We evaluate the performance of several variable selection approaches for clustered binary outcomes to provide guidance for choosing between the methods.Identifying predictors of patient outcomes evaluated over time may require modeling interactions among variables while addressing within-subject correlation. Generalized linear mixed models (GLMMs) and generalized estimating equations (GEEs) address within-subject correlation, but identifying interactions can be difficult if not hypothesized We conducted simulations comparing stepwise selection, penalized GLMM, boosted GLMM, and boosted GEE for variable selection considering main effects and two-way interactions in data with repeatedly measured binary outcomes and evaluate a two-stage approach to reduce bias and error in parameter estimates. We compared these approaches in real data applications: hypothermia during surgery and treatment response in lupus nephritis.Penalized and boosted approaches recovered correct predictors and interactions more frequently than stepwise selection. Penalized GLMM recovered correct predictors more often than boosting, but included many spurious predictors. Boosted GLMM yielded parsimonious models and identified correct predictors well at large sample and effect sizes, but required excessive computation time. Boosted GEE was computationally efficient and selected relatively parsimonious models, offering a compromise between computation and parsimony. The two-stage approach reduced the bias and error in regression parameters in all approaches.Penalized and boosted approaches are effective for variable selection in data with clustered binary outcomes. The two-stage approach reduces bias and error and should be applied regardless of method. We provide guidance for choosing the most appropriate method in real applications. In such cases, application of variable selection techniques can be employed to identify main effects and interactions most strongly associated with the outcome.It has been hypothesized that many common diseases result from interactions among genetic, clinical, and environmental factors –4. Ident effects . In explMethods for selecting variables in regression models fall into two categories: traditional stepwise variable selection methods and sparse model solutions. Traditional variable selection approaches include forward, backward, and stepwise algorithms that select variables for inclusion in a model based on statistical significance or measures of model fit. However, such approaches yield unstable parameter estimates, particularly in data with correlated predictors or outcomes , 10.L1 penalty) which shrinks many coefficients to zero providing a means of variable selection [L1-constrained projected likelihood ratio. Brown et al. [Sparse model solutions simultaneously evaluate all potential covariates and shrink regression coefficient estimates toward zero. Penalized regression techniques penalize coefficients the further their estimated value is from zero by including a penalty term in the likelihood. Lasso regression, a popular penalized regression approach, includes the sum of the absolute values of the regression coefficients in the likelihood , and random effects from the (l-1)th iteration. The predictor, j, that yields the smallest AIC or BIC is updated by adding the resulting product of the jth Fisher matrix and jth score equation to the estimates for the intercept, jth regression parameter and random effects from the (l-1)th iteration. All other parameter estimates are set to the values from the (l-1)th iteration.Boosting was originally proposed to improve prediction performance of weak classifiers , and genequation , 29. Thiequation  propose L1-constrained projected artificial log-likelihood ratio but without necessitating a constrained optimization algorithm. The optimization for both EEBoost and GEEBoost is based on functional gradient descent optimization. GEEBoost can be used for variable selection by choosing the model from the entire path of solutions that yields the smallest QIC, although alternative criterion could also be used.The estimating equation approach has also been recently incorporated within a boosting framework for variable selection, a method referred to as EEBoost  and has Penalized and boosted regression models are known to yield biased regression parameter estimates , 19, 20.X1-X14, yielding 91 possible two-way interactions, a random subject effect, and a repeatedly measured binary response. Correlation for time-varying covariates, Σ, was compound symmetric with ρ = 0.25. Potential predictors and interactions generated in the data included a mix of categorical and continuous variables as well as fixed-time and time-varying covariates to mimic what might be observed in a clinical data set. Binary response variable Y was generated from a Bernoulli distribution where the probability depended on variable X5, the interaction X1X2, and the interaction X2X5. Dependence of Y on the time-varying covariates X1 and X2 created a correlated response within subject. Effect sizes for nonzero parameters in the true model were selected to represent weak, moderate, and strong effects and yield odds ratios of 1.2, 2.0, and 4.5 for a one unit increase in X5, the interaction X1X2, and the interaction X2X5.A simulation study was conducted to evaluate the ability of each method to 1) correctly recover predictors and interactions associated with a repeatedly measured binary outcome, and to evaluate (2) reduction in bias in regression parameter estimates using the two-stage approach, (3) model parsimony and (4) computational efficiency (average time to develop models). Simulation parameters including variable distributions, covariate effect size, number of observations per subject, sample size, the model used to generate the response, and the model fit using each method are defined in Table  correctllme4, glmmLasso, GMMBoost, and threeboost [Y where identified, the two-stage approach was applied to evaluate how well this approach reduced the bias in the parameter estimates. All simulations were conducted in R 64 bit v 3.2.5 using a Quad Opteron 2.8 GHz processor with 96 GB Ram and 3 TB of HDD storage.Five-hundred data sets were generated for each combination of sample and effect size for data with two observations per subject. Similarly, 500 data sets with 10 observations per subject were generated for each sample size for and effect size of 0.2. Forward stepwise GLMM, glmmLasso, GMMBoost, and GEEBoost models were fit to each simulated data set in R v 3.2.5 (Team RDC) using the reeboost –39. Glmmreeboost  considerY and the FDR for null predictors identified in data with two observations per subject for the three effect sizes and four sample sizes. Identification of the correct predictors and predictor interactions for all methods improves with increasing sample size and effect size with the exception of the stepwise selection method. The proportion of times each method correctly identifies the true predictors also increases when the number of observations per subject increases for all methods .Fig. X5 significantly more often than the penalized regression approaches for the smallest effect size (e.g. β = 0.2) for n ≥ 200. However, the stepwise approach identifies the interaction terms in fewer than 20% of models across all sample sizes, effect sizes, and number of observations per subject. When sample size or effect size is small, the glmmLasso approach recovers the correct predictors and interactions more frequently than GMMBoost or GEEBoost for n ≤ 300. However, for large sample size or effect size (e.g. n ≥ 200), glmmLasso, GMMBoost, and GEEBoost exhibit similar ability to recover the true predictors and interactions. For n ≥ 200 at moderate and large effect sizes, glmmLasso, GMMBoost, and GEEBoost all recover the three predictors and predictor interactions in ≥85% of all models. The proportion of times null predictors were selected was similar within method across effect sizes. Stepwise regression and GMMBoost were less likely to select a null predictor than glmmLasso and GEEBoost. GlmmLasso was significantly more likely to select a null predictor at n = 100 than all other methods for The stepwise algorithm correctly identifies the main effect for S1). GlmmLasso had the largest average number of correct predictors across simulations, though GEEBoost was similar for larger sample and effect sizes. When sample sizes and effect sizes were large, GMMBoost often exactly identified the correct model . In contrast, glmmLasso and GEEBoost tended to include more predictors in the model particularly for small sample sizes, which likely explains why these methods recovered the true predictors more frequently than GMMBoost. The FDR for glmmLasso and GEEBoost was between 65–81% and 68–88%, respectively . However, FDR for GMMBoost decreased with increasing sample and effect size achieving FDR = 0 for large n and Among the three penalized approaches, GMMBoost tended to yield more parsimonious models relative to glmmLasso and GEEBoost for all samples and effect sizes Table S. GlmmLasX5, X1X2, X2X5 and the average bias across all null predictors for each approach for simulations with two observations/subject. Bias for the true predictors was generally negative indicating values smaller than the true S2 and Table S2). Stepwise selection was an exception as MSE and bias were consistent with increasing sample size . Among the three penalized approaches, glmmLasso yielded the smallest average bias and MSE for true predictors, followed by GEEBoost and GMMBoost . Results were similar for 10 observations/subject and effect size β = 0.2 though the observed bias was less variable .Fig. S2, respectively.We also evaluated the ability of a two-stage approach to reduce bias in parameters estimates using the penalized selection approaches. We considered two models: (1) the first model imposed the heredity constraint and incorporated all nonzero terms from the penalized model plus all main effects that are zero in the penalized model but are in one or more nonzero interaction term and (2) a second model in which heredity was ignored fitting a GLMM with all nonzero terms ignoring main effects if they are zero in the penalized models. Both approaches performed similarly in terms of bias reduction and thus only results for the second method are shown. The change in bias for each simulation run from stage 1 to stage 2 and the bias in stage 2 for estimates from the glmmLasso, GMMBoost, and GEEBoost in data with two observations/subject are presented in Fig. S2). Fig. n ≥ 300. MSE was similar in the first- and second-stage models for glmmLasso and smaller for GMMBoost and GEEBoost for S1). GlmmLasso exhibited larger bias and MSE in the second-stage models at n = 100 for X5 .The bias in stage 2 was generally smaller for all effect and sample sizes than in stage 1 though similar to stage 1 estimates were still predominantly downward biased  included age, gender, presence of anti-double-stranded DNA antibodies (dsDNA), serum C3 complement levels (C3C), serum C4 complement levels (C4C), urine protein to creatinine ratio (UrPrCr), and estimated glomerular filtration rate (EGFR). Urine samples were collected at two times during the study for all subjects and were analyzed for 15 novel urinary biomarkers. The final data include 22 demographic, clinical, or biomarker variables yielding 22 main effects and 190 two-way interactions. Patient response to treatment, determined using the criterion defined by .Predictors and interactions selected by each method for response in LN are presented in Table This was an observational study examining patient, clinical, and procedural variables associated with occurrence of hypothermia in patients undergoing total hip or total knee arthroplasty (THA and TKA). Participants’ temperatures were evaluated at eight timepoints: (1) leaving holding; (2) operating room arrival; (3) after anesthetic induction; (4) upper-body warmer initiation; (5) incision; (6) every 30 min after incision; (7) leaving the operating room; and (8) arrival to PACU and hypothermia was defined as temperature < 36.0°C. The data included 102 participants with 13 demographic, clinical, or procedural variables and 66 possible two-way interactions. Full study details are provided in Ref. .Similar to the lupus nephritis study, the model selected using forward selection was quite different from the models selected using the penalized regression and boosting approaches. The forward selection model only included main effects, while the other approaches included a mix of main effects and interactions. The glmmLasso model included more predictors than GMMBoost and GEEBoost . GMMBoost recovered the correct predictors and interactions with similar frequency to glmmLasso and GEEBoost at large effect and sample sizes and identified few spurious predictors; thus, GMMBoost is a most appropriate if parsimony is of importance. GMMBoost is the least computationally efficient and may prove difficult in real applications. At smaller sample sizes, GEEBoost had similar performance to glmmLasso and tended to select a more parsimonious model thus offering a good compromise between the computation time and model parsimony. Finally, the two-stage approach effectively reduced the bias for all three methods and thus should be applied regardless of selected method.The simulations provide guidance for choosing an appropriate method to identify predictor interactions without L1 and L2 constraints to yield a sparse solution while facilitating grouping of collinear variables [There are extensions that could be considered to enhance the applicability of each method. Elastic net uses both ariables . Additioariables , 44, 45.a priori hypothesis. Our results demonstrate a strong advantage of penalized and boosted approaches over the traditional stepwise approach in these types of data and provide guidance for choosing the most appropriate method in real applications. Furthermore, the two-stage approach effectively reduces the bias and MSE for all methods and should be applied regardless of choice of method.In this paper, we describe four variable selection approaches for data with repeatedly measured binary outcomes, with focus on identifying predictor interactions without an


Knee and hip arthroplasty are common surgeries within an aging population. Some data has suggested that knee arthroplasty is more traumatic to the body than hip arthroplasty due to the increased complexity and load bearing nature of the joint. Here, we compare the stress of the two surgeries by measuring urinary neopterin and total neopterin as biomarkers of oxidative stress and inflammation. Urinary neopterin and total neopterin  levels were analysed in 28 knee and 22 hip arthroplasty patients pre- and post-operatively to determine oxidative stress and inflammation levels. Total neopterin was 31.1% higher with knee arthroplasty (p<0.05). Urinary neopterin was 32.8% higher in the knee arthroplasty group versus hips. The increase in neopterin and total neopterin following a post-surgical decrease in levels was significant in both groups. Levels of neopterin and total neopterin were varied between patients, but all increased following surgery and subsided by day 28. The increased levels of urinary neopterin and total neopterin from knee arthroplasty indicate that knee osteoarthritis and arthroplasty is a more significant trauma to the body than hip osteoarthritis and arthroplasty surgery. This is also shown by faster inflammatory resolution following hip arthroplasty. Surgery is deliberate but controlled trauma to the body and as a result, sets off a range of stress responses including inflammatory cascades. The levels of tissue disruption from the surgical trauma is expected to be reflected in the scale of the body’s response especially in the level of inflammation. Hip arthroplasty is often reported by surgeons to be less traumatic to the body than knee arthroplasty, which is reflected in poorer functional outcomes after 5 years and decreased reported quality of life ,2. It haThe biochemically measurable difference in physical trauma and inflammation between hip and knee replacement surgery is subtle, with one study showing increased levels of hormonal markers  in knee arthroplasty versus hip, but not inflammatory markers interleukin-6 (IL-6) or C-reactive protein (CRP) . While INeopterin and its unoxidized form 7,8-dihydroneopterin, are relatively sensitive markers of inflammation as they are generated within the site of inflammation. 7,8-Dihydroneopterin is a monocyte/macrophage synthesized antioxidant which is produced upon interferon-gamma stimulation. 7,8-Dihydroneopterin rapidly scavenges superoxide and hypochlorite, both products of the inflammatory response, to generate the highly fluorescent neopterin . These fThe ratio of total neopterin to neopterin can be used to investigate the environmental dynamics between monocyte/macrophage activation and oxidative stress. A low ratio signifies an environment where there is a lot of oxidative stress, relative to immune activation, whereas a high ratio indicates an environment in which there is a high level of immune cell activation with relatively less oxidative stress ,17,18. TNeopterin and 7,8-dihydroneopterin is rapidly cleared by the kidneys and concentrated in the urine thus urinary neopterin appears to provide a clearer report of inflammation than blood analysis when corrected for urine concentration –22. As aTotal neopterin  and neopterin have been used as sensitive biomarkers of inflammation and oxidative stress respectively. They have been used extensively to monitor immune system activation in illness and trauma, allowing for quantitative analysis of patient’s inflammatory recovery ,17,25. AIn this study we have used urinary neopterin and total neopterin to assess differences in oxidative stress and inflammation levels between knee and hip arthroscopy patients over 28 post-operative days as well as assessing the inflammatory recovery for each type of surgery. We hypothesized that firstly, knee arthroplasty would yield higher values for urinary neopterin and total neopterin than hip arthroplasty, and secondly, hip arthroplasty would demonstrate a more rapid reduction in biomarker levels.This was an observational cohort study investigating a diagnostic test with Level III evidence. The surgical cohort comprised of knee and hip arthroplasty patients. Knee arthroplasty patients (n = 28) were aged 68.00 ± 10.37 years (mean ± SD) and 54% were female. The hip arthroplasty patients (n = 22) were aged 66.26 ± 7.77 years (mean ± SD) and 52% female. Ethics approval  and informed consent was gained from the surgical patients. Exclusion criteria were; patients below 18 or above 80 years of age, smokers, or patients having recently received a diagnosis of cancer. The effect of sex or age between the exclusion criteria range has been shown to not affect neopterin levels. Patients provided urine samples before surgery, immediately after surgery, and each day following at approximately midday (± 2 hours) until the patients were discharged. Day 14 and 28 day samples were collected from the patient’s home when possible.For knee arthroplasty, all patients had a tourniquet applied proximal to the surgical site and inflated to 350 mmHg. An anterior midline incision was made over the knee with a medial parapatellar dissection and exposure of the joint. Jig-assisted preparation of the distal femur and tibial plateau was performed with subsequent insertion of the appropriate implants. All components were uncemented in each patient and all wounds were drained for 24 hours. For hip arthroplasty patients the hip was dislocated and the femoral head removed with a power saw. The acetabulum was prepared with power reamers and sized to accept an uncemented shell and a highly cross linked polyethylene liner. No image guidance was used. The femur was reamed by hand and the appropriate uncemented or cemented stem inserted following trial reduction. Leg lengths and off-set were measured and the appropriate femoral ceramic head inserted. Following final reduction, the wound was closed in layers over a pain catheter. No drains were used. All patients for both surgeries received intravenous Tranexamic Acid (1g) following the procedure to reduce blood loss.All anaesthetics were performed as per each anaesthetist’s routine practice. Standard medications used to induce and maintain anaesthesia for all patients were Midazolam, Fentanyl, Propofol, Dexamethasone, Rocuronium, Ondansetron, and Cyclizine. At induction all patients were given intravenous Cefazolin (2g) which was continued for a further 24 hours post-operatively. Post-operative pain management was obtained using Paracetamol, Codeine and Fentanyl.Urinary samples were provided by patients into 70 mL sample bottles in the orthopaedic recovery wards and immediately refrigerated in the absence of light. Samples were taken pre-surgery, as soon as the patient was able post-surgery, and then at 12 pm on each following day until the patient was discharged . The samples were collected daily and transported on ice to the University of Canterbury before being aliquoted and stored in a -80°C freezer.Urine samples were thawed in the dark to remove any ultraviolet light mediated oxidation of 7,8-dihydroneopterin. 5 μL of urine was mixed with 195 μL of 20 mM ammonium phosphate (pH 2.5), with 100 μL transferred into high-performance liquid chromatography (HPLC) vials for neopterin determination. Of the remaining 100 μL of diluted sample, 20 μL of acidic iodide was added, followed by mixing on a vortex mixer and a 15 minute incubation in the absence of light at room temperature to oxidise the 7,8-dihydroneopterin into neopterin for total neopterin analysis. The unreacted acidic iodide was neutralised by addition of 10 μL of 0.6 M ascorbic acid followed by further mixing using a vortex mixer. 100 μL of this solution was transferred into HPLC vials for total neopterin determination.1. Fluorescence wavelengths for neopterin detection were set at 438 nm and 353 nm for emission and excitation, respectively. Peak analysis and result quantification was conducted using Shimadzu Lab Solutions version 5.86.HPLC measurements were made using a Shimadzu 20A HPLC with a Sil-20A autosampler and RF-20Axls fluorescence and M20A SPD absorbance detectors. Separation of neopterin was achieved using a Phenomenex Luna 5 μm Strong Cation Exchange 100 Å 250 mm × 4.6 mm column. The mobile phase consisted of 20 mM ammonium phosphate at pH 2.5 being pumped at 1.0 ml min-1.020 (nM)[neopterin] from pre-surgical values for both groups. From day 2 onwards, urinary neopterin and total neopterin were elevated significantly from the post-surgical measurement in both groups, but not the pre-surgical value. Total neopterin was on average 31.1% higher for knee arthroplasty than hip (p<0.05) over all days , indicatThe TNP/NP ratio gives an indication of inflammation versus oxidative stress levels . In thisThe biomarkers profiles of individual participants are shown as single lines , with grA post-hoc power analysis was conducted to determine the required cohort sizes to detect a statistically significant (α = 0.05) difference between the neopterin levels following knee and hip arthroplasty at day 14 . With 0.Neopterin and total neopterin have been shown to be informative biomarkers in measuring grouped and individual data for oxidative stress and immune cell activation in participants exposed to physical trauma ,25,28,29Elevated levels of urinary total neopterin supports our first hypothesis and indicates that hip arthroplasty results in higher levels of inflammation that knee arthroplasty. . Pre-surThere was a significant post-surgical decrease in both neopterin and total neopterin which has been reported with the use of anesthesia ,31. In tThe ratio between total neopterin  versus neopterin has been used clinically to assess the dynamics between oxidative stress (neopterin) and inflammation  ,23,25. WFurthermore, this result indicates that the dynamics between oxidative stress and monocyte/macrophage activation for hip and knee arthroplasty surgery are equivalent, irrespective of knee arthroplasty generating overall higher levels of oxidative stress and inflammation. The equivalence of the TNP/NP ratio here suggests that even with the varying levels of oxidative stress and inflammation seen between knee and hip arthroplasty patients, the body’s response in upregulation of oxidative stress and inflammatory pathways is equal.Increased inflammation with limited oxidative stress is also more characteristic of tissue destruction and remodeling which would be expected following a physical trauma without infection, such as arthroplasty surgery ,33.The individual data shows substantial variation between patients in, and between, the two groups . Attempt7,8-Dihydroneopterin and its oxidation product neopterin are intimately tied to activity of the immune system and levels of oxidative stress ,20,39. AIn this study we have shown that both knee and hip arthroplasty surgeries cause an increased level of oxidative stress and inflammation following the anesthetic mediated post-surgical dip in urinary neopterin and total neopterin. We have confirmed our hypothesis that this increase is larger for knee arthroplasty versus what was seen from hip arthroplasty surgery. This indicates that osteoarthritis in the knee and the knee arthroplasty procedure cause a higher level of immune activation, likely due to increased complexity and loading on the joint, as well as, increased physical trauma from the surgery. Also we have confirmed that urinary neopterin and total neopterin levels fall faster following a hip arthroplasty, indicating a faster inflammatory recovery. The measurement of urinary neopterin and total neopterin provide a non-invasive measure of patient’s levels of oxidative stress and inflammation in response to surgical procedures and may be valuable in improving the clinical outcomes in these patients.


An increase in intraocular pressure during chronic hemodialysis is linked to ocular complications, such as glaucoma. The behavior of intraocular pressure during hemodiafiltration is unknown. Changes in intraocular pressure with a sodium dialysate concentration fixed at 138 mmol/L and an individualized concentration were studied in 13 patients with end-stage renal disease treated with hemodiafiltration. Up to 31% patients presented an episode of intraocular hypertension without differences between sodium profiles. A large variability in intraocular pressure within patients and a high prevalence of transient intraocular hypertension were found.Ocular complications are common among end-stage renal disease patients and some complications had been linked to increments of intraocular pressure (IOP) during hemodialysis. The changes of IOP during hemodiafiltration (HDF) have been scarcely investigated and the potential influence of the sodium dialysate concentration is unknown. The aim of this study was to compare the IOP changes during HDF with sodium dialysate concentration, either fixed or individualized. Thirteen end-stage renal disease patients participated in the study; they were treated with HDF using a dialysate sodium profile fixed at 138 mmol and another session with an individualized sodium profile. The intraocular pressure was measured before and after each session and every 30 min during HDF. Both groups had a similar HDF prescription, blood pressure, and biochemical parameters. At the end of hemodiafiltration, sodium concentration decreased only in the fixed sodium profile group. The number of patients with at least an episode of intraocular hypertension during HDF ranged from 5 (19%) to 8 (31%) without significant differences between right and left eye nor between dialysate sodium concentration. During HDF, there is a large variability of IOP; transient events of intraocular hypertension are highly prevalent in this sample, and they are not related to the sodium dialysate concentration. End-stage renal disease is a major public health problem in industrialized and developing countries, it is highly prevalent and requires expensive therapies to maintain patients’ long-term survival ,2. ThereIn healthy individuals, an increase in salt consumption is associated to a discrete decrease on IOP . MoreoveThere are several case reports of important IOP increments, glaucoma and retro-ocular pain during hemodialysis ,15,16,17In a previous study, it was shown that the IOP during HD and hemodiafiltration (HDF) has a similar behavior . HoweverThis exploratory study included 13 adult patients with end-stage renal disease, under treatment with HDF three times per week for at least 3 months. Patients were recruited consecutively (non-random sampling). Inclusion criteria were male or female adult patients. Exclusion criteria were previous symptoms of retro-ocular pain, previously diagnosed glaucoma, newly diagnosed glaucoma  or use of ocular hypotensive drugs. Elimination criteria were patients that showed hemodynamic instability during hemodialysis .All patients underwent ophthalmological bio-microscopy and fundoscopy by an ophthalmologist. IOP was measured in both eyes under topical anesthesia with tetracaine 0.5% eyedrops in both eyes. All procedures performed in studies involving human participants were in accordance with the ethical standards of the Research and Ethics Committee of the Instituto Nacional de Cardiología Ignacio Chávez (protocol number 14-891) and with the 1964 Helsinki declaration and its later amendments. Informed consent was obtained from all the participants.3− = 35, Na+ = 138, K+ = 2; (mEq/L) Ca2+ = 3.5, Mg2+ = 1, and polysulfone membranes were used . Two dialysate solutions were used, each with a different sodium’s concentration. Each patient received one HDF session with a profile of dialysate sodium concentration fixed at 138 mmol/L (standard sodium concentration in our center) and another HDF session with individualized sodium. The personalized profile started the HDF session with a dialysate sodium concentration was set to the same value of the venous sodium concentration and gradually moved towards the standard concentration during HDF (one mEq/L every 36 min). Every session lasted 3 h with fixed ultrafiltration profile and HDF were similar in both sessions . Ultrapure dialysate (mmol/L): HCOsessions . All pat+ + K+) + ([glucose]/18) + ([blood urea nitrogen]/2.8).Before the dialysis started, a venous blood sample was obtained in order to measure biochemical parameters . The following dialysis parameters were recorded at 30, 60, 90, 120, 150, and 180 min during HDF and 30 min after the end of the session: systolic and diastolic blood pressure, heart rate, temperature, and oxygen saturation level. At the end of the session, a blood sample was obtained from the venous access to obtain the same parameters as in the first blood sample and the following dialysis parameters were recorded: total filtration volume (mL), ultrafiltration rate (mL/h), blood flow rate (mL/min), arterial pressure (mmHg), venous pressure (mmHg) and final body weight (Kg). The osmolality was estimated with the following formulae: osmolality = 2 × . Baseline IOP was measured before the beginning of dialysis. During dialysis, intraocular pressure was evaluated in millimeters of mercury (mmHg) at the following times: 30, 60, 90, 120, 150, 180 min, and after the end of the session. Intraocular hypertension episode was defined as an intraocular pressure ≥22 mmHg measured during hemodialysis.t test (two group comparisons) or analysis of variance for repeated samples with post-hoc analysis using the Bonferroni method (3 or more groups). The variables without a normal distribution are reported as median (percentile 25–percentile 75) and were compared with the U Mann–Whitney test. The absolute value and percentage were used to report qualitative variables and were compared with Chi-squared test or Exact Fisher’s test. The statistical analysis was performed with SPSS version 15.0 (IBM). Considering that there are no previous reports on the effect of the sodium dialysate profile tested in this study, we could not perform an a priori sample size estimation. However, we estimated an achieved statistical power of 0.56 with the results of the present exploratory study , urate nephropathy (n = 3), IgA nephropathy (n = 2), kidney-transplant rejection (n = 4), focal segmental glomerulosclerosis (n = 1), or collapsing glomerulopathy (n = 1). The hemoglobin measured by routinely laboratory measurements in both groups before HDF sessions was 9.7± 3.1 g/dL and after HDF sessions was 12 ± 3.2 g/dL (p < 0.01). The vascular access was arteriovenous fistula (n = 7) or central venous catheter (n = 6).The study included 13 patients (eight men and five women), with mean age 42 ± 7 years old, median HD vintage was 191 (80–436) weeks. End-stage renal disease etiology was systemic lupus erythematosus  and the severity of these changes (IOP change ≥6 mmHg) to increase towards the end of the HDF in both groups. Despite a significantly lower osmolality in the fixed sodium profile at the end of the HDF compared with the beginning of it, there was no difference compared with the individualized sodium profile and the IOP values. Although the results did show a significant difference of sodium after dialysis (137.7 ± 1.3 vs. 138.9 ± 2.5 mEq/L), this difference might be too small to cause any difference of IOP. Probably the variation in IOP relies with much more importance in the production of aqueous humor rather than the outflow from the eye.A previous study found that increased blood glucose levels decrease IOP in hemodialysis . HoweverIOP is given by the flow rate of the aqueous humor that drains in the trabecular meshwork into the Schlemm canal, the input rate of aqueous flow and the episcleral venous pressure. The modification in any of these variables will modify IOP. Whether the output of aqueous flow rate decreases or the input flow rate or the episcleral venous pressure increases, the IOP will also increase . AlthougDuring HDF, the body fluid volume, plasma osmolarity, and electrolyte concentration is corrected . It has The transient increase of IOP during HDF may be subject to a therapeutic intervention as part of the management of patients during the HDF session. However, the changes are rather heterogenous in hemodialysis ,37, for The measuring of IOP is a technical challenge, although applanation tonometry is the most accurate method, it requires the patient to sit and mount a slit lamp inside the hemodialysis center. Furthermore, changes in the position of the patient are routinely made as part of hypotension management, which is a difficulty for the application of applanation tonometry . TonopenAn important advantage in this study is that the same individuals received both sodium profiles with similar HDF prescriptions; therefore, there was a better control of variability among individuals. Another advantage is the frequency of intra-dialysis IOP measurements, which is likely to allow the identification of transient IOP changes. The limitations in this study include the small number of participants  and the measurement of the data from only one HDF session per group. The limited number of volunteers in this study compromises the external validity of our findings, which should be corroborated with further clinical research. On the grounds that there is a great variability in IOP during hemodiafiltration, IOP measurement should be taken into consideration in patients with risk factors of developing glaucoma. It is known that there is a diurnal variation of IOP intraocular pressure, the mean range of fluctuation is 5 mmHg . TherefoThere is a large variability of IOP during HDF. Transient events of intraocular hypertension are highly prevalent during this HDF pilot study and they are not related to sodium concentration nor osmolarity. The results of this exploratory study highlight the need for more pertinent diagnostic and treatment strategies to prevent further visual field damage in dialysis patients.


This study was a comparison of the outcomes of transcanalicular multidiode laser dacryocystorhinostomy (TCLDCR) and external dacryocystorhinostomy (EXDCR) treatment for patients with acquired nasolacrimal duct obstruction.Thirty-one consecutive patients who underwent TCLDCR (TCLDCR group) and 68 consecutive patients who underwent EXDCR (EXDCR group) due to acquired nasolacrimal duct obstruction were enrolled in the study. Follow-up visits were performed on the first day, and at the first week, first month, third month, sixth month, and every six months thereafter. Surgical success was defined as achievement of a patent osteotomy and a successful bicanalicular silicone intubation during the procedure. Anatomical success was defined by observation of a patent osteotomy on lacrimal irrigation, regardless of epiphora. The surgery time and intra- and postoperative complications were noted for each patient.The TCLDCR group had a significantly shorter mean surgery time (27.9±5.5 minutes) compared with the EXDCR group (58.5±12.0 minutes) (p<0.001). However, the mean anatomical and functional rates of TCLDCR  were significantly lower than those of the EXDCR group  (both p<0.001). Two patients had “cheese wiring” damage of the lower canaliculus and 1 patient in the TCLDCR group had a full-thickness skin defect in the medial canthal region. No serious intra- or postoperative complication occurred in the EXDCR group.Although a TCLDCR procedure decreased the surgical time, it had a significantly lower success rate in the treatment of acquired nasolacrimal duct obstruction compared to EXDCR. The decision of the type of surgery should be made based on the cosmetic and success expectations of the patients and the presence of systemic problems. Since described by Toti in 1904 and improved by Dupuy-Dutemps and Bourguet in 1921, external dacryocystorhinostomy (EXDCR) has been the standard surgical treatment of nasolacrimal duct obstruction  and 68 consecutive patients who underwent EXDCR (EXDCR group) due to acquired nasolacrimal duct obstruction were analyzed in the study. Pre-operative detailed ophthalmic examination and irrigation of nasolacrimal duct were performed in all patients. All patients underwent a rhinoscopy before surgery to detect nasal structural anomalies. Patients who did not accept the potential skin scar and those who have systemic problems that hinder the application of general anesthesia admitted to TCLDCR group. Other patients were included in the EXDCR group. Exclusion criteria were canalicular obstruction, previous surgery or traumatic injury of nasolacrimal system, lacrimal system tumor, nasal septal deviation, and middle turbinate hypertrophy. The study was in accordance with the tenets of the Declaration of Helsinki and it was approved by the Institutional Ethics Committee .EXDCR operations were performed under general anesthesia whereas TCLDCR procedures were executed under regional anesthesia with sedation. Regional anesthesia was performed through blocking infratrochlear and intraorbital nerves using 2% lidocaine with adrenaline 0.0125 mg/ml. Two percent lidocaine with adrenaline 0.0125 mg/ml impregnated sponges were placed in the middle meatus of each patient for 10 min before surgery. Topical corticosteroid (dexamethasone 4 × 1) and antibiotics (moxifloxacin 4 × 1), nasal decongestant sprays (oxymetazoline 3 × 1), and oral antibiotics (amoxicilline+clavulanic acid 2 × 1000 mg) were given to each patient for 10 days, postoperatively.For EXDCR, a 10–12 mm straight skin incision was made medially to the angular vein. Orbicularis muscle fibers were dissected bluntly to expose the periosteum and medial canthal tendon. The medial canthal tendon was cut and separated from the bone and the lacrimal sac was exposed. After dissecting the sac from the bone, an osteotomy of approximately 15 × 15 mm was performed by Kerrison punch. Lacrimal sac and nasal mucosa flaps were formed by an H-shaped incision. Then, posterior flaps were excised, bicanalicular silicone tube  was implanted, and anterior flaps were sutured with 6/0 polyglactin absorbable suture. Finally, the orbicularis muscle and skin were also repaired with the same suture.TCLDCR was performed using a 980 nm diode laser , through a 600 μm silica polyamide laser fiber optic. The laser probe was established in the lacrimal sac through the canaliculus. The transillumination of the aiming beam was seen by a nasal endoscope in the nasal cavity across the middle turbinate. The settings were adjusted for each patient being at 10–13 Watts energy, 450 ms pulse, 450 ms pause, and contact mode. Laser energy was applied until an osteotomy with a diameter of at least 5 mm occurred. After the irrigation and removal of the debris, bicanalicular silicone tube was implanted.rd month, 6th month, and every 6 months thereafter. The follow-up time was 3 years. Surgical success was defined whenever a patent osteotomy and a successful bicanalicular silicone intubation were achieved during the procedure. Anatomical success was defined by a patent osteotomy on lacrimal irrigation regardless of epiphora. Functional success was defined by both the presence of a patent ostium on lacrimal irrigation and the complete resolution of epiphora without any episode of dacryocystitis  years in the TCLDCR group and 54.8±15.9 (range: 18–86) years in the EXDCR group. There were 23 female (74.2%) and 8 male (25.8%) in the TCLDCR group and 51 female (75.0%) and 17 male (25.0%) in the EXDCR group. There were no significant differences between the groups concerning age and gender .The clinical characteristics of the groups are given in Two patients had “cheese wiring” damage of lower canaliculus and one patient had full-thickness skin defect in the medial canthal region, within the 1st week postoperatively, in the TCLDCR group. Reconstruction surgery was performed for patient with skin defect and early silicon tube removal was carried out for the two patients with “cheese wiring.” No serious intra- or post-operative complication including excessive bleeding, infection, or canalicular damage occurred in the EXDCR group.The current study revealed that although TCLDCR had significantly shorter surgery time, success rate was significantly lower compared to EXDCR. The TCLDCR gained popularity in the past two decades to treat nasolacrimal duct obstruction with its advantages such as absence of skin incision and scar, shorter surgery time, minimal risk of bleeding, and easy applicability under local anesthesia. However, several disadvantages of this technique including synechia and granulation tissue formation, and common canalicular damage decreases its applicability to a particular group of patients .rd month after the surgery. However, functional success rate reduced to 67.7%, 63.3%, and 60.3% at the 1st month, 1st year, and 2nd year, respectively. Pinto et al. ; preparation and review of the study ; data collection ; and statistical analysis (AB).


Periodontitis is a chronic inflammatory gum disease associated with systemic diseases such as cardiovascular diseases.Aggregatibacter actinomycetemcomitans (Aa) and Porphyromonas gingivalis (Pg) with the stability, based on the aortic diameter, the growth rate and the eligibility for surgical intervention, of patients with abdominal aortic aneurysm (AAA).To investigate the association of systemic blood biomarkers, C-reactive protein (CRP), levels of lipopolysaccharide (LPS), and IgG levels against periodontal pathogens A. actinomycetemcomitans and anti-P. gingivalis IgG levels were analyzed by ELISA, the LPS analysis was performed by using the limulus amebocyte lysate (LAL) test, and plasma levels of CRP were determined using an immune turbidimetric method. The association between these blood systemic biomarkers, AAA features, periodontal clinical parameters and oral microbial profiles were explored. Regression models were used to test the relationship between variables.Patients with stable AAA (n = 30) and unstable AAA (n = 31) were recruited. The anti-The presence of antibodies against Pg and Aa, LPS and high CRP concentrations were found in all AAA patients. The IgG levels were similar in patients with stable and unstable AAA (both for Aa and Pg). Among investigated blood biomarkers, only CRP was associated with AAA stability. The amount of LPS in saliva, supra, and subgingival plaque were significantly associated with the systemic LPS (p <0.05).post-hoc study emphasizes the presence of antibodies against Pg and Aa, LPS and high CRP concentrations in all AAA patients. The presence of Pg in saliva and subgingival plaque was significantly associated with the blood LPS levels. For further studies investigating periodontitis and systemic diseases, specific predictive blood biomarkers should be considered instead of the use of antibodies alone.This  AAA is a chronic degenerative disorder of the abdominal aorta promoted by genetic and environmental risk factors such as smoking, older age, Caucasian ethnicity, and the male gender . The disTreponema denticola (Td), Porphyromonas gingivalis (Pg), Tannarella forsythia (Tf), and Aggregatibacter actinomycetemcomitans (Aa) have been strongly associated with periodontal tissues destruction  ofin of Aa , can alsin of Aa . The micin of Aa  and diabin of Aa . Indeed,in of Aa , coronarin of Aa  as myocain of Aa . Therefoin of Aa , LPS  in abdominal aortic aneurysm patients. Secondary objectives focused on the relationship between periodontal clinical parameters, microbial profile and periodontitis-related blood biomarkers.The aim of this study was to investigate the relationship between blood biomarkers and serologic immunological profiles related to periodontitis , and was registered on clinicaltrial.gov (file number: NCT03767023).After screening the medical files of eligible AAA patients, the participants were invited to the Department of Periodontology where anamnesis and imaging were recorded. A full periodontal clinical and microbiological examination was performed by a single investigator (LS) and the blood samples were collected by the department nurse (JN M). The included AAA patients were divided into two groups according to the stability of the AAA, defined by The AAA dimensions were assessed by measuring AAA diameters  and volumes , as described previously . More prThe number of teeth, the presence of healthy gingiva or gingivitis, diagnosis, and classification of periodontitis  were recorded for each subject according to g. The obtained pellets were dispersed in 200 µl Instagen. DNA was extracted with InstaGene matrix  according to the instructions of the manufacturer. Five microliters of the purified DNA were used for the detection and quantification of Tannerella forsythia (Tf), Porphyromonas gingivalis (Pg), Aggregatibacter actinomycetemcomitans (Aa), Fusobacterium nucleatum (Fn), and Prevotella intermedia (Pi) by real time quantitative polymerase chain reaction (RT-qPCR). The RT-qPCR assay was performed with a CFX96 Real-Time SystemAll microbiological samples were collected by the same investigator (LS). Saliva samples were obtained by collecting 500 µl to 1 ml of unstimulated saliva. The supragingival samples were collected from the four teeth with the deepest periodontal pockets. The sites were isolated with cotton rolls and gently dried with compressed air . All supIn order to induce bacteremia in blood circulation, patients were invited to have a standardized mastication on paraffin  and to bPg and Aa. The tube was kept at room temperature for 30 min and then the serum was obtained by centrifugation at 2,000g at 4°C for 10 min. Aliquots of serum were stored at −80°C. The antibodies against Pg and Aa were analyzed as previously described  for the detection of IgG levels against Aa; and 8 different strains of Pg  broth  aerobically at 37°C in humidified 5% CO2. The Pg strains were grown anaerobically  at 37°C for 18 h in BHI broth supplemented with hemin (5 mg/l) and menadione (1 mg/l) (Sigma). The bacteria were washed once with phosphate-buffered saline  and then fixed overnight at 4°C in 0.5% paraformaldehyde–PBS. Further, the bacterial suspensions were washed three times in PBS, sonicated and brought to an optical density corresponding to an absorbance of 0.15 at 580 nm in ELISA-buffer . For ELISA, 150 ml of the sonicated mixture from the five Aa or from the eight Pg strains were used to coat Microlon ELISA plates . The unspecific bindings were blocked by 5% bovine serum albumin (BSA) in PBS at room temperature for 30 min. Diluted  serum samples were tested in duplicate. The plates were incubated for 2 h at room temperature and washed three times in ELISA buffer. Horse-radish peroxidase-conjugated, goat anti-human IgG  diluted  were then added and the plates were incubated for 2 h at room temperature. Substrate was then added and absorbance values were measured at 450 nm with a multilabel counter .A first blood sample (10 ml red cap) was harvested to quantify the concentration of antibodies levels against escribed . Brieflyrain 381  for the g at 4°C for 10 min. Aliquots of plasma were stored at −80°C. The LPS analysis was performed by using the EndoLISA kit according to the manufacturer’s instructions . Briefly, all samples were defrosted and vortexed to ensure homogeneity. CSE (endotoxin standard Escherichia coli O55:B5) was diluted for the standard curve with dilution factor 10× and dissolved in endotoxin-free water. In total, 100 µl of each preparation was added in duplicate into the respective wells. A blank control was included as a negative control. Next, 20 µl of 6× Binding buffer was added to each well and was covered in aluminum foil and incubated at 37°C for 90 min at 450 rpm. Samples were washed 3× with 150 µl Wash Buffer before 100 µl of the EndoLISA assay reagent was added to each well. Fluorescent signals were measured at 37°C immediately and after 90 min.A second tube of blood (10 ml EDTA plasma tube) was collected for LPS analysis and immediately placed on ice. Plasma was obtained by centrifugation at 2,000A third blood sample (lithium-heparinate) was used for plasma cell analysis and for high sensitivity CRP (hsCRP), using a commercially available kit . This sample was analyzed within 3 h in a clinical chemistry laboratory, by using standardized and automated procedures.post-hoc analysis showed that at least 30 patients had to be included in both stable and unstable AAA groups. Results were expressed as mean ± SD for quantitative variables and as frequency tables (numbers and percentage) for categorical variables. Non-normal distributed variables were log-transformed to normalize their distribution. Comparisons between stable and unstable AAA groups were done by Student’s t-test or Kruskal–Wallis test for continuous variables and by chi-square test or Fisher exact test for qualitative findings. Regression models were used to test the relationship between study variables and results were expressed as regression coefficients and their standard error (SE). A positive or negative regression coefficient indicated respectively an increasing and a decreasing relationship between the two variables. All regressions were adjusted for AAA stability. When the dependent variable Y was quantitative, classical linear regression was applied. When Y was binary, the logistic regression was used and when Y was ordinal then ordinal logistic regression was applied. Results were considered significant at the 5% level (p <0.05). Calculations were done in SAS  version 9.4.The power calculation of the larger study of which this one is a The periodontal characteristics of study patients with stable (n = 30) or unstable (n = 31) AAA were previously described .p = 0.05).The correlation (in terms of regression coefficients) between clinical periodontal parameters, CRP and LPS blood levels, and antibodies concentrations (anti-Aa and anti-Pg) are presented in p = 0.017) while LPS levels did not differ between the groups. Anti-Aa and anti-Pg antibodies were found in similar concentrations in all blood samples of patients with stable and unstable AAA. The relationship between periodontal-specific blood markers, AAA diameters and volumes are shown in The periodontitis-related blood biomarkers analysis according to AAA stability are shown in The relationships between Aa and Pg in saliva, supra-gingival plaque and subgingival plaque and the levels of CRP, LPS and anti-Aa/anti-Pg antibodies in blood are shown in post-hoc study of a cross-sectional, non-interventional, study focused on the relationship between periodontitis-specific blood biomarkers and the AAA stability.This Pg and Aa and also LPS and CRP) although periodontitis was not an inclusion criterion of the study. Additionally, the LPS in blood was associated with the presence of Pg in saliva and subgingival plaque. Therefore, it seems that periodontitis may have induced bacteremia, endotoxemia and systemic inflammation which may eventually have a role in the physiopathology of AAA  of AAA was observed  and the  and AAA . Therefo and AAA , namely  and AAA . Thus, tPg tended to be slightly lower in unstable AAA patients when compared to those of patients in stable AAA (p = 0.08). It could be hypothesized that virulence factors of Pg such as gingipains , would also be of interest in future research. Findings should therefore be interpreted cautiously.This study suffers from some limitations, particularly because of the post-hoc study emphasizes the presence of antibodies against Pg and Aa, LPS and high CRP concentrations in all AAA patients. However, among investigated blood biomarkers, only CRP was associated with AAA stability. The presence of Pg in saliva and subgingival plaque was significantly associated with the blood LPS levels. For further studies investigating periodontitis and systemic diseases, specific predictive blood biomarkers should be considered instead of the use of antibodies alone.This The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the human subjects’ ethics board of the University Hospital of Liege, Belgium (B707201421977). The patients/participants provided their written informed consent to participate in this study. Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.Conceptualization ; Investigation (LS); Methodology ; Supervision ; Roles/Writing - original draft (LS); Writing – review  Validation .The authors received no financial support for the research, authorship, and/or publication of this article.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Hydrogen cyanide(HCN) is known to react with complex organic materialsand is a key reagent in the formation of various prebiotic buildingblocks, including amino acids and nucleobases. Here, we explore thepossible first step in several such processes, the dimerization ofHCN into iminoacetonitrile. Our study combines steered ab initio moleculardynamics and quantum chemistry to evaluate the kinetics and thermodynamicsof base-catalyzed dimerization of HCN in the liquid state. Simulationspredict a formation mechanism of iminoacetonitrile that is consistentwith experimentally observed time scales for HCN polymerization, suggestingthat HCN dimerization may be the rate-determining step in the assemblyof more complex reaction products. The predicted kinetics permitsfor iminoacetonitrile formation in a host of astrochemical environments,including on the early Earth, on periodically heated subsurfaces ofcomets, and following heating events on colder bodies, such as Saturn’smoon Titan. However, such mechanisms appear to correspond toeither prohibitively high activation barriers31 or unfavorable thermodynamics28 to allow for meaningful  reaction ratesin the interstellar medium. Computational studies of HCN dimerizationon ice grains have predicted similar unfavorable kinetics.32 A reaction network study suggests that the cyanideradical and methylene imine might be important precursors to iminoacetonitrilein molecular clouds.33The formation routes to iminoacetonitrile can be expected to dependgreatly on the chemical and physical environment. Iminoacetonitrilehas recently been detected in the interstellar molecular cloud SagittariusB2, using the Green Bank Telescope, and in the molecular cloud G +0.693, using the Institute for Radio Astronomy in the Millimeter Range(IRAM) 30 m telescope.35 at concentrations ranging from0.01 M to pure HCN (26.2 M).18 However, while it hasbeen hypothesized that iminoacetonitrile is required for the formationof many larger molecules,36 the compound has never been directly observed during HCN polymerizationexperiments. Iminoacetonitrile has been generated by pyrolysis ofcyanoformamide salts and has been well characterized in the gas phaseand in argon matrices by vibrational spectroscopy, mass spectrometry,and UV photoelectron spectroscopy.39 A 1H and 13C nuclear magnetic resonance spectroscopy study has alsoshown that iminoacetonitrile polymerizes rapidly above 233 K.38 Ferris and colleagues have shown that N-alkyliminoacetonitriles in aqueous cyanide solutions reactto form maleonitrile derivatives, which are, in turn, likely polymerintermediates.36In this work,we focus on explaining the formation of iminoacetonitrilein neat liquid HCN. HCN polymerization reactions in polar solutionsare known to be base-catalyzed and to proceed in a wide range of temperatures(195–373 K)40 Thatstudy relied on an implicit solvent model for water and predicteda reaction barrier , which is markedly lower than thecorresponding uncatalyzed pathway , but prohibitively large for reactions at ambient andlower temperatures.To the best of ourknowledge, there has only been one previoustheoretical study of base-catalyzed iminoacetonitrile formation inpolar solution by Kikuchi et al.41 a factor of 1.7 larger than for water at thesame temperature (85.8).42 Moreover, HCNis capable of donating and accepting strong directional hydrogen bonds.These properties of HCN share striking similarities with those ofwater and are suggestive of an unexplored potential for self-catalysis.Finally, the considered reaction mechanism involves charged speciesthat interact both strongly and directionally with the environment.Our studyof this condensed phase chemistry relies on moleculardynamics simulations. The use of dynamics simulations allows for theexplicit consideration and sampling of the solvent environment andis important for three reasons: first, HCN is an exceptionally polarmolecule. The dielectric constant of liquid HCN is 144.8 at 278 K,path-collective variables to define reactioncoordinates. These variables are unbiased in the sense that they areonly based on aspects of the coordination pattern of reactants andproducts. In our exploration, the reaction path is defined by an interpolationbetween the reactant and the product states. We use two path-collectivevariables: the s coordinate, which describes a structure’sposition along the reaction path, and the z coordinate,which describes how distant a given structure is from the path.43 The simulation temperature of 278 K was chosento be in the middle of the liquid range of HCN, which is close tothat reported in recent studies on HCN polymerization.18We here rely ondensity functional theory (DFT) metadynamics simulations to identifyviable formation routes of iminoacetonitrile from HCN while minimizingbias with regard to mechanism or solvent role. Our metadynamics approachrelies on 1 The reaction proceeds throughthe formation of a carbon–carbon bond (dCC) betweenHCN and a cyanide anion that occurs concurrently with a proton transfer(dNH) from a second HCN, which, in turn, regenerates thecatalyzing base. Representative snapshots from our simulations displayinggeometries of reactant I, transition state TS, and product II are shown in TS has been identifiedthrough committor analysis, as the structure from which multiple simulationtrajectories reach reactant and product basins with equal probability. The uncertainty range here refers tothe error due to limited sampling during simulations . The Gibbs energy difference between the two structures is smalland within the accuracy of the methods we use. Consequently, the specificconformation of iminoacetonitrile does not affect our conclusion onthe reaction mechanism nor its kinetics.Our simulations predict a relative Gibbs energyof −7.1± 0.8 kcal/mol for the overall reaction, suggesting that themechanism is thermodynamically allowed  used in typical HCN polymerization experiments tendsto be low (0.03–0.5 M),49 we can conclude that(a) cation coordination is not necessary for iminoacetonitrile formationand that (b) active involvement of solvent molecules (and explicitconsideration of these interactions in modeling) is necessary to explainthe base catalysis of this reaction.Our barrier estimation is markedlylower than that by Kikuchi etal. , who used an implicit solvation model of waterto study the same nucleophilic attack but in the presence of ammoniumand hydronium cations.50 Product compositionsof such experiments also vary significantly with reaction conditionssuch as initial concentrations,51 the presenceof oxygen, and the degree of conversion.52 These often ill-understood products may play important roles inastro- and prebiotic chemistry. As a consequence, definite identificationand characterization of products of HCN polymerization are both importantand exceedingly challenging. Here, we aim to better understand thepotential role of iminoacetonitrile in the formation of HCN polymers.We do so by comparing our theoretical predictions of iminoacetonitrileformation kinetics with observed reaction rates in HCN polymerizationexperiments. Of course, the rate of HCN dimerization is not necessarilythe same as that of its polymerization or processes leading to otherreaction products. Implicit in our following comparison with the experimentare two assumptions: first, that the initial step in HCN’sbase-catalyzed polymerization proceeds through the formation of iminoacetonitrile,and, second, that this initial step is rate-determining. Aside fromits known rapid polymerization into a “red-brown-black material”above 233 K,38 the precise reactivity andscope of iminoacetonitrile chemistry are largely unknown and willbe the subject of future work.Polymerization of HCN typicallyresults in complex reaction mixtureswith many high-molecular-weight products that have poor solubilityin common solvents.In 18 and He et al.19 at room temperature. The temperature dependence of the tetramerizationrate measured at 283–313 K by Sanchez et al.17 suggests an apparent activation barrier of 20.5 kcal/mol,also in close agreement with our barrier estimate. Our predicted reactiontime scale also agrees well with aqueous HCN polymerization experimentsperformed by Miller and colleagues34 aswell as Sanchez et al.2 near 250 K. Wehere remind that aqueous HCN solutions that are close to or belowthe eutectic point (250 K) form a eutectic phase in which HCN is concentrated(to 74.5 mole percent).55 We can speculatethat effects associated with such a phase may be one reason for thesometimes strikingly different experimental results near the eutectictemperature. For example, whereas Sanchez et al. have reported theformation of the HCN tetramer diaminomaleonitrile after days at 251K,2 Marin-Yaseli et al. were unable toobserve meaningful reactivity after a month of reaction time at 253K.49Close to a simulation temperatureof 278 K, a barrier of 22 kcal/molshould proceed on the order of days 4. In oth34 implies a lower reaction barrier in the largely frozensolution compared to that predicted for the liquid . The latter comparison implies that crystalsurfaces and defects in frozen HCN solutions may be better polymerizationcatalysts compared to the liquid system studied herein. Relatedly,reaction kinetics studies performed between 323 and 363 K by Mas etal. suggest that different reaction mechanisms, and barrier heights,are at play at higher temperatures.53As expected, our approximateextrapolation in –8–10–6 cm2/s and much of what was produced is believed to havebeen deposited on Earth’s surface (30 million tons/year).14 However, despite a substantial production inthe atmosphere, the concentration of HCN in Earth’s early oceansis likely to have been much too low to allow for polymerization.56 Eutectic freezing could have occurred in shallowpools or under very harsh glacier conditions and corresponds to aplausible route to concentrated aqueous HCN.23 Our calculations predict that the 250 K eutectic temperature ofa HCN–water mixture should allow for the formation of iminoacetonitrile,and possibly subsequent HCN polymerization, over a time scale of months.The time scale required for the studied dimerizationdrops rapidly with temperature c.f., 4, with i4 haveestablished comets as another environment rich in HCN, and the possibilityof HCN polymerization in comets has been pointed out by many . Th8 The temperatureof Titan’s atmosphere varies drastically with altitude butranges from 200 K at 300 km59 to ∼94K at the surface.60 The formed HCN canbe expected to deposit on the surface and in the hydrocarbon seasof Titan in large amounts.61 The abundance of HCNdissolved in the hydrocarbon seas is uncertain but predicted to below63 and orders of magnitudes smaller than thelowest reported molar concentration used in aqueous HCN polymerizationexperiments (0.02 mole %).17 The cyanideanion, the catalyst in our study, is believed to be the most commonanion in the chemical haze surrounding the large moon.64 However, provided that we assume a similar reactionbarrier in the hydrocarbon seas of Titan as in neat HCN, the low surfacetemperature (<94 K65) would hinder thereaction from occurring within relevant time scales. Whereas solid-stateequivalences to the reaction considered herein might prove operableover very long time scales, our current simulations describe HCN inthe liquid state. In other words, for the iminoacetonitrile formationreaction considered in this work to be feasible on Titan, temporaryheating of HCN surface deposits or melting of atmospheric aerosolswould be required. Such heating might be generated by impact eventsor, possibly, coincide with runaway exothermic polymerization of acetylene,another main product of Titan’s atmospheric chemistry.68HCN is one of the main products of the atmosphericphotochemistryon Saturn’s moon Titan.HCN in the liquid state is complex, featuringan extremely largedielectric constant, strong directional intermolecular interactions,and acid–base equilibria that allows for its participationin catalytic cycles. Our simulation of base-catalyzed dimerizationof HCN explains the formation of iminoacetonitrile in the liquid state.The time scales estimated for iminoacetonitrile formation are similarto those observed for HCN polymerization experiments, which suggeststhat the dimer formation may be the rate-limiting step for a hostof subsequent reaction chemistry, including polymerization. The predictedrate of formation of iminoacetonitrile allows for its formation inseveral environments and supports a potential central role of themolecule in astro- and prebiotic chemistry. Barring detection by nuclearmagnetic resonance and vibrational spectroscopy or successful trappingexperiments, we suggest that the laser desorption ionization of frozenreaction mixtures coupled to time-of-flight mass spectroscopy mayprovide one route to experientially proving the role of iminoacetonitrilein HCN polymerization.69 at thePerdew–Burke–Ernzerhof (PBE)70/DZVP–Goedecker–Teter–Hutter (GTH)71 level of theory, including the D3 dispersioncorrection by Grimme et al.72 The presenceof anions and hydrogen bonds in the simulations merits the inclusionof diffuse functions in the basis set. However, such functions affectrelative energies by 1.1 kcal/mol or less (Supporting Information Table 1) and were omitted in the interest of reducingcomputational costs. The GTH pseudopotential73 was used with a cutoff of 280 Rydberg. Liquid HCN was simulatedin a cubic box with a side length of 15.94 Å subjected to periodicboundary conditions. The number of molecules were tailored to 64,including one cyanide anion, so as to accurately reproduce the densityof HCN at 278 K (0.709 g/cm3).74 The system was propagated in an NVT ensemble at 278 K with a 0.5fs time step. The steered simulations and the subsequent committoranalysis simulations were performed with the canonical sampling throughvelocity rescaling thermostat75 and a thermostattime constant of 50 fs.Ab initio molecular dynamics simulationswere performed with CP2K v6.1s and z path-collective variables were constructedfollowing the exampleof Branduardi et al.43 (see Supporting Information Section 4). The path-collectivevariables used during the metadynamics76 simulations were defined based on the bond topology of the carbonin the cyanide anion, through the distance function by Pietrucci andSaitta.77 The PLUMED library version 2.5.078 was used to steer the dynamics simulations.Two reference structures were used: a cyanide dissolved in HCN anda solvated iminoacetonitrile, leaving the path between those two statesopen for exploration. Following a 5 ps equilibration, the referencestructures were simulated for 20 ps prior to an analysis of theircoordination patterns. The metadynamics simulation was initiated fromthe reactant state.The 79 Ten out of 20 trajectories starting from the TS structure endedup in the reactant basin and in the product basin, respectively. Afree-energy profile of the reaction was then constructed using umbrellasampling80 along the s coordinate using configurations from trajectories obtained in thecommittor analysis as starting points. The umbrella sampling simulationswere run for 11 ps after 1.5 ps of equilibration. The force constants, k, used for the harmonic potentials in these windows (SupportingInformation Table 2) were chosen so that 2 is the varianceof the s coordinate in a typical simulation. Theumbrella sampling windows were spaced at most 0.025 s coordinate unitsapart (corresponding to 3% of the distance between the s coordinates of reactants and products). Together with the chosenforce constants, this separation provided a good overlap of the samplingin different windows . The weighted histogram analysis method (WHAM)81 version 2.0.9 was used to retrieve the free-energyprofile of the reaction. Uncertainties in the energy estimates wereestimated using a block averaging method in WHAM.82The transitionstate (TS) of the reaction observed in the metadynamics simulationwas identified through a committor analysis.E. This conclusion was reached by comparingthe thermal corrections ΔG – ΔE when calculated with PBE-D3 and the more accurate B3LYP84-D3 method (Supporting Information Section 1). Hybrid exchange–correlation functionals, such as B3LYP,represent the most accurate level of theory that can be practicallyimplemented on our system. The B3LYP functional has reported averageerrors of 2 and 4 kcal/mol for association reactions and hydrogentransfer, respectively.48As a necessarycompromise between accuracy and computational cost, all simulationswere done using the PBE-D3 functional. The largest contribution tothe error in the free-energy profile can be attributed to inherenterrors in the potential used to calculate the electronic (Born–Oppenheimer)energy ΔEcorr, was added to the free-energyprofile ΔGPBE, according to ΔGfinal = ΔGPBE + ΔEcorr, where ΔGfinal is our best estimate of the relative Gibbsenergy . The correctionterm, ΔEcorr, was obtained by averaging105 energy evaluations of configurations from the umbrella samplingwindows corresponding to reactant ,transition state , and product  at two levels of theory. The configurationswere taken at 0.1–0.2 ps intervals from the trajectories. ΔEcorr was calculated as ΔEcorr = ΔEB3LYP-D3,avg – ΔEPBE-D3,avg,where the latter two terms are average energy differences calculatedwith B3LYP-D3 and PBE-D3, respectively. The ΔEcorr calculations were performed with the Vienna ab initiosimulation package (VASP) version 5.4.4.85 Standard projected-augmented wave potentials86 were used together with a plane-wave energy cutoff of 800eV and a convergence criterion of 10–4 meV/atom.All VASP calculations were done at the Γ point only.To reduceoverall errors in the free energy, an electronic correction term,Δ


Oncorhynchus mykiss) and maraena whitefsh (Coregonus maraena), have not been adequately studied as a comparative study and are therefore insufficiently understood. For this reason, our aim was to examine muscle growth in more detail and provide a first complex insight into the growth and muscle metabolism of these two fish species at slaughter size. In addition to skeletal muscle composition (including nuclear counting and staining of stem and progenitor cells), biochemical characteristics, and enzyme activity  of rainbow trout and maraena whitefish were determined. Our results indicate that red muscle contains cells with a smaller diameter compared to white muscle and those fibres had more stem and progenitor cells as a proportion of total nuclei. Interestingly, numerous interspecies differences were identified; in rainbow trout muscle RNA content, intermediate fibres and fibre diameter and in whitefish red muscle cross-sectional area, creatine kinase activity were higher compared to the other species at slaughter weight. The proportional reduction in red muscle area, accompanied by an increase in DNA content and a lower activity of creatine kinase, exhibited a higher degree of hypertrophic growth in rainbow trout compared to maraena whitefish, which makes this species particularly successful as an aquaculture species.The growth of fishes and their metabolism is highly variable in fish species and is an indicator for fish fitness. Therefore, somatic growth, as a main biological process, is ecologically and economically significant. The growth differences of two closely related salmonids, rainbow trout ( Coregonus maraena, Bloch 1779) and rainbow trout  belong to the family of Salmonidae and are ecologically important for the Baltic Sea region and economically important for aquaculture production. At the beginning of the 20th century, maraena whitefish was one of the most important food fish in the Baltic Sea region. A combination of extensive fishing, eutrophication of its estuary spawning areas, and habitat fragmentation through anthropic obstruction has brought the population of C. maraena to the edge of extinction. Intensive restocking operations started in the 1990s in Finland and later other Baltic Sea countries joined [C. maraena could be stabilized, but is still on the red list as a vulnerable species [C. maraena, our objective was to examine the skeletal muscles of this fish species in more detail at slaughter size under aquaculture conditions. The slaughter size and with it the carcass weight is fixed by the market and comparing animals at the same body weight  is of greater interest for meat production in different fields [Maraena whitefish,  and red  muscle fibres can be distinguished, whereas pink muscle fibres  represent an intermediate fibre type . The bulMyogenic (stem) cells undergo sequential developmental steps including lineage commitment of progenitor cells, several rounds of proliferation, and initiation and maintenance of the myogenic differentiation program . AlthougThe goal of this present study is a deeper evaluation of the dorsal and ventral myotomal mass and the myogenic properties during fattening stage. To this aim, analyses on nucleic acid content, muscle specific enzyme activity, myofibre type distribution and nuclear density as well as myogenic marker expression were conducted as all factors provide important information for muscle growth and keeping conditions in aquaculture. Altogether, the gained results will give a first insight into muscle metabolism of salmonid species at slaughter size.C. maraena) and rainbow trout . Within this study, samples from adult animals from maraena whitefish  were us4+, NO2- and NO3- concentrations were measured weekly, pH value, temperature and oxygen saturation were constantly recorded. Dissolved oxygen was maintained > 8 mg/l and all physical water parameters were kept in optimal range.Water quality was assured by continuous purification and disinfection . Water parameters including NHAnimals were transported alive in a transport box with an additional oxygen provision from the Institute of Fisheries in Born to the Leibniz Institute for Farm Animal Biology (FBN) in Dummerstorf, Germany. This study followed international, national and institutional guidelines for animal treatment and sacrifice and complied with relevant legislation. Fishes were euthanized according to the animal welfare law Directive 2010/63/EU and TierSchG § 4(3) before slaughtering. Fishes were stunned by a beat on the head and were directly killed by bleed cut in the heart as well as cutting of the spinal cord posterior to the head. Morphometric parameters  were measured and samples of the right epiaxial  and hypaxial  muscles were collected for biochemical analyses. In order to obtain a complete cross-section for the histological and immunohistochemical analyses, the samples were taken from the caudal penduncle.™ BCA Protein Assay Kit  according to manufactures instructions. The supernatant of samples was as well used to quantify the activity of specific muscle enzymes. The CK-NAC-Hit kit  was used to measure activity of creatine kinase (CK). The activity of lactate dehydrogenase (LDH) and isocitrate dehydrogenase (ICDH) were assessed by measuring NAD(P)H conversion using a NADPH standard curve. All amounts and activities are given per g muscle.The dorsal and ventral muscle tissue from 10 animals per species, snap-frozen in liquid nitrogen and stored at -80°C. Subsequently, 100 mg of each sample were pestled and homogenized in 0.01 M potassium phosphate buffer containing 1 mM EDTA in order to analyse the supernatant as previously described in Stange et al. (2020) ; dilutio2 area and in the white region, approx. 1.3–1.7 mm2 were counted and extrapolated to the total area size.Since the red region of the muscle is mainly present in the lateral region, for histological analysis there was no need to distinguish between ventral and dorsal muscles. Histological slices of the cross-section of the postanal area were made from six animals of each species. Tissue was shock frozen for 1 min in a combination of precooled isopentane and liquid nitrogen. Afterwards, samples were stored at -80°C. 10 μm thick serial transversal sections were prepared at the cryostat microtome (MC 1950) at -20°C and mounted on slides. Slices were stained via Haematoxylin and Eosin (H&E) as well as NADH-diaphorase staining by routine procedures . For ill2O2 in methanol solution to inhibit endogenous peroxidase activity and then blocked with 10% goat serum in PBS for 1 hour. After primary antibody staining, the slices were washed three times in PBS for 5 min and incubated with the secondary antibody  and Alexa Fluor 488 goat anti rabbit IgG H&L  or Alexa Fluor 594 goat anti rabbit IgG H&L  for 90 min at RT. After rinsing with PBS twice for 5 min, sections were mounted with Roti Fluor Care DAPI . Fluorescence samples were imaged using a Leica DM400B fluorescence microscope at 100x magnification in combination with Leica DFC320 camera and pictures were analysed by software Cell^D by counting the stained and unstained nuclei with a pen counter. For nuclei count, 1.0–1.8 mm2 of the red region and in the white region, approx. 2.1–2.8 mm2 were analysed.For immunhistochemical staining frozen tissue sections were fixed with 4% PFA for 10 min at room temperature (RT) and washed twice with phosphate-buffered saline (PBS) for 5 min. Slices were incubated in blocking buffer , 0.5% Triton X-100 in PBS) for one hour at RT. Sections were incubated with primary antibodies: rabbit polyclonal anti-Pax3 antibody  or rabbit polyclonal anti-Myf5 antibody  in PBS overnight at 4°C. Slices incubated with mouse monoclonal anti-Pax7 antibody  overnight were treated before staining in 3% HData are presented as mean ± S.E.M. (Standard error of the mean). For statistical analysis of interfibre and interspecies comparision two-tailed Student´s t-test, two sited ANOVA with Tukey´s test for multiples testing was performed with SAS software version 9.4  for the comparison of ventral and dorsal tissue as well as red and white muscle tissue within a species as well as for the interspecies comparison of the same muscle areas and type. Significant differences was determined as p < 0.05.Muscle tissue samples from maraena whitefish and rainbow trout from the dorsal and ventral region were investigated regarding nucleic acid and protein amount , as wellvs. 4.98 ± 0.35°IU/g) and dorsal muscle tissue (7.61 ± 0.76°IU/g vs. 4.99 ± 0.21°IU/g) of rainbow trout compared to maraena whitefish   . In the sh: 63%) . In contsh: 63%) .2 of muscle type, between whitefish and rainbow trout  and in rainbow trout 1.13% ± 0.13 (vs. white muscle: 0.26% ± 0.12). The comparison between the species showed that their muscles are much alike regarding the expression of the selected markers. However, a significantly higher proportion of Myf5+ cells was found in red muscle of rainbow trout (11.63% ± 1.02) vs. maraena whitefish (7.18% ± 1.17)), , scientists aim to compare similar body weights and therefore these animals are grown until this goal is achieved –50. It iTo reach this aim, muscle characteristics like RNA, DNA, and protein content, as well as muscle fibre size and type within the muscle tissue of rainbow trout and maraena whitefish were analysed as well as the cells´ presence, size and myogenic marker expression. Furthermore, we examined muscle specific enzyme activity in order to take a first look at the metabolic cycle through which muscle growth takes place.Rainbow trout and maraena whitefish, the two salmonid species studied here, reach slaughter size very quickly. However, considerable growth differences are present within the salmonids. To compare muscle tissue characteristics of both species, the typical sale size of around 30 cm body length was chosen. Since the size of the animals is crucial for the food industry, the goal is to achieve these sizes in as little time as possible whereas the influences of this selection process on muscle tissue has not been investigated in detail in fish. The trout reached sale size after already one year whereas the whitefish needed two years for reaching the same size. Within this study, both fish species showed nearly identical total length, although in trout the circumference was significantly larger and with 25% more weight also significantly heavier than maraena whitefish. These morphological parameters indicate a faster growth of rainbow trout under the investigated conditions. Results can be explained by different domestication status of both species. Rainbow trout breeding programs for several decades lead to well adapted aquaculture strains with fastest growth rates combined with phenotypic characteristics and vitality. Contrary, adaptations of maraena whitefish for intensive aquaculture generated only from a few generations focus still on optimal rearing conditions, healthy fish stock and optimization of artificial breeding. Fast growing breeding lines will be selected in the next years on this base.In a next step, we investigated whether the rapid growth seen in both salmonid species was mainly caused by hyperplasia or hypertrophy. The presence of small, newly formed muscle fibres is an indicator for hyperplastic growth . In contAdditionally, a significantly higher DNA and RNA content was seen in trout than in maraena whitefish muscle tissue. Consequently, the RNA/DNA ratio in trout ventral and dorsal muscles was double compared to the whitefish. Several studies in recent years have shown that the cytoplasm/DNA ratio can vary during hypertrophic growth and therefore the DNA content is not a fixed factor of fibre size , 53. It Taken together, the larger body circumference and the proportional reduction in red muscle area accompanied by the increase in DNA content and number of cells with high diameter argue for a higher degree of hypertrophic growth in trout compared to whitefish.Coregonus sp. very high LDH activities in the white muscle which the authors explained by their sprinting properties [Naturally, trout are adapted to running water and the red muscle propels the fish at relatively high speeds. The pink or “intermediate white muscle”is not active during low swimming speeds in aquaculture , but becoperties . Our datFor hypertrophic growth, myofibres need to merge and myonuclei contribute to these fibres , 65. Thestatus quo of the cells within fish muscle tissue, we selected these three crucial muscle specific markers for analysis. Pax3, Pax7, and Myf5 could be successfully detected in histological sections, however the percentage of positive cells was rather low. The vast majority of cells did not express one of the selected markers. Both species were mostly comparable regarding myogenic marker expression. Both markers are downregulated during myogenic differentiation process and higher levels are indicative for a higher amount of non-differentiated cells.Pax3/7 and Myf5 expression is found in satellite cells committed to the myogenic lineage as well as in myoblasts , 67 and In accord with that, significantly lower CK values were found in trout muscle compared to maraena whitefish. Whereas the enzyme is not present in myoblasts, CK will be activated within terminal differentiation of myocytes and can be used as a measure for myogenic differentiation . ElevateThis study investigates the muscle characteristics of two fish species when reaching market sale size (slaughter size) reared under commercially used conditions. At carcass weight, the skeletal muscle of these fishes have not been studied in detail until now and previous studies used different methods, complicating the comparison to our findings. The holistic approach shown here allows for a first comparison under practical considerations by including the potential influences of age and growth conditions (e.g. water temperature) on muscle development. The faster growth rate of the trout under the investigated and commercially used rearing conditions makes this species popular and successful as an aquaculture fish. In the future, detailed studies will have to clarify the specific influence each of the aquaculture parameters on growth (kinetics) and identify optimal possibilities for modulation. To unravel underlying mechanisms for differing growth rates and muscle properties is a prerequisite to optimize aquaculture conditions in a species-specific manner. Further investigations on cell properties, including analyses of additional myogenic markers, and their energy metabolism and differentiation potential are needed in the future, as well.