2.1 and CASM: I2) . The meaThe IBM SPSS Statistics software  was used for all analyses.The mean ± SD  of the difference  between the two measurements at each sectional cut was 5±7 μm  for the marginal values , 7±6 μm  for the axial values (ax-b), and 20±29 μm  for the incisal values (inc).The results of the 80 replicas' thickness measurements  are shown in bar charts  for eachThe results of the 80 replicas' thickness measurements  are shown in bar charts  for eachThe ICC for the intra-rater reliability of the LMMs shows very high values and a rather narrow 95% CI. The bias shows values very close to zero for all measurement points. These results indicate high repeatability of LMMs in the current study, regardless of the specific measurement points. Thus, the study's first hypothesis can be accepted, and the third hypothesis can also be accepted for the intra-rater evaluation of the LMMs.,The inter-rater reliability for the LMMs shows similarly high values for the ICC, except for the ma-d measurement, which has a slightly lower ICC (<0.9) and a wider 95% CI for the ICC. The bias shows this dependence on the measurement point more clearly. Although the bias for the ax-d and inc measurement points is still close to zero, the ma-b bias is almost 6 times higher and the ma-d bias is almost 16 times higher than the bias for the intra-rater agreement. The first investigator systematically determined lower marginal values  than the second investigator, which may have been due to the difficulty identifying the exact marginal measurement point. The light-body material thins toward the margins, in contrast to the thicker layers that are found axially and incisally. Thus, the Bland-Altman bias gave a more differentiated view than the ICC, as has been previously claimedThe inter-rater reliability for the CASMs shows very high ICC values with a rather narrow 95% CI. The bias values are closer to zero than the LMMs' bias values for every measurement point and are rather independent of the measurement point. These differences can be explained by the different method of determining the replica's thickness. For the CASM technique, no perpendicular measurementThe influence of the investigators' professional status on the measurement results was not evaluated, which could be considered a limitation of the study. However, for the inter-rater analyses, the measurements were performed by two investigators (postgraduates) with the same professional status (Dr. med. dent. candidates). Thus, a comparison between two individuals' measurements (inter-rater) was possible without a further interfering factor . However, the influence of this possible interfering factor is a further aspect of interest and should be addressed in future studies in this research field.The use of computer-assisted analyses is a constantly growing field not only in dental research. While the conventional replica technique does not require high investment costs and is easily learned, the opposite is true for the CASM technique. The latter technique implies the use of complex software for digitizing and analysis. Fortunately, topical software are becoming more user-friendly.in vitro investigation, this study shows the well-known limitations of in vitro studies compared to in vivo studies; e.g. it is lacking a randomized design in contrast to a randomized, controlled clinical trial (RCT). A specific limitation of in vitro coping fit analyses studies is the artificially superior coping fit compared to the achievable coping fit under in vivo conditions. Clinical factors, e.g. saliva or blood contamination, during dental impression making or margin geometry, lead to unintended modifications in coping fitin vivo studies evaluating the intra- and interrater reliability of measurement techniques usually include different patients,,in vivo conditions, we performed modifications on purpose to create different CAD models. The different CAD models successfully resulted in copings with varying fit and thus varying replicas' thickness between the samples as shown beforein vitro study is the measurement of 80 replicas resulting in a rather high sample size. This enhances the quality of our analyses.As an ,The analyses of the 80 replicas' thickness showed similar values  for both measurements of the first investigator (intra-rater testing). For the inter-rater testing, these values showed higher accordance between both investigators' measurements for the CASM than for the LMM, which is in accordance with the results of the ICC and bias analyses above. The CASMs tended to show higher mean values for the replicas' thickness than the LMMs. This phenomenon has already been described in a study that compared both techniquesFor the LMMs, we used the mean at both sides of each sectional cut for the marginal and axial values and the mean of eight measurements  for the incisal value. This was done to have points analogous to those used for the CASMs, in which no cutting took place, and to reduce errorFor the CASM technique, the replicas were manufactured on duplicate plaster dies due to the stainless steel master dies' reflection. For the LMM technique, the replicas were directly manufactured on the master die. The mean values for the difference between duplicate plaster dies and steel master die are +9.2/-8.5 μm (SD 1.1/0.5) for the identical plaster dies' manufacturing procedure ,Despite multiple studies evaluating the crown fit with the replica technique using LMMsThe same two investigators performed both techniques, LMMs and CASMs, using identical copings. Thus, for the first time, a direct comparison of interrater reliability and agreement between the LMM and CASM techniques was possible, which adds new information to studies in this area. The comparison showed slightly higher reliability and agreement with the CASM technique and no dependence on the measurement points — in contrast to the LMM technique.-,,,,Light microscopy and the computer-assisted replica technique are used in studies to evaluate new materials used and new manufacturing procedures applied for dental restorations for daily clinical practiceThe following conclusions can be drawn for the internal replica technique for evaluating the marginal and internal fit of dental copings.The light microscopy replica measurements showed high intra-rater reliability and agreement (repeatability) and somewhat worse, but still clinically acceptable, inter-rater reliability and agreement at the marginal measurement points.The computer-assisted replica measurement was slightly more objective than the light microscopy replica measurement and was independent from the measurement point.


L and MCL-1, which steers cellular fate towards survival [Cancer cells evade cellular death pathways, such as apoptosis, in order to ensure an uncontrollable growth as well as resistance to various treatments. Dysregulation of the BCL-2 family of proteins that critically regulate the intrinsic apoptotic pathway contributes to the pathogenesis of cancer . A majorc, escape and activate the caspase cascade that orchestrates apoptosis. In the vast majority of cancer cells, BAX is expressed in an inactive conformation or suppressed by anti-apoptotic proteins [Pro-apoptotic BAX is a critical member of the BCL-2 protein family. Following apoptotic stimuli, activating BH3-only proteins interact with the trigger site on BAX resulting in a series of structural changes that culminate in the translocation of BAX from the cytosol to the mitochondria outer membrane (MOM) . Once anproteins , 2. Mutaproteins . TherefoTo assess the therapeutic potential of direct BAX activation, we set out to discover small molecules that mimic the BAX-activating interactions of BIM with the N-terminal trigger site of BAX , 7. We uc release and activation of effector caspases were significantly accomplished at 4 hrs  cell lines that overexpress various anti-apoptotic proteins. BTSA1 promptly and robustly decreased cellular viability in a dose and time-dependent manner. BAX translocation to the OMM, cytochrome in vivo. Lastly, BTSA1 has favorable drug-like properties and will be a useful compound in future studies to assess the therapeutic potential of direct BAX activation in other cancer models as a single agent or in combination with other cancer therapeutics.In summary, we have identified BTSA1 as a potent and selective BAX trigger site activator  (Figure


Poor quality dried blood spot (DBS) specimens are usually rejected by virology laboratories, affecting early infant diagnosis of HIV. The practice of combining two incompletely-filled DBS in one specimen preparation tube during pre-analytical specimen processing  has been implemented to reduce the number of specimens being rejected for insufficient volume.This study analysed laboratory data to describe the quality of DBS specimens and the use of the two-spot method over a one-year period, then validated the two-spot method against the standard (one-spot) method.Data on HIV-1 PCR test requests submitted in 2014 to the Department of Virology at Inkosi Albert Luthuli Central Hospital in KwaZulu-Natal province, South Africa were analysed to describe reasons for specimen rejection, as well as results of the two-spot method. The accuracy, lower limit of detection and precision of the two-spot method were assessed.Of the 88 481 specimens received, 3.7% were rejected for pre-analytical problems. Of those, 48.9% were rejected as a result of insufficient specimen volume. Two health facilities had significantly more specimen rejections than other facilities. The two-spot method prevented 10 504 specimen rejections. The Pearson correlation coefficient comparing the standard to the two-spot method was 0.997.The two-spot method was comparable with the standard method of pre-analytical specimen processing. Two health facilities were identified for targeted retraining on specimen quality. The two-spot method of DBS specimen processing can be used as an adjunct to retraining, to reduce the number of specimens rejected and improve linkage to care. Problems in the pre-analytical stages of specimen collection and processing lead to poor quality specimens and laboratory rejections.3 A meta-analysis of studies between 2001 and 2012 showed that 33.9% of HIV-exposed infants in sub-Saharan Africa are lost to follow up by three months of age.4 Early infant diagnosis in South Africa occurs within a Prevention of Mother-to-Child Transmission continuum of care. Qualitative HIV-1 DNA and RNA PCR testing is done on dried blood spot (DBS) specimens collected from HIV-exposed infants between six weeks and 18 months of age, using the COBAS® AmpliPrep/COBAS® TaqMan® (CAP/CTM) HIV-1 Qualitative test . The current South African national laboratory guideline allows for testing of one completely-filled DBS per assay.5Early diagnosis and treatment of HIV-positive infants significantly reduce HIV disease progression and mortality. Early infant diagnosis of HIV-positive infants is a critical step in the timely initiation of antiretroviral therapy (ART).6 The Department of Virology (DOV) in KwaZulu-Natal province in South Africa provides early infant diagnosis services for the province. Laboratory data from KwaZulu-Natal show a reduction in mother-to-child transmission of HIV which reflects national trends.7 However, there is a challenge of identifying and linking every HIV-exposed infant to care, which contributes to a significant number of HIV-positive infants not commencing ART in a timely manner.8There have been significant successes in the Prevention of Mother-to-Child Transmission programme. However, challenges have been identified in South Africa. Only 70.4% of HIV-exposed South African infants were tested for HIV by two months of age in 2011, reflecting a lack of implementation of policies in the field.10 Specimen rejection may play a role in the failure to link HIV-exposed infants to care. The challenges with DBS specimens in our setting have not been described.Laboratory specimen rejections are an essential part of a laboratory quality management system, allowing the technologist to optimise accuracy of results. However, this practice has also been shown to adversely impact patient care as it can lead to abandonment of the test request.In the standard method of pre-analytical specimen processing, one complete DBS is inserted into one specimen preparation tube. At the DOV, the current practice for DBS with blood spots that are smaller than the recommended size is to use two incomplete DBS and combine them in one specimen preparation tube during pre-analytical specimen processing . Specimen preparation is followed by PCR testing for HIV-1 using the CAP/CTM analyser. The two-spot method is based on the hypothesis that the quality of these specimens may be adversely affected by the small volume of blood they contain. By maximising the amount of blood eluted from each specimen, the correct volume for the PCR can be obtained and a good quality laboratory result can be produced. Neither the two-spot method nor the impact of this method in reducing laboratory rejections have been described in the scientific literature.The objectives of this study were to analyse DOV laboratory data to describe trends in DBS specimen quality, particularly relating to specimens with insufficient volume. In addition, we sought to describe the use of the two-spot method in routine HIV PCR diagnosis. Finally, we performed laboratory experiments to validate the two-spot method.The University of KwaZulu-Natal Biomedical Research Ethics Committee (BREC) approved this research under the approval classes BCA-143/09 and BCA 256/010.11 recommended that all HIV-exposed infants have an HIV-1 PCR test at age six weeks, at six weeks post-cessation of breastfeeding and at any age less than 18 months that the child may be symptomatic . Paediatric guidelines recommend testing infants at the six-week immunisation visit and, for those who test positive, referral for confirmation of HIV infection and initiation of ART. Infants testing negative should be followed up and retested at age 18 months and/or after cessation of breastfeeding.12The study was conducted in the DOV at Inkosi Albert Luthuli Central Hospital in KwaZulu-Natal province, South Africa. The DOV is an ISO 15189:2007 accredited laboratory within an academic quaternary-level hospital. The DOV does early infant diagnostic testing of DBS using HIV-1 PCR on the CAP/CTM analyser for all HIV-exposed infants in public health facilities in KwaZulu-Natal province. During the study period from January 2014 to December 2014, the South African antiretroviral treatment guidelinesWe searched our laboratory database for reports on routine HIV-1 PCR test requests that were rejected during 2014. The database is managed with TrakCare Lab software  and the data were downloaded and stored in Microsoft Excel 2010 files . The data extracted included specimen numbers, reasons for specimen rejections and patient demographics, including the health facility where the DBS was collected (each facility was assigned a two-letter code). We also extracted the results for all HIV-1 CAP/CTM PCR routine diagnostic tests conducted during 2014.13 Blood was pipetted onto pre-labelled SS906 filter paper cards by filling one circle completely (so that it contained 70 µL of blood), then filling two circles incompletely (so that one contained 30 µL and the other 40 µL of blood). Examples of sufficient and insufficient specimens are shown in Residual EDTA blood specimens from previous successful, routine diagnostic HIV-1 PCR tests were selected and anonymised using a number allocated for this experiment. Specimens with insufficient volume and specimens that were collected more than one day prior to testing were excluded. Blood samples from 20 HIV-negative and 20 HIV-positive specimens were selected for the validation study.The complete (70 µL) DBS were processed using the standard (one-spot) method: each blood spot was inserted into a specimen preparation tube with lysis buffer, which had 1000 µL of specimen pre-extraction reagent  added to each tube. The tube was then subjected to thermomixing in a Thermomixer comfort  at 56 °C for 10 minutes at 1000 rpm in order to elute the DBS. The two incomplete (30 µL and 40 µL) DBS were then combined in a single specimen preparation tube and processed using the above method. For both sample types, the DBS were pushed to one side when inserted into the specimen preparation tube, as per the standard procedure.Both types of DBS were then tested in parallel over a period of five days for HIV-1 nucleic acids using the CAP/CTM as per manufacturer’s instructions. Briefly, the specimen preparation tubes were transferred to specimen racks, then loaded into the CAP/CTM analyser, which is an automated closed system with combined extraction, real-time PCR amplification and detection allowing for reduced contamination. Known low-positive and negative controls were included with every batch. The analyser software automatically determined the validity of the run, then determined the presence of HIV-1 nucleic acids based on the Ct value, namely, the PCR cycle at which the signal indicated amplification.13An HIV standard  was used to perform a dilution series and establish a lower limit of detection for the HIV-1 PCR test when using the two-spot method. Replicates of the HIV standard were tested to establish precision.p-value of < 0.05 was regarded as statistically significant.Statistical analyses were performed using IBM SPSS Statistics for Windows, version 22.0 . For the analysis of the laboratory database search, the frequency of each reason for specimen rejection was determined and for each health facility, a percentage of tests rejected as a result of pre-analytical problems was determined out of the total number of HIV-1 PCR tests requested. The HIV-1 PCR results of specimens processed pre-analytically using the standard method were then compared with specimens processed using the two-spot method. The Chi-squared test was used to compare categorical variables. A For the analysis of the laboratory experiment to validate the two-spot method, measures of sensitivity and specificity were used to describe the accuracy of the method. A scatter plot of Ct values was used to compare the two methods. Precision was determined by calculating the standard deviation and coefficient of variance of replicates. A probit regression analysis was used to determine the lower limit of detection of the assay.The analysis of database records for 2014 revealed that 88 481 specimens were sent to DOV for HIV-1 PCR testing, of which 3.8% were rejected or had no results. Ninety-one (0.1%) test requests had no results as a result of analytical problems and 3276 (3.7%) were rejected because of pre-analytical problems .Insufficient specimen volume comprised 48.9% of the total number of rejections resulting from pre-analytical problems . Other rThe data on the percentage of requests that were rejected per health facility were normally distributed . The meaFor all HIV-1 PCR test results in 2014 using the CAP/CTM assay, there was an overall indeterminate rate of 1.7% . There wThe average diameter of the 30 µL DBS was 8.99 mm  and for the 40 µL DBS was 10.08 mm . A comparison of the qualitative results of patient specimens showed 100% agreement in accuracy between the two methods. The comparison of the Ct values for the standard method versus the two-spot method showed that the Pearson correlation coefficient was 0.997 . In the This study is the first to describe the reasons for laboratory rejections of DBS specimens being submitted for HIV-1 PCR testing in KwaZulu-Natal province. Almost 4% of DBS specimens that were submitted to the laboratory were rejected because of pre-analytical problems. Our analysis showed that 48.9% of pre-analytical problems resulted from insufficient specimen volume and we identified two health facilities with high specimen rejection rates.14 and these specimens are therefore rejected by laboratories. Others have also described challenges in the field with specimen collection and processing, as a result of specimen quality factors such as mislabeling or insufficient volume. Rates of specimen rejection vary considerably and there is a paucity of data describing the frequency of each specimen quality issue.16 Data such as specimen quality should be analysed in order to review the performance of HIV-1 PCR testing in the field and to standardise DBS specimen collection methods, storage and testing.14 This study has shown that analysing specimen quality can identify health facilities which require targeted interventions, such as retraining of healthcare workers.Insufficient DBS volume has been shown to affect the yield of HIV-1 nucleic acids7 but problems have been described in the follow-up of mothers and infants.17 It has been shown that delaying HIV diagnosis in HIV-exposed infants results in a failure to link infants to HIV care, significant loss to follow up and mortality.18 At immunisation clinics in KwaZulu-Natal, nurses perform the specimen collection, whereas in hospital wards and clinics, some specimens are submitted by attending doctors. It has been shown that intense training and mentoring of nurses significantly improves the uptake of infant HIV testing and the overall quality of care.19 However, this needs to be expanded and implemented on a much larger scale.21 The need to expand nurse training will increase with the planned implementation of HIV-1 DNA PCR testing of exposed neonates at birth.22 As this is being newly implemented, healthcare workers in labour wards and nurseries may not be as experienced with DBS specimen collection as those in immunisation clinics.The Prevention of Mother-to-Child Transmission program in KwaZulu-Natal has shown significant success, largely because of the implementation of testing pregnant women and starting them on ART,We have described an innovative two-spot method for pre-analytical specimen processing that can reduce the number of DBS specimen rejections resulting from insufficient specimen volume. This method has been implemented in DOV for approximately three years and is used as an adjunct to ongoing telephonic and electronic communication and training of nurses in the public healthcare sector. Personal communications with other laboratories indicate that the two-spot method is being used in other HIV-1 PCR testing laboratories in South Africa. However, to our knowledge the two-spot method has not yet been described in the scientific literature.The specimens processed using the two-spot method in 2014 yielded over 10 000 additional results from specimens that would otherwise have been rejected. Furthermore, the proportion of specimens processed using the two-spot method with indeterminate results was comparable with that of all specimens processed in 2014.The validation study found that the two-spot method correlated well with the standard method and had good precision. The lower limit of detection of the HIV-1 PCR test when using the two-spot method was 3.6 log IU/mL, which is comparable to the 2.7 log IU/mL stated on the package insert for the standard method. Future studies comparing the detection limits between the two methods may be required.23 Another limitation of our study is that the volumes of 30 µL and 40 µL used in the validation study were selected in an attempt to simulate the real-world situation. However, a range of volumes could be present in DBS specimens in the field, for example, 20 µL and 50 µL. A third limitation is that the proportion of positive results using the two-spot method was significantly lower compared with the standard one-spot method. We speculate that this difference may be because of inconsistent selection of specimens for two-spot processing. Practical application of the two-spot method would require an objective assessment of the DBS specimen to reduce the subjectivity of specimen processing in the laboratory. Mean circle diameters for 30 µL and 40 µL spots can be provided to laboratory assistants on a template in order to demonstrate acceptable specimen volumes and aid the selection of patient specimens which are suitable for the two-spot method. We are in the process of implementing the use of such a template based on the findings of this study. A follow-up database analysis will be required to assess whether this intervention is effective. Laboratories that plan to use or are currently using the two-spot method should consider this cautionary measure.A major limitation with respect to analytical sensitivity is that the test is being used in infants within a Prevention of Mother to Child Transmission programme, most of whom have been exposed to ART. The presence of these drugs has been stated as a limitation by the manufacturer on the HIV-1 PCR package insert. Therefore, field evaluation of detection limits of the test may be required in a significantly ART-exposed patient population such as South Africa.In conclusion, we describe real-world problems with DBS specimen quality, which may have major implications for the management of HIV-infected infants in a resource-limited setting. The laboratory database can be used to monitor specimen quality and identify specific health facilities for interventions such as retraining of healthcare workers. As an adjunct to training, the two-spot method of specimen processing may be used to help reduce the number of specimens rejected. This method can potentially be applied to other HIV-exposed infant populations in resource-limited settings to reduce the need for patients having to repeat specimen collection and improve linkage to care.