To determine collagen biosynthesis and prolidase activity radiometric and colorimetric methods were used, respectively. Proline content was determined by applying the liquid chromatography coupled with mass spectrometry. We found that prolidase promoted the proliferation and migration of keratinocytes through stimulation of EGFR-downstream signaling pathways in which the PI3K/Akt/mTOR axis was involved. Moreover, PEPD upregulated the expression of β1-integrin and IGF-1 receptors and their downstream proteins. Proline concentration and collagen biosynthesis were increased in HaCaT cells under prolidase treatment. Since extracellular prolidase as a ligand of EGFR induced cell growth, migration, and collagen biosynthesis in keratinocytes, it may represent a potential therapeutic approach for the treatment of skin wounds.Recent reports have indicated prolidase (PEPD) as a ligand of the epidermal growth factor receptor (EGFR). Since this receptor is involved in the promotion of cell proliferation, growth, and migration, we aimed to investigate whether prolidase may participate in wound healing in vitro. All experiments were performed in prolidase-treated human keratinocytes assessing cell vitality, proliferation, and migration. The expression of downstream signaling proteins induced by EGFR, insulin-like growth factor 1 (IGF-1), transforming growth factor β Wound healing is a physiological process restoring skin functionality. It consists of four phases occurring in proper time and order. This process starts from hemostasis, then inflammation followed by proliferation and eventually tissue remodeling. The sequence of events during wound healing is strictly programmed and any disturbances may impair normal tissue repair. In the microenvironment of the damaged tissue, there are numerous biological factors and cell types involved in this process. One of them is keratinocytes, which proliferate and migrate to the wounded area during the proliferative phase induced by growth factors .Stimulated epidermal growth factor receptor (EGFR) and its downstream signaling proteins are responsible for cell proliferation, differentiation, growth, and migration . Recent 1-integrin receptor is well characterized. This signaling pathway is known to induce prolidase activity -To the best of our knowledge, this study represents the first investigation of the functional significance of prolidase-dependent stimulation of EGFR signaling in a model of wounded keratinocytes. Based on the original observation by Yang et al.  showing In our study, we found that prolidase in a dose-and time-dependent manner induced keratinocyte proliferation and migration both in control and “wounded” cells. However, these processes were more pronounced in “wounded” cells. The difference cannot be attributed to the vitality of the cells since no changes in this parameter were found. We observed that prolidase, added to the culture medium of human keratinocytes, activated EGFR downstream signaling through PI3K/Akt/mTOR proteins. Activated EGFR induces PI3K/Akt/mTOR and Ras/Raf/ERK pathways . As PEPD1 receptor through canonical  and non-canonical  pathways also mediate EMT -proline  into proteins susceptible to bacterial collagenase according to the Peterkofsky’s method . After 2g), the supernatant was transferred to fresh tubes and incubated with Chinard’s reagent  followed by incubation on ice for 15 min. Absorbance was read at 515 nm on TECAN Infinite® M200 PRO . The results were reported as a percent of the control value.The activity of prolidase was determined according to the method published by Besio et al. . Proteinhttps://imagej.nih.gov/ij/) and its rate was calculated according to the following formula.At confluence, HaCaT cells were scratched using a sterile 200 μL pipette tip, washed twice with PBS, and incubated with different concentrations of prolidase in 0.5% FBS-containing DMEM for 24 and 48 h. Every 24 h cells were photographed using an inverted optical microscope  with a 40× magnification for monitoring the wound closure area. The wound closure was measured by ImageJ software (After 24 incubation with prolidase (10 nM), cells were rinsed with prewarmed PBS twice. For fixation, 3.7% paraformaldehyde was used (10 min). Permeabilization with 0.1% Triton for 5 min was performed before FAK and NF-κβ visualization in contrast to that of EGFR where no permeabilization step was conducted. The next step included blocking with 3% fetal horse serum for 60 min at room temperature (RT). Then, cells were incubated with target primary antibodies  overnight at 4 °C at dilutions 1:500 and 1:100, 1:400, respectively. As a secondary antibody were used antirabbit FITC-linked antibody  at a concentration of 5 µg/mL for 1 h in the dark at RT. The cell nuclei were stained with Hoechst (1 ng/mL). Immunofluorescence staining was visualized using a confocal laser scanning microscope  supported with AttoVision software.LC–MS analysis of proline concentration in HaCaT cells was conducted according to the method of Klupczynska et al. . Brieflyt-test were used. Statistical analysis was performed using GraphPad Prism 5.01 . Statistically significant differences were marked as p < 0.05, pp < 0.01, ppp < 0.001, and pppp < 0.0001 and presented by using letters showing the differences between appropriate controls described in the legend of figures.All experiments were carried out at least three replicates and the experiments were repeated at least three times. Data are shown as a mean ± standard error (SEM). For statistical calculations, a one-way analysis of variance (ANOVA) with Dunnett’s correction and The data presented in this report suggest that extracellular prolidase acting through EGFR induced growth, migration, and collagen biosynthesis in cultured keratinocytes . Therefo


To investigate the association between high-normal preconception TSH levels and reproductive outcomes in infertile women undergoing the first single fresh D5 blastocyst transfer.  This was a retrospective study. Euthyroid patients undergoing the first single fresh D5 blastocyst transfer from January 2018 to May 2019 were initially included. The patients were divided into a low TSH (0.27–2.5 mIU/L) group and a high-normal TSH (2.5–4.2 mIU/L) group. The reproductive outcomes were compared between the groups. P=0.234, 0.145, 0.083). No significant differences were observed in terms of gestational age, single live birth rates, birth weight, or birth length.  A total of 824 women were ultimately included, 460 of whom had serum TSH levels less than 2.5 mIU/L and 364 of whom had serum TSH levels between 2.5 and 4.2 mIU/L. The patients were highly homogeneous in terms of general characteristics. High-normal TSH levels had no adverse impact on the clinical pregnancy rate, miscarriage rate, or live birth rate (respectively, aOR = 0.84, 0.65, 0.61, and  High-normal TSH levels did not significantly influence reproductive outcomes in infertile women undergoing the first single fresh D5 blastocyst transfer. Further studies are needed to test whether the results might be applicable to a wider population. Subclinical hypothyroidism (SCH) is defined as a thyrotropin (TSH) level higher than the upper limit of the normal range with normal free thyroxine FT4) levels . There i levels .th day after insemination and were scored according to the Gardner system. The blastocyst was transferred to the uterus under the guidance of ultrasound. Patients with the following criteria were excluded: D6 blastocyst was transferred (n = 3); more than one blastocyst was transferred (n = 1); previous embryo transfers (n = 79); the presence of overt or subclinical thyroid dysfunction (n = 124); and lack of essential data (n = 24). After exclusions, 824 women were transferred with a single D5 blastocyst at the first IVF/ICSI cycle and were divided into two groups based on preconception TSH levels . The study was carried out in accordance with the Code of Ethics of the Declaration of Helsinki.A total of 1056 infertile women receiving fresh blastocyst transfer in the Department of Reproductive Medicine, the Third Affiliated Hospital of Zhengzhou University, from January 2018 to May 2019, were initially included. Generally, the history of infertility in infertile couples is complex, most of which are either male factors or pelvic and fallopian tube factors or both. The women underwent standard controlled ovarian hyperstimulation (COH) with a downregulation protocol using a GnRh agonist or an antagonist protocol according to the patients' conditions. Oocytes were retrieved transvaginally 34–36 h after HCG administration and were inseminated using either the IVF or the ICSI method. Rescued ICSI was performed when necessary to avoid failure of insemination. Variable blastocysts were developed starting on the 5nd–4th morning of menstruation. Samples were centrifuged at 3000 rpm for 10 minutes after half an hour. Then, the serum on the upper layer was used for analysis. TSH, FT3, and FT4 were measured as part of the infertility workup before fertility treatment for first visitors. The measurements were conducted by electrochemical luminescence (ECLIA) on a Cobas 8000 . Generally, thyroid function tests were performed no longer than six months before blastocyst transfer. The provided TSH reference range is 0.27–4.2 mIU/L, the FT3 reference range is 3.1–6.8 pmol/L, and the FT4 reference range is 12.0–22.0 pmol/L. Daily internal quality control and yearly external quality control were carried out by request.Before sample collection, the patients were required to stay calm for at least 30 minutes. All fasting blood samples were collected from patients on the 2β-hCG (>10 IU/L). Clinical pregnancy was defined as the presence of the gestational sac and fetal heart activity following positive serum β-hCG. Early embryo loss was defined as a lack of visible gestational sac or fetal heart activity following positive serum β-hCG. Ectopic pregnancy was defined as a pregnancy that did not occur inside the uterine cavity. Miscarriage, which included early abortion, intermediate abortion, and late abortion, was defined as a pregnancy that did not result in delivery. Preterm birth was defined as a gestational age less than 259 days (37 weeks). Live birth was defined as the delivery of live babies.Pregnancy was defined as positive serum t-test or the Mann–Whitney U test where appropriate. Categorical variables were expressed as numbers or percentages, and the chi-square test or Fisher's exact test was used for comparisons of categorical variables where appropriate. Binary logistic regression was then used to evaluate the impact of preconception high-normal TSH levels on the clinical and obstetric outcomes. Data were analyzed using SPSS 22.0 statistical software. All P values less than 0.05 were considered statistically significant.Our primary outcomes were clinical outcomes, such as clinical pregnancy rate, early pregnancy loss rate, miscarriage rate, and live birth rate. The secondary outcomes were obstetric outcomes, such as the single live birth rate, birth weight, birth length, and gestational age. Continuous data are expressed as the mean (SD), and statistical comparisons were performed by either Student's P=0.279). The frequencies of insemination methods and top quality of transferred blastocysts were similar in both groups (P=0.542). No differences were noted in terms of the type of infertility or scheme of ovarian stimulation . As hysterosalpingography (HSG) affects thyroid function, we further compared the proportion of patients who underwent HSG between the two groups and found a similar proportion (P=0.885).The flowchart of the included women is shown in P=0.036). The mean TSH levels and age were also comparable between pregnant and nonpregnant women .In addition, we also compared the general characteristics of pregnant and nonpregnant women. As shown in P=0.049) and 1.61 (P=0.006), respectively, while younger age had significantly positive effects on the miscarriage rate and live birth rate, with adjusted ORs of 0.91 and 1.10 (P=0.015 and 0.005), respectively. High-normal TSH levels had no adverse impact on the abovementioned outcomes  (As shown in ctively) .P=0.622). The preterm birth rates were similar in both groups (8.1% vs. 8.2%). The single live birth rates were similar between the low and high-normal TSH groups . In addition, the birth weight and birth length of single live births were similar between the low and high-normal TSH groups , as were the birth weight and length of twin live births .We further compared obstetric outcomes between the low and high-normal TSH groups. Of the 824 included women, 247 in the low TSH group had live births, while 195 in the high-normal TSH group had live births . No signIn this retrospective study, we investigated the association between preconception TSH and the reproductive outcomes of 824 infertile women undergoing the first single D5 blastocyst transfer at a university-affiliated hospital and found that a high-normal preconception TSH level (between 2.5 and 4.2mIU/L) was not associated with lower odds of clinical pregnancy and live birth or higher odds of miscarriage. Our results were consistent with those of some previous studies, as the debate regarding the impact of high-normal TSH on ART clinical outcomes still exists.Based on a population of more than eighteen thousand naturally pregnant women in China, Chen et al. clarified that preconceptionally high TSH levels were associated with a small but significantly increased risk of overall adverse events, even within the normal unpregnant range, and concluded that TSH <2.5 mIU/L was more suitable for the assessment of women planning for pregnancy . HoweverOur previous results were in agreement with those of other studies, which demonstrated that mildly elevated preconception TSH levels before treatment had no adverse impact on the clinical outcomes in infertile women undergoing intrauterine insemination (IUI) treatment , 9, 12. According to the molecular basis of TSH and thyroid hormone action during implantation and early development, the thyroid hormone might be involved in endometrium preparation for pregnancy and initial trophoblast development, and thyroid hormones are essential players in the mechanisms regulating implantation and early fetal development . Indeed,In contrast, Grove Laugesen et al. claimed a detrimental effect of high-normal preconception TSH levels on the clinical outcomes of infertile women undergoing their first IVF cycle . In our Baker et al. further demonstrated that a preconception TSH level >2.5 mIU/L was associated with a lower gestational age at delivery and lower birth weight in women undergoing IVF treatment . It shouThere were also some limitations in this study. First, this was a retrospective study with data collected from a single center, and selection bias was inevitable. Second, we did not report the status of thyroid antibodies in infertile women, which might have a role in infertility and miscarriage. However, on the one hand, the existing data are controversial regarding whether thyroid antibodies are associated with infertility or adverse reproductive outcomes , 13, 20.The strength of our study was the highly homogeneous population-based cohort of euthyroid women, as the number, stage, or quality of transferred embryos, as well as the age and ovarian reserve, might potentially affect the results of embryo transfer. It was reported that HSG affected thyroid function . Here, iIn conclusion, we included infertile women as homogeneous as possible to minimize the bias that may lower the power of the impact of TSH on reproductive outcomes. We found that a high-normal TSH level did not significantly influence the reproductive outcomes of infertile women undergoing the first single fresh D5 blastocyst transfer. Further studies are needed to test whether the results might be applicable to a wider population.


The purpose of this study was to examine whether extended use of a variety of screen-based devices, in addition to television, was associated with poor dietary habits and other health-related characteristics and behaviors among US adults. The recent phenomenon of binge-watching was also explored.A survey to assess screen time across multiple devices, dietary habits, sleep duration and quality, perceived stress, self-rated health, physical activity, and body mass index, was administered to a sample of US adults using the Qualtrics platform and distributed via Amazon Mechanical Turk (MTurk). Participants were adults 18 years of age and older, English speakers, current US residents, and owners of a television and at least one other device with a screen. Three different screen time categories  were created for total screen time, and separately for screen time by type of screen, based on distribution tertiles. Kruskal-Wallis tests were conducted to examine differences in dietary habits and health-related characteristics between screen time categories.Aggregate screen time across all devices totaled 17.5 h per day for heavy users. Heavy users reported the least healthful dietary patterns and the poorest health-related characteristics – including self-rated health – compared to moderate and light users. Moreover, unique dietary habits emerged when examining dietary patterns by type of screen separately, such that heavy users of TV and smartphone displayed the least healthful dietary patterns compared to heavy users of TV-connected devices, laptop, and tablet. Binge-watching was also significantly associated with less healthy dietary patterns, including frequency of fast-food consumption as well as eating family meals in front of a television, and perceived stress.The present study found that poorer dietary choices, as well as other negative health-related impacts, occurred more often as the viewing time of a variety of different screen-based devices increased in a sample of US adults. Future research is needed to better understand what factors among different screen-based devices might affect health behaviors and in turn health-related outcomes. Research is also required to better understand how binge-watching behavior contributes impacts health-related behaviors and characteristics. Television (TV) viewing represents one of the most common sedentary behaviors among the US population, with adults watching approximately 34 h per week . WatchinIn part, these outcomes might occur as a result of impacts of screen time on a variety of health-related behaviors, in particular dietary behaviors. Long hours of screen time have been associated with poor dietary patterns among adults, including higher consumption of sugar, especially from soft drinks; higher intake of foods with low nutritional quality like French fries, refined grain products, snacks and deserts; and lower intake of fiber, fish, vegetables, fruits, and whole grains –5. Poor Although associations between television time and health-related behaviors and outcomes is now well-established, the issue of screen time remains a complex and evolving problem. For example, even as Americans continue to consume multiple hours of TV per day, patterns of consumption are changing. Binge-watching, the continuous consumption of screen-based entertainment facilitated in part by media streaming services and television-connected devices, is growing as a phenomenon, potentially contributing to more evening-time screen use with the potential to crowd out other healthful behavioral goals and pursuits . SimultaOnly recently has research begun to explore patterns of modern screen use across devices, times of day, and days of the week . This apA survey to assess screen time across multiple devices as well as dietary habits in a sample of US adults was administered using the Qualtrics platform and distributed via Amazon Mechanical Turk (MTurk). Data collected through MTurk, a crowdsourcing platform that allows researchers and companies to collect data from “virtual workers” in exchange for payment, has been shown to be valid and reliable in previous research .Multiple strategies were followed to achieve high quality data . The study was approved by the Institutional Review Board of Arizona State University.Data collection was conducted during August 2019. Participants qualified for the study if they were: 1) adults 18 years of age and older, 2) English speakers, 3) current US residents, 4) owners of a television and at least one other device with a screen, and 5) noted that they watched television at least 2 h per day on most days. Eligible MTurk workers were asked to electronically sign an informed consent form and complete a survey through Qualtrics that asked about demographics, health characteristics, screen time habits, and usual eating practices. At the end of the survey, workers were provided with a completion code that allowed them to receive their payment through the MTurk website.Screen time viewing time was assessed with an 18-item questionnaire that instructed respondents to estimate total time spent in hours and minutes using TV, TV-connected devices (e.g. devices that stream media content and video game consoles), laptops/computers, smartphones, and tablets. Respondents were asked “Thinking of an average weekday (from when you wake up until you go to sleep), how much time do you spend using each of the following types of screen as the primary activity?” For a more detailed description of this 18-item questionnaire please see Vizcaino et al.  The quesDietary patterns were assessed with the Starting The Conversation (STC) tool, which is an eight-item food frequency instrument for use in primary care and health promotion . The resFamily meal frequency was assessed with the questions 1) “During the past seven days, how many times did all, or most, of the people living in your home eat a meal together without any screens on?” and 2) “During the past seven days, how many times did all, or most, of the people living in your home eat a meal together while watching television?” These were slightly modified from an original question used to examine the relationship between family dynamics, nutrition, and well-being . Fast foSelf-rated health was assessed with the question “How would you rate your health at the present time?” Participants answered in a Likert-type scale ranging from 1 = bad to 5 = excellent. Self-rated health has been found a strong and consistent independent predictor of mortality across a variety of populations in numerous studies . PsycholData were analyzed using the Statistical Package for the Social Sciences (SPSS) version 25.0. Variables under investigation violated assumptions of normality even after transformation attempts; hence, non-parametric tests were conducted. Descriptive data are presented as medians and interquartile ranges for continuous variables, and as proportions for categorical variables.Three different screen time categories were created for total screen time. Similar to other research on screen time use , individKruskal-Wallis tests were conducted to examine differences in dietary habits between screen time categories. Significant results were followed up with pairwise comparisons using the Dunn’s procedure with a Bonferroni correction for multiple comparisons. Differences in demographics and health characteristics between screen time categories were examined also with Kruskal-Wallis tests for continuous variables, whereas chi-square tests were used for categorical variables. Spearman correlation was used to explore for associations between binge watching and variables under investigation. Significance was set at alpha < 0.05.X2 (2) = 822.68, p < 0.000. Total screen time for light, moderate, and heavy users had a median of 7, 11.25, and 17.5 h per day, respectively. The only significant demographic differences between total screen time categories were age and race/ethnicity; X2 (2) = 10.32, p = .006 and X2 (8) = 28.26, p = .000, respectively. Heavy users tended to be slightly younger and have a greater proportion of African-Americans and Asians/Pacific Islander. See Table p < 0.000  = 18.96, p = .000. Heavy users also reported the greatest number of days eating a meal together as a family while watching television (X2 (2) = 9.49, p = .009), and conversely, the least number of days eating a meal together as a family without any screens on (X2 (2) = 9.31, p = .009) as compared to moderate and light users. In addition, heavy users reported the highest frequency of fast food consumption compared to both moderate and light users; X2 (2) = 10.58, p = 005.Descriptive data for dietary habits and health-related variables can be found in Table X2 (2) = 13.02, p = .001 and X2 (2) = 20.23, p < .000, respectively); however, no significant differences in dietary patterns were observed between groups across TV-connected devices, laptop/computer, or tablet (X2 (2) = 2.63, p = .268; X2 (2) = .09, p = .954; X2 (2) = 1.66, p = .435). Consumption of fast food was consistently higher among heavy users of all screen devices , except for laptop/computer (p = .75). Please see Table Interestingly, unique dietary habits emerged when examining dietary patterns by type of screen separately. For example, only heavy users of TV and smartphones reported the least healthful dietary patterns as compared to the other groups (X2 (2) = 17.67, p = .000), the highest perceived stress scores (X2 (2) = 25.63, p = .000), and the least healthful behavioral patterns including the lowest number of hours of sleep (X2 (2) = 12.21, p = .002), poorest sleep quality (X2 (2) = 6.82, p = .033) and lowest amount of physical activity (X2 (2) = 13.56, p = .001). In addition, both heavy screen users and moderate screen users reported a significantly higher BMI as compared to light users (X2 (2) = 14.96, p = .001). Similarly, unique patterns emerged when examining by type of screen separately. For instance, physical activity was significantly lower only for heavy users of TV (X2 (2) = 11.39, p = .003), whereas self-rated health was significantly lower for heavy users of TV, TV-connected devices, and smartphones (X2 (2) = 6.84, p = .033; X2 (2) = 7.54, p = .023; X2 (2) = 10.52, p = .005; respectively) , frequency of fast food consumption , eating family meals in front of the television , and perceived stress . Please see Table Lastly, binge watching was significantly associated with least healthful dietary habits . These “heavy users” reported the least healthy dietary patterns (e.g. they consumed few fruits/vegetables and regularly consumed sodas/sweet tea), the lowest frequency of meals shared with the family without screens on, and the highest frequency of fast food consumption. When analyzed separately by type of screen, only “heavy users” of television and smartphones showed statistically different scores in dietary patterns compared to the other groups. At the same time, both “heavy users” of TV and TV-connected devices reported a statistically higher frequency of family meals while watching TV, and “heavy users” of all devices except laptops showed a statistically higher frequency of fast food consumption. These results highlight the importance of separately exploring the impact of different screen-based devices on dietary habits instead of focusing only on an aggregate measure of total screen time.Further, while frequency of fast food consumption, poor intake of fruits and vegetables, and excessive intake added sugars have all been connected to risk factors, and incidence of, chronic disease, the role of family meals is also important to health outcomes , 8, 9. RMore specifically, it may be possible that each screen-based device is associated with distinct factors that influence diet. For instance, TV viewing has been associated with poor dietary choices among children in part because of heavy advertising of candy, chips, sugary beverages, and fast foods , 29, andAlternatively, associations found in this study between poor dietary habits and TV-connected devices may be related to the long hours of dedicated viewing that adults engage in through binge-watching, given that longer hours of TV-watching are associated with worse dietary patterns –5. Such The present study also found significant differences in other health variables among different screen time categories. Heavy users of all screens reported the lowest physical activity, self-rated health, hours of sleep, sleep quality, and highest perceived stress. Heavy and moderate users of all screens reported higher BMI scores compared to light users. Similar to dietary habits, unique patterns emerged depending on the type of screen analyzed; for example, “heavy users” of TV, TV-connected devices, and smartphones reported the poorest self-rated health compared to the other groups, whereas only “heavy users” of TV reported statistically lower physical activity compared to “intermediate” and “light users.”Prior studies have found long hours of television viewing associated with premature mortality and cardiovascular disease (CVD) after adjusting for numerous covariates such as diet , physical activity, and sleep duration , 11. It While future research is needed to further characterize screen-time use across multiple screen-based devices, studies should also be conducted to better understand which factors across various devices might be associated with adverse health behaviors and in turn poor health-related outcomes either differentially or in combination. This line of inquiry is of priority especially for TV-connected devices given the increased popularity of video-on-demand services among Americans. A recent report indicated that 66% of the general population pays for a subscription of such services, and the percentage rises to 87% for households that consume more family-oriented movies and programming . As the As with any study, important limitations existed in this work. The sample used to gather data for analyses was a convenience sample derived through MTurk; nonetheless, previous research has found that MTurk workers more closely resemble the US population compared to college samples and other internet samples . AnotherPoorer dietary habits seem to be associated with extended use of screen-based devices, particularly television and smartphones. Physical activity, sleep and sleep quality, and stress might also be affected in different ways by different types of screen-based devices. Because the landscape of screen use in the US continues to evolve towards ever more complex configurations of usage and time commitments, future studies should continue to investigate how various screen-based devices might affect health behaviors and subsequently health-related outcomes in the long-term. Research may also incorporate considerations in relation to the impact of major life disruptions, such as the recent COVID-19 pandemic, which could greatly alter utilization of screens, potentially for better or worse. Further, it may be important to explore strategies for managing different types of screen use depending on the health-related characteristic or outcome of interest.


The causality between the use of alcohol and cigarettes and atrial fibrillation (AF) remains controversial. We conducted a Mendelian randomization (MR) study to evaluate the association of genetic variants related to tobacco and alcohol use with AF.Single nucleotide polymorphisms (SNPs) related to smoking initiation (N = 374), age at initiation of regular smoking (N = 10), cigarettes per day (N = 55), and smoking cessation (N = 24) were derived from a genome-wide association studies (GWAS) of tobacco use . SNPs related to heavy alcohol use (N = 6) were derived from a GWAS of UK biobank . The genetically matching instrumented variables were obtained from the GWAS of AF . The estimates between tobacco and alcohol use and AF were combined by inverse-variance weighted (IVW), simple median, weighted median, MR-robust adjusted profile score method, MR-PRESSO, and multivariable MR.P = 3.35 × 10−6) for AF. Genetically predicted age at initiation of regular smoking, cigarettes per day and smoking cessation were not associated with AF. The IVW estimate showed that heavy alcohol consumption increased AF risk . The results were consistent in complementary analyses and multivariable MR.A total of 65,446 AF patients and 522,744 referents were included. In the IVW analysis, the odds ratio per one-unit increase of smoking initiation was 1.11 (95% CI, 1.06–1.16; Our MR study indicated that regular smoking was associated with increased risk of AF, no matter the age at initiation of regular smoking, or the number of cigarettes smoked per day. Genetically predicted heavy alcohol consumption increased the risk of AF. Cigarette smoking and alcohol intake are potential modifiable risk factors for atrial fibrillation (AF), which is the most common sustained arrhythmia and associated with an increased risk of stroke, heart failure and even death . SeveralMendelian randomization (MR) analysis is a method using instrumental variables associated with exposure to infer causality and interactions in observational studies , which cr2 > 0.8; identified using online tool SNiPa, available at: http://snipa.helmholtz-muenchen.de/snipa3/).The diagram of the MR assumptions underpinning a MR analysis of the association of tobacco and alcohol use on AF was shown in the Additional file P < 5 × 10−8). Specially, smoking phenotypes associated instrumental variables were defined as a 1-Mb region nearby the top P-value.In this study, publicly available data were used to conduct the MR analyses. All participants of the original studies included in the GWASs had provided informed written consent. The data of tobacco and heavy alcohol use were extracted from a large-scale genome-wide association studies with smoking initiation, cigarettes per day, smoking cessation, age at initiation of regular smoking and heavy alcohol intake  with the specified SNPs were used. Finally, 374 out of 378 SNPs were included in the analyses . The GWAS on smoking initiation identified 378 single nucleotide polymorphisms (SNPs), explaining 2.3% of the heritability in up to 1,232,091 individuals. While 10 SNPs were unavailable in the AF database, 6 proxy SNPs in LD  was not found in the AF database and no appropriate proxy SNP was available. Thus, 9 SNPs were selected as instrumental variables  in 337,334 individuals were estimated to explain 1.1% of the variation in cigarettes per day. There was 1 SNP (rs4886550) not found in the AF database and we failed to match the appropriate proxy SNP. Therefore, 54 SNPs associated with cigarettes per day were used as instrumental variables in the MR analyses  were identified as instrumental variables in 547,219 individuals, which explained 0.1% of the variation in smoking cessation  identified 6 SNPs, explaining 2.0% of the variance in heavy alcohol use, in up to 21,967 cases and 103,282 controls of European ancestry from UK Biobank. All 6 SNPs were included in the analyses  of 4 consortiums and databases, including AFGen consortium , Broad AF study , UK Biobank , and Biobank Japan  . In addition, there was a genetic correction between smoking and alcohol consumption (rg = 0.34) [P < 0.05/5 = 0.01 for 5 exposures  corrected for multiple comparisons using the Bonferroni method. A two-sided P value of < 0.05 was considered suggestive for significance. MR analyses were conducted using the TwoSampleMR and MR-pleiotropy residual sum and outlier R packages. All analyses were conducted in R version 3.6.1.A fixed-effects inverse-variance weighted (IVW) meta-analysis was used to combine the estimates. Complementary analyses using the simple median, weighted median, MR-robust adjusted profile score , and MR = 0.34) . To inte = 0.34) , using p = 0.34) . For eacThe analytic sample included 65,446 atrial fibrillation patients  and 522,744 referents from case–control and population-based studies from 4 large-scale consortiums and databases  per one-unit increase  of smoking initiation without detected pleiotropy bias  .Genetically predicted smoking initiation was associated with a higher risk of AF in the standard IVW analysis, with an OR of 1.11 , and there was moderate heterogeneity among SNPs (I2 = 46.1%) of smoking cessation. The IVW showed that associations of genetically predicted tobacco use with AF were consistent using an LD of r2 < 0.1 and in European-ancestry individuals  , and a low heterogeneity between heavy alcohol use and AF (I2 = 22.5%). The significant association between heavy alcohol use and AF was stable when restricting the analysis to an LD of r2 < 0.1 and individuals of European ancestry  Fig. . The ass01) Fig. . There wThis MR analyses indicated that regular smoking (current or former) was associated with increased risk of AF, no matter the age at initiation of regular smoking, or the number of cigarettes smoked per day. Genetically predicted heavy alcohol consumption increased the risk of AF.I2 = 78%) among studies. In the Atherosclerosis Risk in Communities (ARIC) study, the incidence of AF was lower in people who quitted smoking as compared with current smokers, although statistically nonsignificant  [Our findings are in line with a recent meta-analysis of 29 prospective studies demonstrating that current, former and ever smoking were associated with a 32%, 9% and 21% increase of AF risk compared with the never smoking . We foun65–1.17) . What’s 65–1.17)  showed tOn the one hand, smoking regularly can lead to myocardial ischemia by increasing systemic catecholamine, reducing oxygen-carrying capacity and promoting coronary artery contraction . On the Larsson et al.  performeP = 0.005).In a recent prospective, open-label, randomized, controlled trial conducted by Voskoboinik et al.  using daOur study is the first to use MR analysis to explore the relationship between tobacco and alcohol use and atrial fibrillation. One of the strengths of this study is the large sample size of more than half a million individuals and up to 65,446 AF cases; the other is biases that potentially exist in conventional observational studies could be reduced. However, our MR analyses were subject to some limitations. First, the study lacked complete data such as sex, we therefore cannot evaluate the association between alcohol intake and AF in different genders, considering some observational studies reporting an association in males and AF at high levels of alcohol intake, but not females –36. SecoIn conclusion, our MR study indicated that regular smoking might be associated with increased risk of AF, no matter the age at initiation of regular smoking, or the number of cigarettes smoked per day. The result also showed that heavy alcohol intake was linked to an increased risk of AF.Additional file 1. Supplementary methods. Table S1. Descriptive information of the studies and datasets included in the analyses. Table S2. Characteristics of the genetic variants associated with smoking initiation. Table S3. Characteristics of the genetic variants associated with age at initiation of regular smoking. Table S4. Characteristics of the genetic variants associated with cigarettes per day. Table S5. Characteristics of the genetic variants associated with smoking cessation. Table S6. Characteristics of the genetic variants associated with heavy alcohol drinking. Table S7. Summary of atrial fibrillation cases and referents by ancestry. Figure S1. Diagram of the Mendelian randomization assumptions underpinning a Mendelian randomization analysis of the association of smoking and alcohol use on atrial fibrillation. Figure S2. Mendelian randomization association of genetically predicted smoking and heavy alcohol use with atrial fibrillation, using a linkage disequilibrium threshold of r2<0.1. Figure S3. Mendelian randomization association of genetically predicted smoking and heavy alcohol use with atrial fibrillation in multiple ancestry and European ancestry


Microfluidic applications have expanded greatly over the past decade. For the most part, however, each microfluidics platform is developed with a specific task in mind, rather than as a general-purpose device with a wide-range of functionality. Here, we show how a microfluidic system, originally developed to investigate protein phase behavior, can be modified and repurposed for another application, namely DNA construction. We added new programable controllers to direct the flow of reagents across the chip. We designed the assembly of a combinatorial Golden Gate DNA library using TeselaGen DESIGN software and used the repurposed microfluidics platform to assemble the designed library from off-chip prepared DNA assembly pieces. Further experiments verified the sequences and function of the on-chip assembled DNA constructs. In recent years, researchers in different fields have developed a diversity of microfluidics technologies. Microfluidics systems have been applied to parallel gene synthesis; PCR; DNA purification, separation, and sequencing; Next Generation Sequencing sample preparation; DNA sizing by single molecule detection; DNA hybridization arrays; drug discovery; pathogen identification; cellular analysis; biochemical clinical assays; protein arrays; and biosensors –8. MicroOne possible alternative, pursued herein, is to start with an existing microfluidic device designed for a specific function, and attempt to repurpose it  for an alternative application. Here, we begin with a microfluidic formulation device, which was previously designed to perform functions such as the combinatorial mixing of 16 buffers and 16 precipitation agents with a purified protein sample. This chip showed the possibility and practicality to access a vast number of chemical conditions and to accurately dispense and mix fluids on the picoliter scale .E. coli and sequence validated and functionally tested. The DNA design and construction workflow on a microfluidic device is shown in Previously, we developed PR-PR –15, a hiTo control the repurposed microfluidic device, we integrated PR-PR , 15 withhttps://teselagen.com/design-build-platform/) to specify the library, as shown in To demonstrate the application of the repurposed microfluidic device to DNA construction, we chose to pursue the combinatorial Golden Gate assembly of the library shown in gfp, BCD21-gfp, and the vector backbone (mixed with ligase); were dispensed into the input wells. To assemble the construct Promoter1-BCD1-gfp (for example), valves 1, 3, and 5 were open and valves 2, 4, and 6 were closed, as shown in Our repurposed microfluidic chip was designed specifically for formulation and so has the requisite hiearical valving design need for combinatorial DNA assembly. Additionally, the chip features a metering scheme that allows for sequential injection of precise sample aliquots from a single microfluidic channel into an array of reaction chambers through a positive displacement crossinjection (PCI) junction. The PCI junction is formed by the combination of a three-valve peristaltic pump and a four-port crossinjection junction with integrated valves on each port . Each cygfp [gfp gene (We selected to pursue a subset of 4 constructs (of 308) from a previously published library (combinations of 22 constitutive promoters with 14 bicistronic devices followed by gfp ). We usegfp gene . The TesE. coli DH10B, and grew the transformed cells on selective LB plates overnight. One transformant colony from each assembly reaction was picked for further screening. Colony PCR and Sanger sequencing validated the expected sequences of all 4 cloned plasmids , for the plasmid sequence region spanning the spacer, insulator, promoter, BCD, gfp, and terminator as shown gfp, which contains strongest promoter and BCD, exhibited highest GFP fluorescence, followed by Prom11_BCD1-gfp, Prom1_BCD21-gfp, and Prom11_BCD21-gfp. A slight delay of ~80 mins to kickstart the fluorescence was observed for construct Promoter11_BCD1_GFP. As observed in Mutalik et al [E. coli e.g. in case of Promoter11_BCD1_GFP, a small change in the strength of promoter could result in a quantitatively measurable change in the translation initiation which explains the delay in time for fluorescence to kick-start. We only used a subset of 4 constructs from a library of 308 constructs to illustrate the DNA construction capabilities on a pre-existing microfluidic chip. To understand the engineering of expression cassettes, more details can be found in the Mutalik et al publication [We transformed off-chip each of the resulting 4 DNA assembly reactions into ik et al  where thlication .E. coli and not to the platform used for DNA construction.These results importantly demonstrate that the repurposed microfluidic device, in combination with controller programed by PR-PR operated by MATLAB , can be More testing is needed to further establish the efficiency and repeatability of the microfluidic device. We do not anticipate any new challenges will emerge while applying the device to other DNA assembly strategies (such as Gibson Assembly), considering that the pump and valves controlled by PR-PR script on a microfluidic device will remain the same.All DNA oligos were purchased from Integrated DNA Technologies . DNA assembly reagents and restriction enzymes were purchased from New England Biolabs . Unless otherwise specified, all chemicals, solvents, and media components were purchased and used without modification from Sigma-Aldrich , Fisher Scientific , or VWR .E. coli using Spin Miniprep kits , served as DNA templates for the PCR amplification of promoter fragments Promoter1, and Promoter11, respectively. Similarly, pFAB4876, and pFAB4883 served as DNA templates for the PCR amplification of the two BCD variant fragments BCD1_gfp, and BCD21_gfp, respectively. pFAB4876 served as the DNA template for the PCR amplification of the vector backbone. Primers  used for the PCR amplifications are listed in https://public-registry.jbei.org/folders/427) and are physically available from the authors and/or addgene (http://www.addgene.org) upon reasonable request.pFAB4876, and pFAB4932, extracted from Following PCR amplification, residual (methylated) DNA template in each PCR reaction was DpnI digested at 37°C for 1 h. Each 110 μL digest reaction consisted of 95 μL PCR product, 11 μL 10x Fast Digest buffer, 1.5 μL Fast Digest DpnI , and 2.5 μL deionized water. DpnI was inactivated at 80°C for 5 min, and DNA purification of each DpnI reaction was conducted with a PCR purification kit  according to the manufacturer’s protocol, each purified sample eluted with 50 μL of elution buffer.Following DpnI digest and purification, 70 μL BsaI digestion reactions consisting of 50 μL purified DpnI reaction, 7 μL NEB4 buffer, 0.7 μL BSA, 5 μL BsaI, and 7.3 μL deionized water, were performed overnight at 37°C. BsaI was deactivated at 65 °C for 20 min. Digested samples were run on a 0.8% agarose gel followed by gel purification kit  according to manufacturer’s protocol. Golden Gate protocols were derived from the previously reported articles, in which the DNA fragments to be assembled are BsaI digested and gel purified prior to ligase DNA assembly , 18.The Golden Gate DNA assembly scripts see  were comE. coli DH10β competent cells [7 μL of each DNA assembly reaction was mixed with 50 μL nt cells  (one assgfp variant with the strongest promoter and RBS emits the brightest green color as can be seen in 600 approx. 0.1) of LB-kanamycin media in a 96 well black clear bottom micro-plate reader plate. DI water and LB media were used as blanks. Measurement of GFP fluorescence was performed using the Tecan microplate reader with excitation/emission at 485 nm/ 530 nm. Captured data were transferred directly into an Excel file for direct data analysis. As can be seen in Liquid cultures and bacterial colonies of all variants were qualitatively analyzed by inspection under the day light and UV light. The Promoter1_BCD1-Colony PCR was performed for each transformant using 2 μL overnight growth culture as template with primers Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Tablehttps://public-registry.jbei.org/folders/427) and are physically available from the authors and/or addgene (http://www.addgene.org) upon reasonable request.Plasmids and strains used in this study, along with annotated DNA sequences, have been deposited in the public instance of the Joint BioEnergy Institute Registry  (https:/(DOCX)Click here for additional data file.S1 Raw image(TIF)Click here for additional data file.S1 Promoter(PR)Click here for additional data file.S2 Promoter(PR)Click here for additional data file.S3 Promoter(PR)Click here for additional data file.S4 Promoter(PR)Click here for additional data file.


Femoral neck fracture (FNF) is among the commonest fractures affecting the geriatric population. Hemiarthroplasty (HA) is a standard treatment procedure and has been performed by hip surgeons for decades. Recently, primary total hip replacement has proved advantageous for the treatment of such fractures.The aim of this study is to retrospectively review all causes of failure of all patients who underwent HA in our institute and reevaluated whether HA remains a favourable choice of treatment for patients with displaced FNFs.A total of 4516 patients underwent HA at our centre from 1998 to 2017. The HA implants included unipolar and bipolar prostheses. Patients diagnosed with displaced FNF, underwent primary HA initially, required second revision procedures, and followed up for a minimum of 36 months were included in this study. Data were collected and comprehensively analysed.n = 30, 30.3%), femoral stem subsidence , periprosthetic fracture , infection , and recurrent dislocation . The mean follow-up period was 78.1 months. The interval between failed HA and revision surgery was 22.8 months.In 4516 cases, 99 patients underwent second surgeries. The revision rate was 2.19%. Reasons for failure were acetabular wear (HA has a low revision rate and remains a favourable choice of treatment for patients with displaced FNFs.Level III, Retrospective Cohort Study, Therapeutic Study. Femoral neck fracture (FNF) is among the commonest fractures affecting the geriatric population. In displaced fracture types, treatments include closed or open reduction and internal fixation, hemiarthroplasty (HA), and total hip replacement (THR). HA is a frequently recommended treatment and has been performed for decades . NeverthThis was a retrospective cohort study and was performed at a single trauma centre. From 1998 to 2017, 4516 patients underwent hemiarthroplasty in our institute following a diagnosis of displaced FNF. The HA implants included the Austin Moore (unipolar monoblock) prosthesis and various bipolar systems   approach or the posterior (Moore or Southern) approach. The prosthesis system was chosen according to the preference of the surgeon, and the use of cement fixation depended on bone quality and was decided intraoperatively. A portable radiograph of the hip joint was examined before the patient was transferred back to the ward unit. Oral analgesic agent and intravenous morphine (PRN) were administered for pain control if not contraindicated. Intravenous antibiotics were continuously administered every 8 h after surgery for 1 day and prolonged depending on the patient’s clinical condition.Each patient had his or her own chart with detailed records, including personal data, the mechanism of injury and associated conditions, fracture type and classification, course of management, implantation details, fixation technique, surgical approach, and functional recovery process. Regular follow-ups were arranged after discharge for all patients. The anteroposterior and lateral radiograph views of the wound condition were evaluated during each outpatient department visit.t test, chi-square test, and multinomial logistic regression analysis were used. P < .05 was used to indicate statistical significance .Statistical analysis was performed using SPSS version 24.0 statistical software . An independent 2 (range: 16.9–30.9). Of the fractures, 54 were left sided and 45 were right sided. The interval between injury and surgery was 1.8 ± 1.6 days (range: 1–8). The mean follow-up period was 78.1 ± 55.8 months (range: 40–219). Fourteen patients expired during the follow-up period due to infections , malignancies, or cardiovascular diseases. All patients’ demographic data are summarised in Table Of 4516 patients, 99 were found to receive second revision surgery, including 5 open reductions and internal fixations, 18 revision hemiarthroplasties, and 76 conversion THRs. The revision rate of failed HA and the conversion rate of THR were 2.19% (99 of 4516) and 1.68% (76 of 4516), respectively. The average age of the patients at the time of the injury was 76.4 ± 8.7 years (range: 44–93 years). Of the patients, 57 were male and 42 were female. The physical health and associated medical conditions of the patients were rated based on the American Society of Anaesthesiologists’ (ASA) physical status classification: 32 patients were in class II, 41 patients were in class III, and 26 patients were in class IV. The mean body weight index (BMI) was 22.8 kg/mn = 30, 30.3%), femoral stem subsidence , periprosthetic fracture , infection , and recurrent dislocation   approach and 35 underwent the posterior (Moore or Southern) approach. The mean femoral cup size was 47.3 ± 3.6 mm (range: 40–54). Reasons for failure included acetabular wear . The aggressive prevention of postoperative trauma is ostensibly more critical than is long-term acetabular wear.Some concerns in relation to HA have been discussed in other studies: The reoperation rate for failed HA is reportedly as high as 24%, and the problem of acetabular wear has been noted as the primary cause of HA failure –15. ThesNo significant difference was noted in the comparison among the groups for the 5 HA failure types in terms of age, sex, BMI index, ASA classification, prosthesis use, fixation technique, surgical approach, and femoral cup size. The risk factor of HA failure was not identified. Peter et al. found that higher ASA scores and BMI indexes (> 40) are strong predictors of revision THR requirement, but similar results were not obtained in our data analysis. Further studies are required to determine the major predictors of HA failure .P = 0.282, CI = 0.532–8.736) is noted. The result of analysis is presented in Table The risk of periprosthetic fractures when using cemented or cementless stems are currently discussed. Olof GS et al. stated cementless femoral stems are not recommended for the treatment of FNFs in geriatrics high number of due to late-occurring periprosthetic fractures . HoweverThis study has limitations. First, it was a single-centre retrospective cohort study. Second, surgeries were performed by different surgeons and using different surgical approaches, fixation methods, and prosthesis systems. More comprehensive research and randomised control studies are required to elucidate these results.On the basis of the encouraging mid- to long-term outcomes in this population, we consider that hemiarthroplasty remains a favourable choice of treatment for patients with displaced FNFs.


Functional abdominal pain (FAP) disorders are one of the most common gastrointestinal disorders in children. We aimed to define the association between obesity and functional abdominal pain (FAP) disorders and to assess differences between overweight/obese children and normal weight children with FAP disorders.We conducted a retrospective study of children (2–18 years old) with a clinical diagnosis of FAP who were followed-up in our pediatric gastroenterology unit between 1/2016–10/2018. FAP disorders were defined according to the ROME IV criteria. Body mass index (BMI) percentiles were defined by CDC standards. Patients with BMIs ≥85th percentile were designated as being overweight/obese. A population control group was obtained from the 2015–2016 Israel national health survey.Data from 173 children with FAP disorders  were included. Seventy-one children (41%) were classified as having functional abdominal pain-NOS, 67 (38.7%) as having irritable bowel syndrome (IBS), and 35 (20.2%) has having functional dyspepsia. Fifty-three children (30.6%) were classified as being overweight/obese.p = 0.04). Children with FAP and overweight were older  and had more hospitalizations due to FAP  compared to Children with FAP and normal weight.Adolescents with FAP disorders had a significantly higher prevalence of overweight/obesity compared to controls (39.5% vs. 30%, respectively, Adolescents with FAP had higher prevalence of overweight/obesity compared to controls. Future studies are warranted to raise awareness of weight issues in FAP and determine the effect of weight loss on FAP. Functional gastrointestinal disorders (FGID) are very common clinical conditions encountered in day-to-day clinical pediatric gastroenterology practice . IncreasChildhood obesity is a serious and urgent public health problem that is associated with physical and psychological comorbidities . The bidAll study procedures were approved by the institutional review board of the Tel Aviv Medical Center (Helsinki Committee), and parental informed consent was obtained for all participants.https://www.cdc.gov/growthcharts/clinical_charts.htm). Patients with BMIs ≥85th percentile were designated as being overweight or obese.We conducted a retrospective study of 2- to 18-year-old children with a clinical diagnosis of FAP disorders that were followed-up in the Pediatric Gastroenterology Unit at the “Dana-Dwek” Children’s Hospital, Tel Aviv, Israel, between January 2016 and October 2018. The Pediatric Gastroenterology unit is an outpatient clinic at our hospital, which is a tertiary referral center for pediatric gastroenterology. The patients were identified through the local electronic health records database. After the patients with clinical diagnosis of FAP were identified from the medical records, we performed an objective evaluation of the compatibility of their diagnosis with the ROME IV criteria . Patienthttps://www.health.gov.il/UnitsOffice/ICDC/mabat/Pages/default.aspx). The Israeli health survey is a national survey, conducted every 3–4 years by the ministry of health of Israel, that aims to investigate health and nutrition status among the Israeli population by a representative sample of children and adults. The data in the survey is presented according to age groups (2–11 and 12–18 years of age) and gender.A population control group was obtained from the 2015–2016 Israel national health survey  as well as anthropometric data . We also reviewed imaging and endoscopies studies, hospitalizations, dietitian consultations, nutrition recommendations, and follow-up durations.p < 0.05 was considered statistically significant. SPSS was used for all statistical analyses .Categorical variables were reported as frequency and percentage. Continuous variables were evaluated for normal distribution using a histogram and Kolmogorov–Smirnov test. Since none of the continuous variables were normally distributed, they were reported as median and interquartile range (IQR). Overweight proportion was compared to the published data using the one-sample binomial test. Characteristics of overweight and non-overweight children were compared using the Chi-square test or Fisher’s exact test for the categorical variables, and the Mann-Whitney test was used for the continuous and ordinal variables. All statistical tests were two-sided, and n = 71, 41%), IBS , and FD . No abdominal migraine cases were identified. The median duration of the abdominal pain was 8 months (IQR 4–20), the median BMI percentile was 64 (IQR 26.7–86.5), and the median follow-up duration was 2.1 months (IQR 0–6). The demographic and clinical data are presented in Table A total of 274 children were identified as having the diagnosis of FAP. Forty-eight children who did not meet the ROME IV criteria and 53 children with missing data were excluded, leaving a total of 173 children for final analysis. Of these children, 114 (65.8%) were females, and the group’s median age was 11.5 (IQR 7.9–14.5) years, nearly all of them were from Israeli Jewish origin . The functional disorders were functional abdominal pain NOS (P < 0.001). Twenty children (11.5%) were hospitalized due to FAP. In addition, 105 children (60.6%) were given general balanced dietary recommendations from the physician and 46 (26.5%) children received formal dietitian consultations. Specific dietary recommendations were given as follows: high fiber , reduced simple carbohydrates  or lactose free .Sixty children (36.4%) underwent gastroscopy, 13 (7.5%) underwent colonoscopy, and 132 (76.3%) underwent imaging studies, of whom 115 (66.5%) had an abdominal ultrasound and 17 (9.8%) had an abdominal computerized tomography or a magnetic resonance enterography. Ninety-nine children (57.2%) received medications: 50 (28.9%) were treated with stool softeners, 38 (22%) with proton pump inhibitors (PPI), and 11 (6.4%) with probiotics. No gender difference was noticed in medication use. However, significantly more children with FD were treated with PPI compared to children with IBS and functional abdominal pain NOS , had more hospitalizations due to FAP (20.8% vs. 7.5% p = 0.01), were treated more with PPI  and had more dietitian consultations  compared with children with FAP and normal weight  were accounted as overweight/obese. Among these 53 children, 20 were truly obese (37.7%), 15 (28.3%) boys and 38 (71.7%) girls. The distribution of FAP disorders among the children with overweight/obesity was as follow; 22 (41.5%) with IBS, 16 (30.2%) with functional abdominal pain-NOS and 15 (28.3%) with FD (Table p = 0.04) (Table p = 0.04), and specifically in girls  in that age range. There was no significant difference between prevalence of overweight/obesity of boys with FAP in any age group compared to controls.The 6432 controls were divided into two age groups, one 2–11 years of age and one 12–18 years of age. The younger age group included 1792 children, of whom 919 (51.3%) were boys and 873 (48.7%) were girls. There were 299 children (16.7%) in the young age group who were designated as having overweight/obesity. The older age group of 4640 children included 2223 (47.9%) boys and 2417 (52%) girls, among whom 1433 (30.8%) were designated as having overweight/obesity  that may improve symptoms and prevent or at least minimize the need for medications and hospitalizations.To the best of our knowledge, this is the first study to show an association between obesity and FAP diagnosed in children according to the updated ROME IV criteria for diagnosis of FAP, which was made by a pediatric gastroenterologist in a clinic setting and not by collecting information from a self-administered questionnaire. This study is limited by its retrospective nature, missing more precise data on skinfold measurements, body composition, and other parameters. In addition, our control group might include children with FAP disorders, although we believe that due to the high number of children included, the percentage of children with FAP disorders would be similar to the general population. Lastly, a part of children with diagnosis of FAP in the medical records did not fulfil the ROME IV criteria and therefore were excluded. In real life, some degree of incompatibility between clinical diagnosis and formal criteria may be anticipated. Another explanation is the difference between ROME IV and ROME III criteria, that was one of the rationales to conduct the current study.Adolescents with FAP disorders have a higher prevalence of overweight/obesity compared with controls in the general population. Children with FAP and overweight/obesity have more hospitalizations and medical treatment with PPI compared to children with FAP disorders who are of normal weight. Heightened awareness of these findings might improve treatment planning and management to meet the needs of children with overweight/obesity and FAP. Further studies should focus on the interrelationship between FGID and obesity and specifically on the pathophysiologic mechanisms between these conditions, and also to investigate the effect of weight reduction on FAP symptoms.


Transpancreatic biliary sphincterotomy (TPBS) is an advanced cannulation method for accessing common bile duct (CBD) in endoscopic retrograde cholangiopancreatography (ERCP). If CBD cannulation is difficult, an endoscopist can open the septum between the pancreatic and biliary duct with a sphincterotome to gain access. Long-term results of this procedure are unclear. We wanted to evaluate the short- and long-term complications of TPBS on patients with native papilla and benign indication for ERCP.ERCPs performed in Helsinki University Hospital between 2007 and 2013 were reviewed. The study group comprised 143 consecutive patients with TPBS and 140 controls (CG). Data were collected from patient records and a phone survey was performed as a follow-up ≥ 4 years after the index ERCP.p = 0.067). The rates of other acute complications were similar between the groups. ERCP ended with no access to CBD in four cases (2.8%) in TPBS. The median length of follow-up was 6 years in TPBS and 7 years in CG. During this period, three patients (2.1%) in TPBS and six patients (4.3%) in CG suffered from acute pancreatitis (AP) (p = 0.238). One (0.7%) patient in CG and none in TPBS developed chronic pancreatitis (CP). Abdominal pain was suffered by ten patients (6.9%) in TPBS and twelve patients (8.6%) in CG daily, whereas by six patients (4.2%) in TPBS and twelve patients (8.6%) in CG weekly.Post-ERCP pancreatitis (PEP) developed in seven patients (4.9%) in TPBS and one patient (0.7%) in CG (TPBS is a useful procedure, with acceptable complication rates. No significant difference occurred between the groups when evaluating the short-term or long-term complications with a follow-up period of four to 10 years. Additionally, no significant differences occurred in upper abdominal pain, episodes of AP, or development of CP. ERCP (endoscopic retrograde cholangiopancreatography) is one of many methods to examine and treat diseases of the bile duct and the pancreatic duct. Cannulation of the papilla (papilla Vater) is often performed with a guidewire and a sphincterotome. According to the European Society of Gastrointestinal Endoscopy (ESGE) guidelines, biliary cannulation is defined as difficult if cannulation lasts longer than five minutes, success requires more than five attempts, or the guidewire accidently passes the pancreatic duct at least twice . TherefoOne advanced additional cannulation method is transpancreatic biliary sphincterotomy (TPBS). TPBS was first described by Goff in 1995 . The ratERCP is an invasive procedure with an overall complication rate of approximately 4–11% –11. The PEP was mild or moderate in 90% of the cases. However, 10% of the PEP patients suffered from a severe form of the disease and risk of death among these patients is 3–10% , 16. TheRisk factors for PEP can be classified as patient- or procedure-related factors. Patient- related factors include a patient’s young age, female sex, history of PEP, recurrent acute pancreatitis (AP), diseases such as primary sclerosing cholangitis (PSC) and sphincter of Oddi dysfunction (SOD) , 18, 19.This study aimed to evaluate acute and long-term complications of TPBS. The primary outcomes were development of episodes of PEP, AP during the follow-up period, development of chronic pancreatitis (CP) and post-sphincterotomy stenosis. The secondary outcomes were other ERCP complications and re-ERCPs performed during the follow-up period.The study design was a retrospective case–control study with a phone survey as a cut point. Calculation of the sample size involved estimated PEP rates of 2.2% and 10.4% in regular wire-guided cannulation and in TPBS, respectively . According to this calculation, we aimed to both include a minimum of 137 participants in both groups and have a long-term follow-up period lasting at least four years after the first ERCP.We reviewed the ERCPs performed between January 2007 and December 2013 in the Endoscopy Unit of the Helsinki University Hospital (HUS). Indications of the needs for ERCP were defined from the procedure data. The exclusion criteria identified were primary sclerosing cholangitis (PSC), chronic pancreatitis (CP), biliary or pancreatic malignancies, being aged under 18 and deceased patients. Remaining in the study after exclusion was 170 consecutive TPBS patients with native papilla. Indications for ERCPs in this study were CBD stones, benign biliary stricture, problems related to post-liver transplantation (stricture), bile leakage after cholecystectomy and SOD. For these TPBS patients, we searched for 170 consecutive age- (+ /− 10 years) and sex-matched controls with the same inclusion criteria but having successful biliary access without requiring TPBS. Of these groups, 143/170 (84.1%) TPBS patients and 140/170 (82.6%) patients in control group (CG) participated in the phone survey and comprised the final study groups.Patients and controls received a letter describing the survey and were interviewed by phone within the following two weeks. This study was approved by the Committee on Ethics of the Helsinki and Uusimaa Hospital District.The hospital`s patient data system provided medical records and ERCP procedure information.ERCP procedures were performed with a guidewire and a regular sphincterotome . The TPBS was performed towards the axis of the CBD, in the 11 or 12-o’clock direction. The length of the cut was the same as for a regular biliary sphincterotomy when removing small (< 1 cm) biliary stones. If needed, additional NK cut was performed obliquely towards 10 o’clock, starting from the upper end of the previous pancreatic sphincterotomy. The intention was to cut the CBD and expose the lumen.Collected were the following patient demographic data: age, sex, BMI (body mass index), ASA  and laboratory results . Additionally collected were ERCP procedure data: cannulation method, duration of the ERCP and performed procedures. ERCP procedures were graded by the complexity of procedures criteria . If a paA phone survey collected follow-up data: recurrence of biliary symptoms , episodes of AP, abdominal pain during the previous year, use of medication , pancreatin), use of alcohol and tobacco, symptoms related to pancreatic insufficiency such as steatorrhea, development of CP, hospital admissions and re-ERCPs or abdominal surgical procedures. Additionally collected were the number of re-ERCPs and both indication of and procedures during ERCP. Biliary stones were defined as remnant if new ERCP was required within the same hospitalization period and less than one week after a previous ERCP; otherwise, the stones were defined as recurrent stones.Data analysis involved IBM SPSS Statistics for Macintosh, Version 25.0. Fisher`s exact test was used for categorical variables and Wilcoxon-Mann–Whitney U test for continuous variables when comparing both the two groups and interrelations to different variables and PEP. Probability below 0.05 was considered statistically significant. Risk factors for PEP were evaluated with binary logistic regression as an odds ratio (OR) and 95% confidential interval (CI).p = 0.122).All the 283 ERCP procedures were therapeutic. Three patients in TPBS (one of whom with NK) and two patients in CG (one with NK) had undergone previous failed ERCP attempts: native papilla was present in 142 and 139 patients, respectively. ERCP ended with a final failure, that is no access to the biliary tract was achieved, in four cases (2.8%) in TPBS, whereas all cases in CG were successful, as defined in the patient selection, (Table The primary cannulation method in the TPBS group was an NK precut in two cases 1.4%) due to a stone in the papilla. After precutting, access was possible only to PD, thus requiring further TPBS. Cannulation methods in TPBS are presented in Fig. .4% due tp < 0.001).The median duration of ERCP procedure was 27 min (7–87 min) in TPBS and 15 min (7–65 min) in CG (p = 1.000). One case in the TPBS group had no access to CBD, only to PD, and resulted in final failure after bleeding. One case was unsuccessful in TPBS + NK, although successful performance of ERCP and BS occurred three days later. In CG, one patient had suspected biliary leakage; however, due to both normal findings and normal outflow of contrast medium, BS was not performed. BS was not performed in another case involving a young patient (33 years).Biliary sphincterotomy (BS) was performed 141 times (98.6%) in TPBS and 138 times (98.6%) in CG  of TPBS and 131 (93.6%) of CG, were classified as difficulty grade 2 (20).p = 0.067, OR for TPBS 7.15, 95% CI for OR 0.869–58.93). Complications related to anesthesia or patients’ comorbidities were pneumonia, acute transient brain ischemic attack and worsening of chronic liver dysfunction. One patient in both groups suffered from late-onset AP, both of whom were classified as mild.Table Table p = 0.495). Table CP was diagnosed only in one patient (0.7%) in CG and none in TPBS, p = 0.95. Tablep = 0.238). Abdominal pain was a common symptom in both groups: 58 patients (40.6%) in TPBS and 50 patients (35.7%) in CG mentioned upper abdominal pain in the phone survey. Of these patients, ten (6.9%) in TPBS and twelve (8.6%) in CG suffered daily from abdominal pain, while six patients (4.2%) in TPBS and twelve patients (8.6%) in CG suffered weekly. Abdominal pain was categorized as heartburn by 32.1% vs. 30.0%, as upper abdominal pain by 32.1% vs. 22.0%, as biliary-attack-type pain by 28.3% vs. 22.0% and as other unspecific pain by 7.5% vs. 26.0% in TPBS and CG, respectively.Three patients (2.1%) in TPBS and six patients (4.3%) in CG suffered from AP during the follow-up period  and in CG 22 patients (15.7%) had taken some medication for abdominal pain or symptoms within the previous year. The most common medications used were PPIs, metamizole-pitofenone, paracetamol and NSAIDs .Our evaluation pertained to abdominal pain-related contacts to hospitals after index ERCP. Re-ERCP data are shown in Table p = 0.505) numbered 16 in TPBS, and 5 in CG. Other re-ERCPs were scheduled control procedures.Performance of re-ERCPs with an acute indication (p = 0.445), of whom normal findings occurred in 31 (75.6%) and 30 (65.2%), respectively.Patients who underwent gastroscopy during the follow-up period numbered 41 patients (28.7%) in TPBS and 46 patients (32.9%) in CG  research group and later by ESGE was published in 2014 and 2016 [Only few studies have evaluated the long-term complication risks of TPBS , 23 or pWe have performed TPBS in difficult cannulation situation for several years and collected the data to provide a large patient series to confront the long-term complication issue. We wanted to explore if patients with TPBS have more CP or symptoms related to papillary stenosis, for example AP episodes. Therefore, we needed the follow-up period to last for several years. However, we found no relations between TPBS and CP or papillary stenosis. Concerns with TPBS included increased risk of complications, additionally long-term consequences of PS in cases when the target is not PD, and a risk of papillary stenosis that may lead to recurrent pancreatitis –29.Late complications like papillary stenosis and ductal strictures are only encountered at least 3 months after the procedure indicating a need for long term follow-up .In difficult cannulation, if advanced cannulation methods, such as TPBS, are needed, the risk of PEP arises , 2. By tIn HUS, prophylactic pancreatic stents were seldom used during the study period. The ESGE has suggested that placement of prophylactic stents might reduce the PEP rate , 12. SixOur follow-up study found no patients with stricture or CP in TPBS group. Notable, however, are limitations regarding patient selection and retrospective manner. Patients in both groups suffered from upper abdominal pain. However, no differences existed in the frequency or severity of the pain. Most of the patients had symptoms related to recurrent biliary stones or heartburn. Painkillers or PPIs were equally used in both groups. The most common reason for evaluating the follow-up contacts to healthcare were upper abdominal pain and symptoms related to biliary stones. Due to these symptoms, the most common procedures were cholecystectomy and gastroscopy.According to a previous study in HUS, the TPBS method has been successful and is therefore commonly used as a rescue method in our unit . We suggPossible bias in this study relates to the patient selection process. We wanted to interview all the patients; therefore, the inclusion criteria considered only benign indications for ERCP. PEP rates are slightly lower in the present study than in previous studies in the HUS endoscopy unit in ERCP patients with TPBS , 33. NevTPBS is a useful rescue method in difficult cannulation, having an acceptable complication rate. No significant difference occurred between groups when evaluating the short- or long-term complications. Additionally, no significant differences occurred during the follow-up period concerning upper abdominal pain, usage of painkillers, episodes of AP or development of CP.


It is unclear whether increased left ventricular (LV) thickness is associated with worse clinical outcomes in severe aortic stenosis (AS). The aim of this study was to determine the effect of increased LV wall thickness (LVWT) on major clinical outcomes in patients with severe AS.p < 0.001). Furthermore, in patients with increased LVWT, the all-cause and cardiovascular death rates were significantly lower in the AVR group than in the non-AVR group . Multivariate analysis revealed that increased LVWT, age, dyspnea, and AVR surgery were significantly correlated with cardiovascular death.This study included 290 severe AS patients  between January 2008 and December 2018. For outcome assessment, the endpoint was defined as death from all causes, cardiovascular death, and the aortic valve replacement (AVR) surgery rate. During follow-up (48.7 ± 39.0 months), 157 patients had AVR, 43 patients died, and 28 patients died from cardiovascular causes. Patients with increased LVWT underwent AVR surgery much more than those without LVWT (60.0% vs. 39.0%, In patients with severe AS, increased LVWT was associated with a higher AVR surgery rate and an increased rate of cardiovascular death independent of other well-known prognostic variates. Thus, these findings suggest that increased LVWT might be used as a potential prognostic factor in severe AS patients. Left ventricular hypertrophy (LVH) is known as an adverse clinical factor in cardiovascular disease –4. LVH iFrom January 2008 to December 2018, all echocardiographic studies performed at Dong-A University Hospital were reviewed to identify severe native AS patients. A total of 290 patients were enrolled in this study. The data collection finished in December 2019 for the evaluation of AVR and death. The clinical characteristics and laboratory values of the patients were extracted from the electronic medical records. The clinical data included age, gender, height, weight, body surface area (BSA), body mass index (BMI), and blood pressure (BP). The symptoms were defined as the presence of angina, syncope, or dyspnea NYHA class ≥ II. Coronary artery disease (CAD) was defined by > 70% luminal reduction in major coronary arteries. The incidence of AVR, death from all causes, and cardiovascular death were compared between patients with increased left ventricular wall thickness (LVWT) and normal LVWT during the follow-up period. This retrospective study was approved by the Institutional Review Board of Dong-A University Hospital.2 in women and > 115 g/m2 in men [2, or indexed AVA (AVAI) < 0.6 cm2/m2 [Two-dimensional and Doppler examinations were performed using a commercially available echocardiographic system  equipped with a 2.5-MHz transducer. The LV end-diastolic dimension (LVEDD), LV end-systolic dimension (LVESD), interventricular septum (IVS), and posterior wall thickness (PWT) were measured, and LV ejection fraction (LVEF) was assessed using modified Simpson method. The LVM and LVM index (LVMI) were calculated from the parasternal M-mode measurements using validated formula. The left atrial (LA) volume was measured using the biplane Simpson method at end-systole from apical 4- and 2-chamber views. The LA volume index (LAVI) was determined as the LA volume divided by the body surface area . Increas6 cm2/m2 . Grading6 cm2/m2 .p-values of 0.2 in univariate analyses were included in multivariate analysis. A P < 0.05 was regarded as the statistical significance in all tests. All analyses were conducted using SPSS version 20.0 .Continuous variables are presented as the mean ± SD. Differences between two groups were compared with t-test. Categorical variables are presented as numbers with percentages and were compared with the chi-squared test. To find out the independent risk factors for cardiovascular death in severe AS, logistic regression analysis was performed. Variables with p < 0.001). Although the all-cause death rate was not significantly different between the normal LVWT and the increased LVWT groups , the cardiovascular death rate was significantly higher in patients with increased LVWT than in those with normal LVWT . However, the all-cause death rate in patients with increased LVWT was significantly lower in the AVR group than in the non-AVR group .Compared to patients with normal LVWT, patients with increased LVWT showed higher number hypertension and dyspnea, respectively, and a higher mean NYHA class at baseline Table . During p = 0.66, Fig. Compared to the group with normal LVWT, the increased LVWT group showed significantly higher IVS, PWT, LVM, and LVMI and significantly lower mitral E. The AV peak pressure gradient (PG) and the mean PG were significantly higher in the group with increased LVWT than in the group with normal LVWT. AVA and AVAI were significantly smaller in the group with increased LVWT than in the group with normal LVWT Table . Using tTable p = 0.028), age , dyspnea , AVR surgery , and CAD  were independent predictors. However, the LVEF was not a significant risk factor for cardiovascular death (p = 0.285).Table The major findings of the present study were as follows: (i) increased LVWT was associated with a higher rate of AVR surgery, (ii) In patients with increased LVWT, the all-cause and cardiovascular death rates were both significantly lower in the AVR group than in the non-AVR group, and (iii) Increased LVWT was an independent predictor for cardiovascular death in patients with severe native AS.Although the presence of extreme LVH in severe AS indicates poor prognosis, current guidelines do not consider LVH an indication for surgical replacement in patients with AS . The indp = 0.001, Table Because clinical decisions should be based on the risk of mortality, AVR might not a suitable endpoint in conservatively treated patients with severe AS. However, AVR is also the most important decision in the course of treatment for severe AS from a doctor’s point of view. In our study, the main causes of AVR were the development of symptoms 92/157, 58.6%) and LV dysfunction . Although LV hypertrophy was not considered a risk factor at the time of AVR, this retrospective study showed that patients with increased LVWT underwent AVR much more frequently than those without LVWT . Honda et al. have reported that concomitant AR in severe AS patients had significantly worse clinical outcomes [p = 0.661). The prevalence of CAD in death patients was significantly higher than that in survived patients . Likewise, mortality of CAD patients was significantly higher than that of patients without CAD in severe AS . This study had limitations. First, because this was a retrospective study, the clinical data may be incomplete. Second, the follow-up period was clearly too short and the absolute number of patients was too small to have strong statistical power. Third, LVH may be a consequence or reflection of the AS severity, not be the independent factor, because it was obtained by narrow-range analysis of LV wall thickness. Forth, we tried to incorporate LVH as an indication for AVR with classic symptoms and LV dysfunction, but this approach is incomplete comparison, because considerable patients with symptoms or LV dysfunction did not have AVR due to their very old age and poor health condition.The multivariate regression analysis in this study showed that increased LVWT was independently correlated with cardiovascular death in patients with severe AS. In this study, 108 patients 108/208, 51.9%) with increased LVWT had no or mild symptoms. In addition, 36 patients 36/125, 28.8%) with increased LVWT who underwent AVR had no or mild symptoms. This may imply that symptom-based decisions for interventions in severe AS might miss a chance for surgery in patients about 30%) with no significant symptoms. Thus, LVH could be used as a more sensitive marker than symptomatic criteria for AS surgery decisions. Previous studies reported that LVH was associated with an increased rate of cardiovascular events in severe AS independent of other prognostic covariates, even in asymptomatic patients , 20, 21.8, 51.9% , 28.8% w% with noIn conclusion, increased LVWT was an independent predictor of cardiovascular death. Thus, AVR should be strongly considered in all AS patients with increased LVWT, even in asymptomatic patients.