This paper presents a rapid HPLC method for the separation of isomaltulose  from other common edible carbohydrates such as sucrose, glucose, and maltodextrins, which are commonly present in food and dietary supplements. This method was applied to determine isomaltulose in selected food supplements for special diets and athletic performance. Due to the selectivity of the separation system, this method can also be used for rapid profiling analysis of mono-, di-, and oligosaccharides in food. O-α-d-glucopyranosyl-d-fructose) or Palatinose is a disaccharide where glucose and fructose are joined by a 1,6-glycosidic bond  into glucose and fructose as quickly as sucrose. This has prompted ongoing clinical trials on isomaltulose in dietary supplements for physical performance ,13,14. IIn such mixtures of saccharides, determination of isomaltulose is difficult not only because of a limited choice of separation techniques that should separate many isomers of strongly polar compounds, but also due to the absence of good UV-chromophores for their detection.A common choice is a refractive index detector (RID) which, however, has a low sensitivity and runs can also be time-consuming due to the isocratic elution required. Alternatively, an evaporative light-scattering detector (ELSD) can be advantageously applied for sugar analysis. The ELSD has become a widely utilized detector due to its extraordinary properties; unlike the RID, the ELSD is compatible with gradient elution, which is necessary for chromatographic analysis of complex samples  . With thMethods for determination of carbohydrates are briefly summarized in the following paragraphs. However, polysaccharides analysis is excluded because it is outside the main purpose of this work and related methods can be found elsewhere ,30.Traditional methods for mono- and disaccharides determination (sum of saccharides) are volumetry and polarimetry; after the analyte derivatization, photometry or fluorimetry can be applied . A disadHigh-performance thin-layer chromatography (HPTLC) of oligosaccharides from inulin has been shown by Prošek et al. . VojvodiDetermination of monosaccharides in complex sugar mixtures by gas chromatography (GC) after derivatization to silyloximes is often applied . GC withGel electrophoresis methods for fluorophore-labeled carbohydrates were reviewed by Jackson . DetermiNowadays, separation of low molecular weight sugars is usually performed by HPLC in two prevailing modes: (i) anion or cation ion-exchange chromatography , or (ii) HILIC on amino-propyl modified silica gels.2+–cycle. The method typically uses a strongly basic eluent on ion-exchange resins with pulsed amperometry detection (HPAEC-PAD). HPAEC-PAD with isocratic elution has been recently applied for fermentable short-chain carbohydrates profile (FODMAP) in cereal-product ingredients [In the case of ion-exchange chromatography, the analytes are retained between the hydroxyl groups of the saccharides and ionic components of the column stationary phase. In 1980, Mopper et al. reported on the determination of various sugars after separation on the IEC column after complexation with borate, and post-column derivatization with ethylenediamine for fluorometric detection . Recentlredients , with grredients .Under the HILIC mode, separation methods on amino-propyl stationary phase usually operate with a mobile phase consisting of an excess of acetonitrile (less than 20% water). The excess of acetonitrile is also typical for LC-MS compatible buffers, which are also suitable for detection by ELSD. The buffers may be formate, acetate, or carbonate with an ammonium counter-ion. In 1998 Hernández et al.  comparedGrembecka et al.  developeA recent review on disaccharide determination did not cover isomaltulose , perhaps2+–catex column [Since Japan was the first to introduce Palatinose commercially, the first article on isomaltulose separation and determination appeared there in 1992 . The autx column . The autx column  reportedChromatographic separation saccharides and alcoholic sugars, including isomaltulose, in 60 min on zeolites for preparative purposes has been reported . RecentlThe above-described growing use of isomaltulose in human medicine, as well as in the field of nutritional and physical performance supplements, raises the need for separation methods for the rapid determination of isomaltulose in samples where other common carbohydrates  are present.This paper presents a fast gradient elution method for determination of isomaltulose by HPLC under Hydrophilic Interaction Liquid Chromatography mode (HILIC) on a polyol stationary phase with ELSD. The retention time of isomaltulose is 6 min, which is a shorter time than in most separation methods published so far.The presented method (HILIC-ELSD) has several advantages: (i) derivatization of the sample is not necessary, (ii) the polyol stationary phase is able to strongly retain analytes without the need for significant concentrations of either salts or ion-pairing reagents, (iii) saccharides (often isomers) are separated from a mixture (can be identified and determined), (iv) gradient elution necessary for real samples can be easily applied (using MS-compatible buffers), and (v) the buffers are compatible with mass-flow dependent detectors. The applicability of the method is demonstrated on determination of isomaltulose in twenty dietary food supplements (gels and drinks) available on the market.An HPLC system Dionex Ultimate 3000 , with a Varian 380-LC  evaporative light-scattering detector was employed. The column was HALO Penta-HILIC , 150 × 4.1 mm, with particles diameter 2.7 μm. No precolumn was used.Chemicals were purchased from various sources: acetonitrile of a gradient-HPLC grade, ammonium formate, ammonium bicarbonate, ammonium acetate, formic acid, acetic acid, standards of glucose, isomaltulose, saccharose, cellobiose, trehalose were from Sigma-Aldrich . Maltodextrin DE 12-19 was purchased from 4fitness.cz .Samples containing isomaltulose available in the market of the Czech Republic were bought from various producers: Rossmann—Burgwedel, Germany; Heaven Labs—Prague, Czech Republic; Penco—Prague, Czech Republic; Nutrend—Olomouc, Czech Republic; Extrifit—Dolní Újezd, Czech Republic; Amix—Mnichovo Hradiště, Czech Republic; Energikakan—Skeppsbron, Sweden; Vitamin center—Bologna, Italy; for additional information, see Stationary phase: HALO Penta-HILIC 4.6 × 150 mm column (AMT), particles 2.7 µm. Mobile phase: 35 mM ammonium formate with acetonitrile. The elution gradient : 0–5 min 18% B, 5–8 min 18–35% B, 8–10 min 35% B, 10–12 min 35–18% B, 12–15 min 18% B.Flow rate 2.0 mL/min, thermostat temperature 10 °C, injection volume 4 µL. The detector was an ELSD with the following parameters: gain 2, smooth width 10, evaporator temperature 40 °C, nebulizer temperature 40 °C, nitrogen flow-rate 1 slm (standard liter per minute).Statistical calculations  were carried out in MS Excel .Initial experimental conditions for the method optimization were based on recently published papers on study of monosaccharides and disaccharides retention on a polyol stationary phase ,26,27,55v/v).In HILIC mode, in contrast to reversed phase HPLC, water (or aqueous buffer solution) exhibits the highest elution strength. Firstly, retention behavior of isomaltulose under isocratic elution with a mobile phase containing a mixture of acetonitrile and water was studied. Water content varies in the range of 8–30%  was in accordance with previous results : it was y-axis) in On the other hand, dotted lines (right φ is the volume fraction of a stronger solvent (in this case water) in the mobile phase, S is the slope when fitted with a linear regression model, and Wk’ is the retention factor for the weaker component (organic solvent) only. Thus, a plot of log k’ vs. φ should yield a straight line. If, on the other hand, the retention is due to adsorption then a plot log k’ vs. log φ should yield a straight line, since the best equation describing this situation is the Snyder-Soczewinski equation:Bk’ is the retention factor with the pure solvent B, SA and Wn are the cross-sectional areas occupied by the solute molecule in the surface and solvent molecules, respectively (the ratio SA/Wn can be considered as a constant).To clarify which of the mechanism prevail under our experimental conditions, we carried out an analysis of the retention data based on the following idea: if retention is governed by the partition mechanism, then an empirical equation can describe the process :(1)logk′Since the courses observed on k′ and 1/T should be linear according to van’t Hoff equation:H° is standard retention enthalpy change, ∆S° is standard retention entropy change, R is the gas constant, T is the column absolute temperature.Additional information about thermodynamics of the separation process can be obtained from a partitioning chromatography model: if retention is controlled by partitioning between the mobile phase and the immobilized layer of water on the stationary phase, then the relationship between ln φ < 0.15). Negative retention enthalpy values indicated an exothermic process. The enthalpy values derived decreased as the polarity of the mobile phase (water content) decreased, which could indicate the existence of strong specific (secondary) interaction between analytes and functional groups of the stationary phase. Calculated negative standard enthalpy changes were from −0.1 (φ = 0.3) to −17 kJ/mol (φ = 0.1); so they were maximized  at higher water content, where they exceeded −10 kJ/mol, which is a value typically observed in reversed-phase chromatography [Van’t Hoff plots in tography .From previous experiments, we can conclude that the water content in the mobile phase and the column temperature are important parameters that affect the retention of polar analytes in HILIC. A low water content and a low temperature would mean a high retention and resolution, and would also produce a dramatic drop in peak area of isomaltulose which is critical for quantitative analysis (limit of detection would be worse). Therefore, a compromise between high retention (high resolution) and sufficient peak area must be chosen. To find better experimental conditions we followed our own experience ,55; in tA trend of higher retention due to increasing buffer concentration is related to a hydrophilic partitioning process, because the partitioning model for HILIC assumes the presence of a water-rich liquid layer on the stationary phase. High levels of organic solvent in the mobile phase could cause the salt to be preferentially in the water-rich liquid layer. This would result in an increase in the volume or hydrophilicity of the liquid layer, leading to stronger retentions . HoweverIn our experiments, water in the mobile phase was replaced for acidic formate buffer (pH adjusted with formic acid). Although dissociation of saccharides negligible is under these conditions , it affects the adjacent water-rich layer (HILIC mixed-mode).φ = 0.15, isocratic elution). An increase in retention time was observed with increasing salt concentration in the buffer   . Peak areas (comp. dotted lines) at high volume fractions are also comparable.Next, isomaltulose was tested at three different temperatures and five different buffer volume fractions .08–0.3) B. A compφ = 0.15 the peak areas are 3–5 times higher), which is important for quantitative analysis .However, a key finding is that with decreasing volume fractions, the peak areas do not fall so steeply  in the buffered mobile phase , (2), and (3), respectively.k’ vs. φ) shows a similar non-linear course at all three temperatures. Similarly, courses at temperatures 25 °C and 40 °C, respectively, in k’ vs. log φ , we can conclude that in our system the separation mechanism can be considered mixed, controlled by both partitioning and adsorption, which is consistent with the mixed-mode partitioning-surface mechanism from the literature [φ = 0.3) to −20 kJ/mol (φ = 0.1), and were maximized  at higher buffer content, very similar to the acetonitrile-water system.Despite the limited range of variables tested , ammonium acetate at pH = 5.9, 4.8, and 4.0, respectively, and ammonium bicarbonate (pH = 7.7). The number of theoretical plates, resolution isomaltulose–sucrose and peak area of isomaltulose, respectively, were used as comparison criteria.For ammonium acetate and ammonium bicarbonate, temperatures of ELSD nebulizer and evaporator had to be raised (60 °C) to stabilize the baseline (bicarbonate) and improve run-to-run repeatability (acetate). However, none of these four buffers provided a better efficiency or resolution for isomaltulose–sucrose. Ammonium formate (pKa = 3.75) exhibited practically the same efficiency  at all the three tested pH levels; at pH = 4.8 slightly higher peak areas were observed, which could lower LOD if this aspect was critical. However, this was not the case , so pH = 3.75 was finally selected to maximize the buffer capacity with respect to different matrices of real samples.In the following experiments, the mobile phase of the acetonitrile-ammonium formate buffer was always used. To improve resolution, a temperature of 10 °C was chosen. The selectivity of the separation system is so high that is possible to resolve even anomers of saccharides . TherefoBased on previous findings, to separate isomaltulose from sucrose, the starting composition of the mobile phase selected was 18% ammonium formate (35 mM) and 82% acetonitrile at 10 °C. Due to the subsequent applications of this method for real samples containing maltodextrins, a gradient elution was applied to elute the oligosaccharide from the separation column. The elution gradient was as follows : 0–5 min 18% B, 5–8 min 18–35% B, 8–10 min 35% B, 10–12 min 35–18% B, 12–15 min 18% B. For a complete list of experimental conditions see above, A is the peak area, k is the calibration slope, c is the analyte concentration. Plots before and after the linearization procedure are shown in To establish a calibration curve with ELSD, one must consider that a directly obtained calibration curve  is not linear, and a mathematical transformation should be applied to obtain a linear dependence. A possible approach is to find an exponent m < 1 that the peak area is powered to. Typically, the value lies in the range 0.65–0.80 ,55. The 2, or quality coefficient QC) is a purpose parameter and is unnecessary to report.Validation parameters based on recommendation of ICH Q2 (R1) protocol  were obtAccuracy (recovery) for isomaltulose was tested with a food supplement Carbo snack gel  as a matrix, because its composition is very similar to other running gels and the declared carbohydrate contents were only glucose and oligosaccharides  without isomaltulose or other disaccharides. The results from all calibration levels was 97.7–103.8%, which excludes interference of the matrix and suggests that for calibration the method of external standard curve can be utilized. All the validation parameters are summarized in A sample preparation procedure should provide a final concentration of isomaltulose at the center of the calibration range ≈ 1 mg/mL.Samples with a simple matrix  were dissolved in distilled water only: typically, 0.1–0.5 g of a gel (or a powder) was weighed and dissolved into a 25 mL volumetric flask, sonicated for 3 min, and then filtrated through a 0.45 μm syringe filter.Samples with declared proteins or amino acids  are added for long-term physical performance; the content of oligosaccharides  in high-energy gels is also important.The current legislation, the European Parliament Regulation and Council Regulation No. 1169/2011 , requireIsomaltulose was determined in various commercially available products containing isomaltulose and the results were compared with the declared amounts. The samples contained different levels of isomaltulose in the range 0.9–92.8%. Some of the results were partially presented .Instant tee Babydream Früchte-Tee , with a declared content of 93.9% Palatinose, is in a good agreement (within the method precision) with the determined value of 92.8%, and the chromatogram shows only single peak see .For the Mana non-flavored drink , 10.1 g saccharides and 2.2 g of sugars, respectively, are declared; the amount of isomaltulose determined was 2.1%. In chromatograms see  there isAmong all the supplements tested, the highest content of isomaltulose 32.0% was found in the long-lasting energy gel Enduro snack, apricot flavor . Although isomaltulose is not declared, it corresponds to 37% of sugars declared (if 5% belongs to glucose). In the chromatogram , 20% isomaltulose is declared, which is in a good agreement with the amount determined: 19.0% and 21.4% . The declared content of saccharides is 90% and 67% of sugars, respectively. The chromatogram see A shows tVitargo gel  exhibits also a high content of isomaltulose, 19.3 and 19.4%, respectively, which is in a good agreement with declared values of 18.9%. In chromatograms, there is a dominating peak of trehalose see B, which Energy gel long trail , 70 g in tubes (or 35 g in packs), various flavors : the declared value is 10%, determined as 9.6–10.9%. These samples also exhibit fructose, glucose, and maltodextrins on the chromatogram see , which iExpress energy gel  contains a variety of additives including caffein, taurine, tyrosine; the declared value of isomaltulose is 5.0%, determined as 5.3%. The largest peak in the chromatogram see  is glucoThe Isogel Carbohydrate Smart Snack juicy orange, green apple , 70 mL bag, contains a high signal of fructose and glucose that can be explained by a high declaration of sugars = 42% , 60% of saccharides  see . The proIsogel Energy shock, juicy orange, lemon lime flavor , 70 mL bag, contains the greatest number of peaks on the chromatograms among our samples, caused by many declared additives like amino acids, caffein, xanthan gum (a polysaccharide), and starch, which corresponds to a high content of saccharides (60%) see . The proRock’s! energy gel, lemon lime flavor , 32 g bag, was very similar to Isogel Energy Shock , with an even higher declared content of saccharides (78%) (xanthan gum), with no amino acids or caffeine. The extremely high signal of fructose and glucose can be explained by the high declaration of sugars = 47%  has a unique declaration of fructose syrup which is a mixture of glucose and fructose (declared as 2%). Trehalose was also present (declared as 1%) and relatively high signals of maltodextrins. The declared content of isomaltulose is 1%; the amount found is 0.9% see D.A rapid separation method was developed and applied for determination of isomaltulose in the presence of sucrose, glucose, and oligosaccharides. The application of the method to commercially available samples of beverages and dietary supplements for athletic performance mostly confirmed declared amounts of isomaltulose within the experimental precision (≈2%). If there was amount of isomaltulose not declared, the determined value was lower than a declared amount of “sugars”.This method can be used to determine isomaltulose (or other saccharides) in food supplements of so-called healthy food, and also in food supplements for physical exercise and athletic performance, the assortment of which is currently under development; eventually to determine isomaltulose as a potential drug against diabetes. The method could be also used for sugar profiling of food (after appropriate sample preparation). Baseline separation of sucrose and isomaltulose would allow this method to be used to control or monitor adulteration of isomaltulose.


Restorative proctocolectomy (RPC) is the most common operation in ulcerative colitis. Nevertheless, permanent ileostomy will sometimes be unavoidable. The aim was to evaluate the reasons for pouch failure and early morbidity after pouch excision.The number and the reasons for pouch failures were analysed in patients undergoing RPC 1985-2016.Out of 491 RPC patients, 53 experienced pouch failure ; 52 out of 53 underwent pouch excision. The cumulative risk for excision at 5, 10 and 20 years was 5.6, 9.4 and 15.5%, respectively. The reasons for failure included septic events such as fistula in 12 (23%), chronic pouchitis in 11 (21%) and leakage in 8 (15%) patients. Functional reasons for pouch failure were recorded as poor function in 16 (30%), incontinence in 12 (23%) and stricture in 12 (23%) patients. Multiple causes for pouch failure were recorded for individual patients. Seven cases of Crohn’s disease were found among the failure cases: two before pouch excision and five after. Altogether, 15 Crohn’s disease diagnoses were set in the RPC cohort, giving a percentage of 47% of pouch failure in this disorder. A complication occurred in 23 (44%) patients within 30 days after surgery; 16 were mild (Clavien-Dindo grades I–II).Eleven percent of RPC patients suffered pouch failure: more men than women. The reasons were multiple. Crohn’s disease created a risk of failure, but a half of these patients maintained the pouch. Morbidity after pouch excision was moderate, but in most cases slight. Since its introduction by Parks and Nicholls in 1978 –4. The mAll consecutive patients with UC undergoing RPC at Tampere University Hospital between 1985 and 2016 were identified from the patient records using the ICD-9 and ICD-10 codes for UC and NCSP  codes for the types of operations. A database to form an RPC registry was collected from patient files including details on patient history, surgical technique, postoperative morbidity and follow-up. On patients with pouch failure, we collected details on patient history, reasons for failure, treatment prior to failure and on the pouch excision procedure. Seventy-five patients had moved elsewhere in Finland. Data on the pouch excision of these patients were retrieved from the register of the National Institute of Health and Welfare (NIHW). The patients were identified by the NSCP procedure codes. A widely used prognostic model, the Charlson Comorbidity Index, was used to categorise pouch failure patients’ comorbidities, a method based on the diagnostic codes . Each coAll pouches in our RPC registry are of J-type and the anastomosis is either handsewn or stapled. Pouch failure was defined as the need for a permanent ileostomy with or without pouch excision. Early complications were defined as occurring within 30 days of the operation.. Results are shown as odds ratios (ORs) with their 95% confidence intervals (CIs). The cumulative probability of pouch excision was estimated by the Kaplan-Meier method. Statistical significance was set at p ≤ 0.05.The data were analysed using SPSS . For categorical variables, the results were given as frequencies and percentages, and for continuous variables as means and standard deviations or as medians. Chi-square or Fisher’s exact test was used to assess differences in categorical variables. The binary logistic regression analysis was used to determine the predictors of pouch failureThe study group comprised 491 RPC patients: 209 women and 282 men. The median follow-up time of the RPC cohort was 11 years (0–33). The data was gathered and analysed during 2017. The baseline characteristics of the patients in the RPC cohort and the pouch failure cohort are presented in Table Altogether 53 10.8%) pouch failures were recorded: in 10 (4.7 %) out of 209 women and in 43 (15.2 %) out of 282 men OR 3.58 (95% CI 1.75–7.37). Of these, 52 underwent pouch excision and one permanent loop stoma was built due to a leakage. The median time from the operation to the failure was 4.7 (0–26) years, in women 5.0 (0.7–17) and in men 4.7 (0–26) years. Half of the pouch failures occurred within the first 5 years and 70% within 7 years of RPC surgery  prior to excision, inflammation was seen by inspection in 55%, fistula in the pouch in 4%, fistula in the anastomosis 11%, stricture in the longer limb in 11% and stricture in the anastomosis in 11%. Pouch biopsy histology was available for 42 patients and showed mild inflammation in 29%, chronic active inflammation in 55% and granulation tissue in 2.4% of the patients; in 14% the histology was interpreted to be normal. Of the 48 operation specimens, histology active inflammation was seen in 37 (77%), fistula in three (6.3%), abscess in three (6.3%), stricture in two (4.2%) and mucinotic carcinoma in one (2.1%) and one was interpreted to be normal. In four cases, it was no longer possible to find the pathology statement and one patient died soon after the operation and the specimen was not analysed.Altogether, 15 Crohn’s disease diagnoses were set in the RPC cohort after the initial diagnosis of UC; pouch failure occurred in seven (47%) while the frequency in UC was 9.7%. In five of these seven cases, the diagnosis of Crohn’s disease was made after pouch failure. The median age was 47 years (21–72), and 1 (14%) of these patients were female. In those with final diagnosis of UC and pouch failure, the median age was 44 years (26–53), and 8 (17%) were female.A closer inspection of pouch endoscopy and operation histology of these Crohn’s patients revealed two longer limb strictures and pouch strictures while histology showed mainly chronic active inflammation. Postoperative histology showed inflammation in all patients.Of the eight patients with their pouch in place and a diagnosis of Crohn’s disease, four (50%) had died: one died of breast cancer, and the cause of death of the three patients remained obscure. Five of the patients were taking azathioprine and one was taking biologic drugs, and all were satisfied with their pouch function.p = 0.896, respectively.Although pouch failure was more common in men than in women, there were no significant gender differences in the causes of pouch failure, medication prior to failure or operations performed prior to failure. In the whole RPC cohort, there was no difference in leakage 15 (7.7%) vs. 31 (11%) or in the occurrence of pelvic sepsis, 32 (15.3%) vs. 43 (15.2%) between women and men.Median BMI at the time of RPC did not differ between those retaining the pouch and those suffering pouch failure 24.2 vs. 25.0%. BMI was drawn from the patients’ anaesthesia forms: in 11 subjects, the information was missing data. In the pouch failure group, the BMI could not be analysed in three cases.None of the five laparoscopically treated patients had experienced pouch failure during their rather short follow-up.We divided the RPC patients into ≥ 65 and younger and there was no difference in pouch failure for these groups 2 (11.8%) vs. 51 (10.8%) p < 0.001, and also pelvic sepsis, 15 (28.3%) vs. 60 (13.7%) p = 0.005, respectively. In the pouch failure group, early relaparotomy was carried out on 11 (20.8%) and of those with functioning pouch on 31 (7.1%), p = 0.003. Covering stoma had been used in 19 (35.8%) in the pouch failure group and 250 (57.2%) patients in the pouch in place group, p = 0.003. The anastomoses were handsewn in 45 (84.9%) and in 243 (55.5%) patients, p = <0.001, respectively. The univariate and multivariate analyses of the demographic, surgery related and complication data are presented in Table p = 0.022 and male gender OR 3.83 (95%CI 1.80–8.12) p ≤ 0.001.More early leakage occurred in failure patients than in those with functioning pouch; 15 (28.3%) vs. 32 (7.3%) All pouch excision operations were performed as open surgery and median blood loss per operation was 325 mL (50–2300 mL). Median hospital stay after pouch excision was 9 days (2–24). Readmission to hospital was recorded for 13 (25%) of the pouch excision patients.An early complication was recorded in 23 (44%) of the patients after surgery but 16 (70%) of these were Clavien-Dindo grades I–II. Early complications after pouch excision surgery are shown in Table This study investigated the frequency and reasons for pouch failure after RPC for UC in the second biggest IBD surgery centre in Finland in a period spanning more than three decades. The overall frequency of pouch failure was 10.8%. In a previous report with a large number of patients and in a meta-analysis, similar pouch failure percentages of 9.7  and 8.6%The reasons leading to pouch failure were often multifactorial: one complication leading to another and subsequently to functional difficulties. The reasons reported in different studies may vary according to how pouch problems are categorised or recorded. The most common reasons for failure in our study were septic events in 2140%) , pouchitis in 13%, chronic pouchitis in 21%, Crohn’s disease in 13% and functional reasons: poor function was seen in 16 (30%), incontinence in 12 (23%) and stricture in 12 (23%) patients. In a British multicentre study comprising 94 pouch failure patients, the causes were similar: septic complications in 33 (35.1%), poor function in 29 (30.8%), pouchitis in 9 (9.6%) and Crohn’s disease in 5.3% [0%  d enough .The differences in the uni- and multivariate analyses for the use of covering stoma and type of anastomoses are explained with the changes in practice through the years: handsewn anastomoses were performed mainly in 1985–2005 and covering ileostomy when considered necessary in 1985–2005 and as a routine after that. The year of IPAA has also been taken into account there and it obviously affects the follow-up time.In our study, all but one of the pouch failure patients 52, 98%) underwent pouch excision. In earlier reports, this percentage has been smaller: in the study by Foley 40% , Tulchin2, 98% unn = 22) and found no dysplasia or cancer in histopathology in pouch biopsies; none of the patients requested further surgery and the majority had satisfactory ileostomy function. Altogether, dysplastic transformation in the ileal pouch has been estimated to be rare [In this study, morbidity of the excision was frequent but most of the complications were mild and thus acceptable. There are very few studies on morbidity after pouch excision. Karoui et al. concluded that early and especially late morbidity is high and pouch excision RPC patients should be initially counselled on this . Prudhom be rare . The preThe study was limited due to its retrospective nature and lack of systematic follow-up of the pouch failure patients.Although most of the RPC patients operated on for UC had satisfactory functional outcome, 10.8% experienced pouch failure in the long run. The most common reasons for failure were deterioration of functional results after septic complications. Pouch excision is a major surgery and associated with moderate morbidity, necessitating precise planning and patient counselling. The possibility of Crohn’s disease should be considered in UC patients with pouch failure who have been operated on.


Chloroflexi and Cyanobacteria taxa in the biofilms near leachate streams indicated the selective enrichment of Ktedonobacteria and Oxyphotobacteria members.The 16S rRNA amplicons from biofilms inhabiting rocks near various water bodies of Marsberg Copper Mine  reveal the diversity of their microbial communities. The abundance of  Chloroflexi and Cyanobacteria taxa in the biofilms near leachate streams indicated the selective enrichment of Ktedonobacteria and Oxyphotobacteria members.The 16S rRNA amplicons from biofilms inhabiting rocks near various water bodies of Marsberg Copper Mine  reveal the diversity of their microbial communities. The abundance of  Marsberg Kilianstollen Copper Mine  is known for its copper-rich Lower Carboniferous alum shales and lydites as well as copper remineralizations; previous investigations revealed the geochemistry of heavy metals, copper in particular, in (acidic) sulfidic mine waters . In thishttp://ebg.ucalgary.ca/metaamp/) with optimized parameters. The minimum length of overlap was set to 24 bp, and the maximum number of mismatches in the overlap region and primer sequences was adjusted to 4 (https://mothur.org/wiki/silva_reference_files/) by Mothur , biofilms growing near or in diverse mine drainages, ranging from fresh stream water to copper-rich acidic leachate, were aseptically collected and stored at −20°C. The biofilm samples MB1 through MB6 were collected from colonized rocks of the adit wall, within 25 to 35 cm of an acidic leachate stream, MBS18 was taken directly from the acidic leachate outflow stream sediment, samples MBS1 through MBS4 were collected from colonized rocks near the fresh spring water stream, and the biofilms from unconsolidated mine rocks and a wooden plank used for timbering were labeled as MBS11, MBS13, and MBS10 . The micActinobacteria (27%), Chloroflexi (17%), Proteobacteria (16%), Cyanobacteria (12%), Acidobacteria (10%), and Bacteroidetes (6%) in all samples . According to the UniFrac weighted algorithm (P = 0.034), while the unconsolidated rock group microbiome shared similarity with both the spring water and leachate samples (P > 0.097). Ktedonobacteria, Cyanobacteria, and Actinobacteria contributed 26%, 23%, and 16%, respectively, to the biofilm community (MB1 through MB6 and MBS18) growing near the copper acidic leachate stream. The genus-level analysis of all samples identified 10 distinct uncultured Ktedonobacteria genera; 80% of these could be classified within the Ktedonobacteraceae family. Marsberg Kilianstollen offers a large reservoir of uncultured novel heavy metal-resistant microbial strains, especially belonging to the class Ktedonobacteria of Chloroflexi.The prevalent phylum taxa were acteria 1%, CyanobPRJNA670497, and the individual samples under the accession numbers SRR12876542, SRR12876543, SRR12876544, SRR12876545, SRR12876546, SRR12876547, SRR12876548, SRR12876549, SRR12876550, SRR12876551, SRR12876552, SRR12876553, SRR12876554, and SRR12876555 are publicly published.The 16S rRNA gene amplicon raw read data sets have been deposited in the NCBI Sequence Read Archive (SRA) under BioProject accession number


MHC-independent αβTCRs (TCRs) recognize conformational epitopes on native self-proteins and arise in mice lacking both MHC and CD4/CD8 coreceptor proteins. Although naturally generated in the thymus, these TCRs resemble re-engineered therapeutic chimeric antigen receptor (CAR) T cells in their specificity for MHC-independent ligands. Here we identify naturally arising MHC-independent TCRs reactive to three native self-proteins  involved in cell adhesion. We report that naturally arising MHC-independent TCRs require high affinity TCR-ligand engagements in the thymus to signal positive selection and that high affinity positive selection generates a peripheral TCR repertoire with limited diversity and increased self-reactivity. We conclude that the affinity of TCR-ligand engagements required to signal positive selection in the thymus inversely determines the diversity and self-tolerance of the mature TCR repertoire that is selected. The ligand recognition specificity of the αβT cell receptor (TCR) repertoire is established during T cell differentiation in the thymus. The recombination activating genes Rag1 and Rag2 induce random TCR gene re-arrangements in immature thymocytes and those with productively rearranged TCRα and TCRβ genes express αβTCR protein complexes on their cell surfaces , 4.The mature TCR repertoire that is positively selected in normal mice is specific for linear antigenic peptides bound to Major Histocompatibility Complex (MHC)-encoded molecules, a recognition feature known as “MHC restriction” –7. In coRelatively little is known about the specificity and thymic selection requirements of MHC-independent TCRs because only two individual TCRs from QuadKO mice named A11 and B12A) have been identified and characterized in detail  and B12A–15. HoweWe undertook the present study to determine if MHC-independent TCRs required high affinity TCR-ligand engagements to signal positive selection and, if so, to determine the consequences of high affinity thymic selection on the mature TCR repertoire. We now identify and characterize MHC-independent TCRs that recognize new MHC-independent ligands: TCR-38 is specific for CD48 and TCR-146 is specific for ICAM-2 (CD102). Like CD155, CD48, and CD102 also function as low affinity ligands for cell adhesion receptors. We focused on TCR-146 which binds exclusively to ICAM-2 (CD102) and found that it bound with high 1.6 μM affinity independently of LFA-1. In the thymus, TCR-146 strictly requires ICAM-2 to signal positive selection, indicating that MHC-independent TCRs generally require high affinity ligand engagements to signal positive selection which is very different from conventional MHC-restricted TCRs. Importantly, we discovered that the requirement for high affinity ligand engagements in the thymus selects a peripheral MHC-independent TCR repertoire with markedly limited receptor diversity and increased self-reactivity. In contrast, dependence on CD4/CD8 coreceptors allows conventional MHC-restricted TCRs to signal positive selection with very low affinity ligand engagements which generates a peripheral TCR repertoire that is both highly diverse and self-tolerant. We conclude that the affinity of TCR-ligand engagements that signal positive selection in the thymus profoundly affects the diversity and self-reactivity of the selected TCR repertoire.KO (H2-Ab−/−B2m−/−Cd4−/−Cd8−/−) mice, we began by generating T-hybridoma cell lines from QuadKOBcl-2Tg (QB) LNT cells that express the pro-survival Bcl-2Tg to minimize loss of TCR specificities from in vivo signaled cell death   reacted against both MHCKO and CD155KO spleen APC stimulators indicating that their MHC-independent ligands were molecules other than CD155 (We generated T-hybridomas from QB LNT cells that had been stimulated with platebound anti-TCRβ/anti-CD28 antibodies and screened them for recognition of MHC-independent ligands expressed on MHCs (APCs) . Three Tan CD155 . TCR seqan CD155 . Completin vivo CD155 expression in QB mice. To answer this question, we performed parallel T-hybridoma fusions with LNT cells from CD155-sufficient (CD155+/+) and CD155-deficient (CD155−/−) QB LNT cells (+/+ and CD155−/− QB mice expressed MHC-independent TCRs that reacted against MHCKO spleen APC stimulators (KOCD155−/− APCs (p < 0.05)   right. Tin vivo ligand expression is limited only to TCRs specific for CD155 or if it extends to TCRs specific for other MHC-independent ligands as well. However, no other MHC-independent TCR ligands have yet been identified. Consequently, we embarked on identifying the MHC-independent ligands recognized by the three T-hybridomas that we had selected to study. We first verified that all three T-hybridomas reacted against ligands expressed on the murine CH27 B cell line but did not react to ligands expressed on the human 293T cell line  protein, while T-hyb 25 reacts to plate-bound CD155 protein but not CD102 protein . MoreoveInterestingly, the stimulatory ligand for each of these T-hybridomas  is an adhesion molecule that can also bind with low affinity to counter-receptors on lymphocytes, as ICAM-2 (CD102) binds to LFA-1 and Mac-1 , 18; CD4D of 1.6 μM and 0.5 μM, derived from equilibrium and kinetic fittings, respectively. No detectable binding of TCR-146 was observed to immobilized human CD102 under the same condition  and 5.12 × 10−3 (1/s), respectively, for TCR-146/mCD102 binding  by surface plasmon resonance (SPR) in a completely cell-free assay . The solondition . The kin binding . Compareted TCRs , 23. Simted TCRs   is also a low affinity ligand for the adhesion molecule LFA-1 which consists of a dimer composed of CD11a and CD18 chains on endothelial cells, monocytes, platelets and lymphocytes. The cellular adhesion molecule LFA-1 binds to several adhesive ligands  –27 to prTg) that is specific for a novel MHC-independent ligand  MHfficient , and  host mice but proceeds successfully in LFA-1-deficient (CD11aKO) host mice as determined both by thymocyte expression markers  and by LNT cell generation (Tg LNT cells in CD11aKOCD102WT mice (~20–25 × 106) is unaffected by LFA1-deficiency . We. WeTg mein vivo clonal deletion which was prevented in mice expressing the pro-survival Bcl-2Tg. Surprisingly, however, positively selected CCR7+ TCR-146 thymocytes appear in both Bcl-2Tg+ and Bcl-2Tg− mice  numbers in Bcl-2Tg− mice  in in vitro mixed lymphocyte responses as measured by CFSE dye dilution  stimulator cells as well as against third party B6 and B10.A spleen stimulator cells (Tg T cells are self-reactive as they are unreactive against syngeneic (B6) spleen stimulator cells  and QB mice as quantified by the Inverse Simpson Index (ISI) (KO mice (p < 10−15), and the repertoire diversity of MHC-restricted TCR in B6.Bcl-2Tg mice is far greater than the repertoire diversity of MHC-independent TCRs in QB mice (p < 10−12 to 10−15). Based on these findings, we conclude that positive selection by high affinity TCR-ligand engagements severely reduces TCR repertoire diversity and increases self-reactivity.To evaluate this prediction, we compared the diversity of TCRα and TCRβ sequence repertoires from the periphery of MHC-restricted B6, B10.BR, BALB/c, and B6.Bcl2 mice vs. TCRα and TCRβ sequence repertoires from the periphery of MHC-independent Quadex (ISI) . Note thex (ISI) . On averKO mice that were reactive against three native self-proteins which otherwise functioned as low affinity cell adhesion molecules. We focused on TCR-146 which was reactive against ICAM-2 (CD102), a low affinity ligand for the cell adhesion molecule LFA-1, and which bound soluble ICAM-2 (CD102) independently of LFA-1 with relatively high 1.6 μM affinity. To assess in vivo positive selection signaling by TCR-146, we constructed TCR-146 transgenic mice and found that TCR-146 strictly required its high affinity ligand ICAM-2 to signal positive selection in the thymus, demonstrating that high affinity positive selection signaling was not unique to CD155-specific TCRs  tyrosine kinase molecules must be recruited to ligand-engaged surface TCR complexes. So the different TCR affinities required by MHC-restricted and MHC-independent TCRs to signal positive selection reflect the different efficiencies with which Lck is recruited to different ligand-engaged TCR complexes. In CD4/CD8 coreceptor-sufficient mice, Lck is associated with the cytosolic tails of CD4/CD8 coreceptors that bind to surface pMHC ligands together with MHC-restricted TCRs, so that coreceptors efficiently bring Lck to ligand-engaged TCRs. In contrast, in CD4/CD8 coreceptor-deficient mice, Lck is coreceptor-free and must be passively captured within surface TCR clusters, a process which is highly inefficient and requires high affinity TCR-ligand engagements that persist long enough to passively capture free Lck within TCR-ligand clusters. Thus, the weak TCR signals that induce positive selection are generated either by low affinity coreceptor-dependent TCR-ligand engagements or by high affinity coreceptor-independent TCR-ligand engagements, with the former generating an MHC-restricted TCR repertoire and the latter generating an MHC-independent TCR repertoire.The requirement for high affinity TCR-ligand engagements to signal positive selection is unique to MHC-independent TCRs as MHC-restricted TCRs signal positive selection by engaging very low affinity ligands. The obvious difference in TCR-ligand affinities required to signal positive selection of immature thymocytes is quite surprising because positive selection requires uniquely weak TCR signals–and this should be the case regardless of ligand specificity. While TCR signal strength is often equated with TCR-ligand affinity, this study requires that TCR signal strength and TCR affinity be conceptually separated. When this is done, the positive selection requirement for weak TCR signaling means that only few activated p56in vitro. Indeed, their self-reactivity is not limited to in vitro assays as MHC-independent QuadKO mice have in vivo lymphocytic infiltrations into multiple organs beginning at ~6 months of age . Instead, we think that peripheral MHC-independent T cells become self-reactive in the periphery because mature T cells increase their intra-cellular content of Lck during their differentiation from immature thymocytes which generates stronger TCR signals in the periphery.The requirement for high affinity TCR-ligand engagements to signal positive selection profoundly effects the self-reactivity and diversity of the mature TCR repertoire. Regarding the self-reactivity of the mature TCR repertoire, high affinity positive selection necessarily generates a peripheral MHC-independent TCR repertoire with high affinity for self-protein ligands. Even so, it is surprising that MHC-independent TCRs are incompletely self-tolerant to their own self-proteins as revealed by their self-reactivity in mixed lymphocyte cultures s of age . As explFailure of high affinity MHC-independent TCRs to induce clonal deletion can provide insight into how much CD4/CD8 co-receptors contribute to the strength of intracellular signal transduction during thymic selection. In our thinking negative selection in the thymus requires that sufficient Lck be recruited to ligand-engaged TCR complexes to transduce strong intracellular signals that induce immature thymocyte death. In the absence of CD4/CD8 coreceptors, recruitment of coreceptor-independent Lck to ligand-engaged TCRs is inefficient and requires long duration TCR binding to the negatively selecting ligand as best measured by dwell time . In contRegarding the limited repertoire diversity of MHC-independent TCRs, we think limited diversity is a necessary outcome of the positive selection requirement for high affinity TCR engagements. Because affinity is a measure of how well-receptor and ligand fit together with high affinity indicating a near-perfect fit, a high affinity requirement for positive selection limits positive selection signaling to only the few MHC-independent TCRs able to bind an individual ligand with sufficient affinity to signal positive selection. In contrast, a low affinity positive selection requirement allows many different poorly fitting TCRs to engage an individual ligand and signal positive selection. As a result, a high affinity positive selection requirement severely limits the number of different TCRs that can be selected into the mature repertoire by an individual self-ligand in the thymus, whereas a low affinity positive selection requirement allows multiple, even millions, of different TCRs to be selected into the mature repertoire by an individual self-ligand in the thymus . An impoBased on this study, we suggest that MHC-restricted TCRs with low affinity positive selection requirements arose as a result of evolutionary pressure to generate a maximally diverse and, therefore maximally protective, TCR repertoire. Selection of a highly diverse and more self-tolerant TCR repertoire was achieved during evolution by employing CD4/CD8 coreceptors with cytosolic tails that bound Lck and extracellular domains that bound to MHC, since CD4/CD8 coreceptors efficiently recruited Lck to MHC-engaged TCRs which markedly lowered the affinity of TCR-ligand engagements required to signal positive selection in the thymus. Notably, since CD4 and CD8 coreceptor external domains only bind to different classes of MHC proteins, the involvement of CD4 and CD8 coreceptors in thymic selection necessarily resulted in low affinity positive selection of a peripheral TCR repertoire that was MHC-restricted, highly diverse, and self-tolerant.It is a curious feature of the MHC-independent TCR repertoire that the ligands identified so far are involved in cell adhesion. We suspect this is because cell adhesion ligands are highly expressed on cells in the thymus which is necessary for sufficient numbers of TCR-ligand engagements to form and signal positive selection. In addition, we previously noted that cell adhesion molecules like CD155 are downregulated during T-hybridoma fusions which decreases the fratricide of T-hybridomas that bear TCRs with those ligand specificities and increases their relative recovery in T-hybridoma fusions .Finally, naturally arising MHC-independent TCRs generated by Rag-mediated gene rearrangements resemble re-engineered therapeutic CAR T cells in recognizing self-ligands independently of MHC , 38. TheIn conclusion, this study provides novel insights into the relationship between the required affinity of TCR-ligand engagements that signal positive selection in the thymus and critical features of the mature TCR repertoire in the periphery. This study suggests the novel concept that the required affinity of positive selection signaling in the thymus determines, inversely, the diversity and self-tolerance of the peripheral TCR repertoire.B2m−/−H-2-Ab1−/−), RAG-deficient (Rag2−/−), Quad-deficient (B2m−/−H-2-Ab1−/−Cd4−/−Cd8a−/−) (Cd155−/−) were generated as previously described (Cd102−/−) deficient mice (Cd11a−/−) (MHC-deficient (Cd8a−/−) , and QuaCd8a−/−)  were breescribed , as wereent mice  and LFA-d11a−/−) . Animal New transgenic mouse strains constructed for this study were generated by cloning full length TCR cDNAs for TCRα and TCRβ into the human CD2 transgenic vector to obtain T cell specific expression.KO and QuadKOBcl-2Tg (QB) mice were stimulated with plate-bound anti-TCRβ/CD28 (5 and 2 μg/ml) for 48 hr. fused to TCR-null BW5147 cells, and subcloned at <1 cell/well (Lymph node αβT (LNT) cells from Quadell/well . IL-2 seell/well .MAbs with the following specificities were used in the present study: CD4 (GK1.5 or RM4.4), TCRβ (H57-957), CD5 (53-7.3), CD8α (53-6.7), CD69 (H1.2F3), CCR7 (4B12) were obtained from BD Biosciences . LEAF-purified antibodies against mouse CD155 (clone 4.24.1) were obtained from Biolegend. Functional grade antibodies against mouse CD102 (clone 3C4 (mIC2/4), mouse CD54 (clone YN1/1.7.4), mouse CD11a  were obtained from eBiosciences, mouse CD48 (clone HM48-1) was obtained from Biolegend. Purified anti-CD3 antibodies [clone 7D6 ] were geFlat-bottom 96-well plates were coated overnight with recombinant proteins in 50 μl of PBS. Hybridomas were added overnight, after which supernatants were assessed for IL-2.E. coli (~150 clones/well) were inoculated overnight into 96-well format culture blocks (10 blocks). Plasmids were purified with a Qiaprep 96 Turbo miniprep kit (QIAGEN) and were transfected to HEK293T cells with Lipofectamine 2000 (Invitrogen) in 96-well flat-bottom plates and left overnight. Hybridomas were cocultured with cDNA-transfected 293T cells for 24 h, after which mIL-2 amounts in the supernatants were obtained. Positive clones were selected for secondary and tertiary screenings. Subpool libraries  and clone libraries  were prepared and screened. Positive clones were sequenced to identify the specificity of the transfected cDNA.Total RNA was prepared from the murine CH27 cell line by RNeasy Maxi (QIAGEN Inc.) and was purified with FastTrack MAG Maxi mRNA isolation kit (Invitrogen) to obtain poly(A)+ RNA. cDNA was synthesized with the SuperScript system (Invitrogen) and was cloned into SPORT6 vector with SalI and NotI restriction sites. ElectroMAX DH10B competent cells (Invitrogen) were transformed by electroporation, and after titration, Cells from thymi and lymph nodes were analyzed on an LSRII (BD Biosciences) and dead cells were excluded by forward light-scatter and propidium-iodide uptake.DNA encoding extracellular portions of TCR α and β chains were cloned into pET30a vectors as described . ReceptoSurface plasmon resonance measurements were performed using a BIAcore 3000 instrument and analyzed with BIAevaluation 4.1 software (Biacore AB). Murine ICAM-2-Fc, human ICAM-2-Fc and NKp44-Fc fusion proteins (R&D systems) were immobilized on carboxylated dextran CM3 chips (Biacore AB) to 4000–7000 response units (RU) using a primary amine-coupling in 10 mM sodium acetate (pH 5.0). The analytes consisted of serial dilutions of TCR-146 between 8 and 0.5 μM in a buffer containing 10 mM Hepes (pH7.4), 0.15 M NaCl, 1 mM CaCl2 and 0.5 mg/ml BSA. The dissociation constants were obtained by kinetic curve fitting for the binding of TCR146 to murine ICAM-2 using BIAevaluation 4.1 (BIAcore Inc.).To test T cell reactivity against stimulator cells from different mouse strains, we labeled purified LNT responder cells with CFSE and cocultured them with irradiated (2000 rads) LPS-activated splenic B cell blasts. Cultures were analyzed on day 4 by multicolor flow cytometry.Chimeric CD155 molecules were generated as described . BrieflyTCRα and β repertoire sequences for all indicated mouse strains were obtained by high throughput next generation RNA sequencing through Adaptive Biotechnologies Corp. and iRepertoire, Inc. Three or more animals from each strain were sequenced. Sequence diversity was estimated for each overall repertoire and for each individual Vα- and Vβ-gene family using Inverse Simpson Index (ISI) as calculated by AAfrequency, an in-house developed program .https://clients.adaptivebiotech.com/pub/lu-2019-natcomms] with doi: 10.21417/JL2019. Both TCRα and TCRβ sequences are also available upon request.TCRβ sequences used for diversity analysis are available through Adaptive Biotechnology's ImmuneAccess database [The animal study was reviewed and approved by NCI Animal Care and Use Committee.FV, IS, PS, and AS conceptualized and designed the study and interpreted the results. FV performed the experiments, analyzed data, and drafted the manuscript. IS, JL, AB, XT, TG, AA, MR, and JA performed experiments and contributed to the analysis of the data. KH, JY, and PS provided conceptual and technical support. BE provided an experimental animal model. AS conceived and supervised the research program and experiments, and wrote the manuscript. All authors listed have approved the work for publication.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.


Arteriovenous malformations (AVM) of the cervical spine can present with symptoms resulting from their mass effect, vascular steal, or subarachnoid hemorrhage (SAH). While ruptured cerebral aneurysms bleed fast and usually cause severe headache, AVM bleed slowly; moreover, when the location is extracranial, the presentation might be even more confusing. For these reasons, the clinical course can be misleading. We present the case of a woman who had bleeding from an AVM of the cervical spine and discuss the classification and treatment options of AVM. Arteriovenous malformations (AVM) of the cervical spine can present with symptoms resulting from their mass effect, vascular steal, or subarachnoid hemorrhage (SAH) . RuptureWe discuss here the case of a woman who came to the hospital with two complaints: chest pain consistent with acute coronary syndrome and headache for four days. The workup for the chest pain led to normal findings. Further evaluation of the headache, given its persistent nature and subsequent development of sensorimotor deficits, led to the diagnosis of a spinal hematoma secondary to a cervical AVM.A 59-year-old woman presented to the emergency department with chest pain radiating to the left shoulder, arm, and back, and two days of headache. Her chest pain virtually resolved on admission, and the workup for acute coronary syndrome was negative. Her headache, however, fluctuated in intensity, and became worse on the third day, when she also reported some numbness on her left fingertips. A computed tomography (CT) of the head was normal.Her blood pressure was 157/68 mmHg, pulse 67/minute, respiratory rate 18/minute, temperature 36.2ºC. Her neck was supple, with no paraspinal tenderness. There was no focal neurologic deficit.On the fourth day, the headache level increased. Her paresthesia level was reportedly more proximal, affecting the left forearm, and she developed difficulty with flexion and abduction of that arm. No deep reflexes were elicited on that limb. A lumbar puncture performed under radiologic guidance produced a bloody fluid. Its analysis showed 2,440,000 red blood cells/μL, 1,100 white blood cells/μL , glucose 14 mg/dL, protein level of 376 mg/dL, and negative Venereal Disease Reseach Laboratory (VDRL) test. Magnetic resonance imaging (MRI) showed a C5-C6 spinal hematoma and abnormal vessels adjacent to the dorsal aspect of the cervical canal (Figure AVMs cause less than 5% of all SAH . Most phThe classification of AVM has sustained multiple changes. Currently, type IV AVM represents those with a vascular nidus that involves the spinal parenchyma with or without the involvement of an axial artery or vein . OverallSAH can sometimes present with an indolent course, especially when it results from low-flow lesions, like AVM, particularly those with extracranial location, such as the spinal subarachnoid space that has a larger capacity for blood accumulation. A non-resolving or progressive headache, with or without neurologic deficit, should raise suspicion for such presentation.


We have previously demonstrated that high concentrations of glutamate induce excitotoxicity and elicit a transient increase in the intracellular concentration of Mg2+ due to the release of Mg2+ from mitochondria, followed by a decrease to below steady-state levels. Since Mg2+ deficiency is involved in neuronal diseases, this decrease presumably affects neuronal survival under excitotoxic conditions. However, the mechanism of the Mg2+ decrease and its effect on the excitotoxicity process have not been elucidated. In this study, we demonstrated that inhibitors of Mg2+ extrusion, quinidine and amiloride, attenuated glutamate excitotoxicity in cultured rat hippocampal neurons. A toxic concentration of glutamate induced both Mg2+ release from mitochondria and Mg2+ extrusion from cytosol, and both quinidine and amiloride suppressed only the extrusion. This resulted in the maintenance of a higher Mg2+ concentration in the cytosol than under steady-state conditions during the ten-minute exposure to glutamate. These inhibitors also attenuated the glutamate-induced depression of cellular energy metabolism. Our data indicate the importance of Mg2+ regulation in neuronal survival under excitotoxicity.Magnesium plays important roles in the nervous system. An increase in the Mg Although it is the most abundant neurotransmitter in the mammalian brain, excessive accumulation of glutamate around neurons induces neuronal cell death in a phenomenon known as excitotoxicity i [2+]i turns to decrease. Given that Mg2+ deficiency is involved in neuronal diseases, the decreasing [Mg2+]i phase likely has detrimental effects on neuronal survival, and thus plays a key role in excitotoxicity. However, the mechanism causing the decreasing [Mg2+]i phase and its effect on excitotoxicity has not yet been elucidated.At the cellular level, Mgembranes ,14,15,16sduction , cellulasduction ,19, and sduction . These strations ,22. In a neurons ,24,25,26toxicity . Toxic c2+]i are involved in neuronal cell death via excitotoxicity in rat hippocampal neurons. We investigated the mechanisms involved in the phase in which the transient [Mg2+]i increase in response to glutamate is reversed, and the effects of [Mg2+]i on cellular energy metabolism. Quinidine and amiloride were used to inhibit cellular Mg2+ extrusion. Because those inhibitors are not specific for Mg2+ transport, it is important to confirm that both inhibitors show similar effects and that the effects were mediated via the changes in [Mg2+]i. Glutamate excitotoxicity results from continuous activation of the NMDA receptors on neurons, which leads to excessive Ca2+ influx into the cytosol and overload in the mitochondria, resulting in depolarization of the mitochondrial membrane potential and the release of cell death signals i and intracellular Ca2+ concentration ([Ca2+]i) were measured by simultaneous imaging using the probes KMG-104-AM , the Fura-Red data were presented and analyzed as F0/F.Fluorescence measurements were performed using fluorescence microscope, ECLIPSE TE300  equipped with 10× and 20× objective lenses, S Fluor (Nikon). Excitation light with a wavelength of approximately 488 nm was selected from a 150W Xe lamp using a monochrometer unit . The fluorescence signals that passed through a 510 nm dichroic mirror were separated using a 590 nm dichroic mirror and detected with a CCD camera, HiSCA (Hamamatsu photonics) using a 535/55 nm band pass filter for KMG-104 and a 600 nm long-pass filter for Fura-Red, respectively. Time-lapse images were acquired every 5 sec. Fluorescence intensity (F) was calculated as the mean intensity in a region of interest (ROI) containing the entire cell body by using Aquacosmos software (Hamamatsu photonics). The values of F recorded during time-lapse imaging were normalized by the initial fluorescence intensity . The neurons were washed with HBSS and then incubated for 20 min in HBSS containing 25 nM TMRE. Next, the dye was diluted to 2.5 nM in HBSS, and the neurons were incubated for an additional 10 min to equilibrate the dye. For measurements under high Mg0 of each cell, and the data were presented and analyzed as F/F0.Fluorescence of the TMRE was measured using the fluorescence microscope, ECLIPSE TE300. Excitation light with a wavelength of approximately 560 nm was selected from a 150W Xe lamp using the monochrometer unit. The fluorescence signals that passed through a 580 nm dichroic mirror and a 600 nm long-pass filter were detected with the CCD camera. F was calculated as the mean fluorescence intensity in a ROI containing the entire cell body. The values of F recorded during time-lapse imaging were normalized by the F2+ for measurements under high Mg2+ conditions, the fluorescence was measured.To compare intracellular ATP concentrations, genetically encoded ATP sensor, ATeam , was indFluorescence imaging of the neurons was performed using a confocal laser scanning microscope system, FV-1000  with a 40× oil-immersion objective lens. ATeam was excited at 440 nm from a diode laser. The fluorescence signals were separated using a 510 nm dichroic mirror and observed at 460–500 nm for cyan fluorescent protein (CFP) and 515–615 nm for yellow fluorescent protein (YFP). Fluorescence intensity was calculated as the mean intensity in a ROI containing the entire cell body. The intracellular ATP levels are represented by the YFP to CFP fluorescence ratio (ATeam ratio).p < 0.05 was considered significantly different. For multiple testing, analysis of variance (ANOVA) was performed, and then Dunnett’s test was used to compare the control group with the other groups or Tukey’s test was used to compare all possible combinations of groups, assuming parametric distribution. 2+ extrusion mechanisms, and quinidine, amiloride, and imipramine are used to inhibit these Mg2+ extrusion mechanism i i .2+]i and [Ca2+]i were well correlated with each other immediately after the application of glutamate stimulus, and the correlation coefficient decreased with time (2+]i increase and the increasing phase of [Mg2+]i, we examined the correlation of the [Mg2+]i and [Ca2+]i maxima observed within 1 min of glutamate stimulus (2+]i is triggered by the increase in [Ca2+]i as previously shown [2+]i response, without affecting the Mg2+ response, immediately after the stimulus.Under all three conditions, the changes in i response, and the [Mg2+]i increase was completely abolished. However the decrease in [Mg2+]i remained (2+]i increasing phase is Ca2+-dependent event. Both quinidine and amiloride suppressed this Mg2+ decrease to a similar extent (2+]i increase evoked by the glutamate stimulus, and amiloride partially attenuated it (2+ mobilization other than Ca2+ influx from extracellular medium. This might have some contribution to the attenuation of glutamate-induced Ca2+ response by these inhibitors shown in 2+]i does not require the preceding [Mg2+]i and [Ca2+]i increases. This means that the [Mg2+]i-decreasing phase is not a homeostatic cellular response attempting to maintain a normal [Mg2+]i, but rather a glutamate-induced activation of Mg2+ extrusion via channels and/or transporters, and that this occurs in response to a neuronal excitation or an intracellular signal other than Ca2+. Taken together, these results indicate that glutamate stimulus induces both Ca2+-dependent Mg2+ release from the mitochondria and Mg2+ extrusion from the cytosol, simultaneously.Next, we investigated whether the Caremained a. Combinr extent b. Quiniduated it c. This s2+ extrusion had any effect on cellular energy metabolism. Since mitochondria play a central role in neuronal ATP synthesis, changes in mitochondrial membrane potential during glutamate stimulus were measured. In response to the glutamate stimulus, mitochondrial membrane potential decreased gradually (2+]i increase to the same extent (2+ for maintenance of mitochondrial membrane potential, glutamate stimulus-induced changes in the mitochondrial membrane potential under high Mg2+ conditions were compared to those under normal conditions. High Mg2+ partially attenuated the decrease in mitochondrial membrane potential (2+]i high. This supports the idea that Mg2+ has a protective effect on depolarization of mitochondrial membrane potential.Next, we investigated whether inhibitors of Mgradually a. It wasradually b. Thus, e extent , amiloriotential c,d, pres2+ extrusion and the resulting high [Mg2+]i lead to the maintenance of cellular ATP concentrations during the excitotoxicity process. Intracellular ATP concentrations were compared using a genetically encoded ATP sensor, ATeam [2+ also suppressed the glutamate-induced decrease in ATP level (2+]i resulting from the inhibition of Mg2+ extrusion contributes to the maintenance of intracellular ATP levels.We also investigated whether the inhibition of Mgr, ATeam . The preTP level b. These 2+]i activates mTOR in cultured hippocampal neurons i.In the previous study, we demonstrated that increase in i response (2+ from other than extracellular medium (2+]i transient (2+-dependency (2+ increase (2+]i increase by the amiloride does not affect glutamate excitotoxicity. Therefore, the neuroprotective effect of the inhibitors is probably due to the maintenance of high [Mg2+]i during the glutamate stimulus. This idea is also supported by the results that glutamate-induced decrease in mitochondrial membrane potential and cytosolic ATP level were partially attenuated under high Mg2+ conditions (2+]i high. Our data therefore indicate the importance of Mg2+ regulation in neuronal cell death and survival.In this study, we demonstrated that inhibitors of Mg neurons . A toxicmiloride . These imiloride . The promiloride . While qresponse e,f, in pr medium , they diransient c, in spipendency . Amiloripendency , despiteincrease e,f. Givenditions b, presum2+ in ensuring cell survival involves the regulation of mitochondrial functions [2+ concentration results in a downregulation of mitochondrial membrane potential and ATP synthesis [2+ suppresses mitochondrial Ca2+ uptake [2+]i contributes to the maintenance of cellular ATP levels was also supported by our previous work i, inhibited the Akt/mTOR pathway and cancer proliferation [2+]i within the physiological concentration range is correlated with mTOR activity [2+ is a key regulator of mTOR and mTOR-related signal transductions. Inhibition of the Akt/mTOR pathway in cultured neurons in response to toxic concentrations of glutamate has been reported [2+]i demonstrated in this study (2+]i prevent neuronal cell death. In this study, we showed that inhibition of mTOR abolished the attenuation of glutamate excitotoxicity by inhibiting Mg2+ extrusion (2+]i protect neurons against excitotoxicity via the mTOR signal.One of the ways in which Mgductions . In partions i protect cells from neurodegenerative disorders, the Mg2+ extrusion mechanisms might be a therapeutic target for neuronal diseases. Among Mg2+ channels and transporters, SLC41A1 is considered to be the major transporter for cellular Mg2+ extrusion i affects multiple aspects of the process of excitotoxicity, even though it is not a main signal for inducing neuronal cell death (2+ inside cells is important for neuronal survival under conditions of cellular stress. It may protect neurons from neurodegeneration also in vivo because it has been reported that gain-of-function mutation in SLC41A1, that leads to enhanced Mg2+ release from cytosol, and mutation in a Mg2+ and Ca2+ permeable channel, TRPM7, that attenuates cellular Mg2+ uptake, are associated with the pathogenesis of neurodegenerative diseases in human brain [2+ dynamics in vivo are needed. Our results demonstrated in this study suggest the potential of cellular Mg2+ homeostasis and the Mg2+ transport system for therapy and the prevention of neurodegenerative diseases.As discussed above and summarized in xtrusion ,48, whilsion [2+ . Preventll death . In presan brain ,51. To s


CA activators may be thus useful for improving cognition as well as in diverse therapeutic areas , for which activation of this enzyme was recently shown to be involved.Carbonic anhydrases  activators were shown to be involved in memory enhancement and learning in animal models of cognition. Here we investigated the CA activating effects of a large series of histamine based compounds, including histamine receptors (H Here we report the first such study, including in our investigations 28 such derivatives which have been assayed as activators of four pharmacologically significant isoforms, hCA I, II and VII (cytosolic isoforms) and hCA IV (membrane-anchored enzyme).Thus, histamine was the main compound used to obtain new CAAs2.2.1.1 and compounds 2–30 were commercially available, highest purity reagents from Sigma-Aldrich, Milan Italy.Histamine 2.2.2 hydration activity with Phenol red as indicator, working at the absorbance maximum of 557 nm, following the initial rates of the CA-catalysed CO2 hydration reaction for 10–100 s. For each activator, at least six traces of the initial 5–10% of the reaction have been used for determining the initial velocity. The uncatalyzed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of activator (0.1 mM) were prepared in distilled-deionized water and dilutions up to 0.1 nM were done thereafter with the assay buffer. The activation constant (KA), defined similarly with the inhibition constant (KI), was obtained by considering the classical Michaelis–Menten f is the free concentration of activator.A stopped-flow methodM ([S]≪KM), and considering that [A]f can be represented in the form of the total concentration of the enzyme ([E]t) and activator ([A]t), the obtained competitive steady-state equation for determining the activation constant is given by Equation0 represents the initial velocity of the enzyme-catalysed reaction in the absence of an activatorWorking at substrate concentrations considerably lower than K3.1 (L- and D-enantiomers), other histamine derivatives were not yet assayed for their potential CA activating effects.As mentioned above, histamine 1  was one 1R-H4R) as well as the huge number of agonists/antagonists developed for the management of various disorders, among which allergies, gastritis and gastric ulcers, narcolepsy, acute unilateral vestibulopathy, and atopic dermatitis,Considering the relatively large number of histamine receptors (H2–30 (1 and are: the H1R agonist 2-(2-aminoethyl)thiazole 6; the H2R agonists impromidine 16 and nordimaprit 19; the H3R agonists Nπ-methylhistamine 3, α-methylhistamine 4, methimmepip 8, proxyfan 9, imetit 14, VUF16839 23; the H1R antagonists pyrilamine 24, loratadine 29; the H2R antagonists metiamide 12, cimetidine 13, ranitidine 17, tiotidine 18, zolantidine 20; the H3R antagonists ciproxifan 10, clobenpropit 15, ABT239 22, GSK189254A 28, GSK334429B 30; the H4R antagonists JNJ39758979 25, JNJ7777120 26, A940894 27; the mixed modulators of the histaminergic system Nτ-methylhistamine 2, 4-methylhistamine 5, 1-methylhistidine 7, burimamide 11, betahistine 21.Some of these compounds, possessing structures 2–30 , were in2–5, the thiazolyl derivative 6 or τ-methyl-His 7, the histamine chemotype is readily observable, whereas the remaining compounds incorporate more drastic changes of the basic structure, but all of them possess moieties which can in principle shuttle protons in the pH range of 6–8 which, as mentioned earlierIn some of these compounds, such as the methyl-histamines Compounds 17, 20, 22, 25–30 did not induce any activation of the tested CA isoforms (KAs >100 µM). Consistently, these derivatives do not possess the histamine chemotype in their structures and/or other moieties that clearly make CA activation possible. As a unique exception, compound 30 reported a 5.06 µM selective activation of hCA VII. It should be stressed that other derivatives, such as 18, 19, 21, 23 and 24, do not directly include imidazole-like scaffolds in their chemical structure, but showed however to possess significant CA activation profiles in a low micromolar range (KAs between 1.36 and 45.5 µM) and are thus included in the SAR discussion. These compounds possess however protonatable moieties of the secondary amine or guanidine type in their molecule, which like the imidazole may shuttle protons and thus act as CAAs.The cytosolic and ubiquitous hCA isoforms I and II were quite efficiently activated by most active derivatives , in a low micromolar to submicrolar range. Intriguingly, derivatives 15, 21 and 23 did not produce any hCA I activation up to 100 µM, whereas 18 did not activates neither hCA I nor hCA II. The methylation of histamine 1 at position Nτ (2), α (4), 4 (5) and the imidazole/thiazole swap (6) increased up to one order of magnitude the hCA I activation profile (from 2.1 to 0.11 µM). The presence of an extra proton transfer group (COOH) as in 7 (Nτ-methyl-histidine) further improved 2-fold the KA (52 vs 110 nM) against hCA I compared to compound 2. The Nπ-methylation of histamine  slightly decreased the activation efficacy of the molecule, situations also encountered for the inclusion of the aliphatic amine into a cycle  or the amine/ether swap . Substituting the amine group with N-linked thioureas as in 11 and 12 improved 2-fold the KAs towards hCA I with respect to histamine. Among the remaining derivatives, only the bis-imidazole 16 reported an improved KA over the lead towards this isoform (KA of 0.72 µM). In fact, the presence of S-linked thioureas worsened the activation efficacy 2-  or 4-fold  with respect to the lead histamine. Oddly, the S-linked thiourea 19 showed a 2-fold improved KA compared to histamine, although bearing a dimethylamino group in place of the imidazole ring. As an exception, the N-rich compound 24 interestingly activated hCA I just two times less than histamine, in spite of a completely diverse structure. On the contrary, all histamine derivatives here reported showed a superior hCA II activation efficacy with respect to the lead (KA > 100 μM). Among mostly low micromolar CAAs (KAs in the range 2.1–13.5 μM), derivatives 3, 4 and 7 stood out as the most potent hCA II modulators of the study (KAs in the range 82 nM–0.57 μM). In particular, the α-methylation of histamine, as in 4, induced the largest increase of efficacy, up to a KA of 82 nM, when compared to the ineffective (as CAA) lead molecule.No submicromolar KA values was measured for 1–30 as hCA IV activators. Indeed, all KAs are in a rather flat low micromolar range (KAs in the range 1.02–13.9 μM), making this membrane-associated isozyme the less activated one by the compounds tested in this work. Interestingly, with the exception of compound 18, all derivatives were more efficient CAAs than the lead histamine, which showed a KA of 25.3 μM. Of note, the imidazole/thiazole swap led to the most effective activation increase with respect to the lead, with a KA of 1.02 μM in case of derivative 6.The other cytosolic isoform investigated here, hCA VII, was the most effectively activated one by the compounds investigated in this study. Indeed, a wide subset of KAs were detected in a submicromolar range (from 0.10 to 45.5 μM) for some of these derivatives. All of them showed much better activation profile than the reference compound histamine (KA of 37.5 μM) towards hCA VII. Contrariwise to hCA I and II, the most efficient CAAs were not detected among the methylhistamine derivatives 2–5: Nτ-methylhistidine 7, N-methylpiperidine 8 and the aryl ether 10, showed KAs ranging between 110 and 190 nM. The bis-imidazole 16 stood out as the most effective hCA VII activator of the study with a KA of 100 nM. Intriguingly, compound 23 did not activate hCA VII below 100 μM, whereas derivative 30, previously classified among the inactive compounds for the other CA isoforms, weakly activated this CNS-associated CA (KA of 5.06 μM). In fact, this isoform is one of the most widely spread in the brain, probably being involved in crucial metabolic/pH regulation processes, while it is not expressed in other tissues. It is thus relevant that a rather wide set of compounds was detected here , which showed a promising isoform selectivity towards hCA VII over the ubiquitous CAs (up to 100-fold over hCA II).The following structure-activity relationship (SAR) can be worked out from the data reported in Table 2 also include the literature references regarding the compounds ability to cross the BBB, which presumably should also lead to brain CA activating effects, as well as evidences for their action at central level.4.2–30 in 17, 20, 22, 25–30), not having the histamine chemotype in their structure, did not report any activation effect for the tested CA isoforms (KAs >100 µM). hCA I and II were effectively activated by methylhistamine derivatives (2–7), whereas more intriguing SAR were observed for hCA VII with more lipophilic groups  promoting greater and more selective isoform activation. Of note, a subset of selective hCA VII activators was identified, that could serve to drive the identification and optimisation of new brain specific CAAs. We are currently witnessing a second youth period for CAAs, because of innovative pharmacological studies spurring researchers to take into account these lately neglected agents for their potential clinical relevance in the treatment of emotional memory disorders, including the improvement of the clinical efficacy of exposure-based treatments of obsessive-compulsive disorders, phobias, generalised anxiety, and post-traumatic stress disorder. The here gathered data might also provide more insights on the pharmacodynamics of therapeutically used histamine modulators, whose therapeutic action and/or side effects could be related to polypharmacology. Overall, this work might bring new lights on the intricate relationship between CA activation and brain physiology.In the present study, we investigated the CA activating effects of a series of histamine receptors agonists/antagonists (compounds


Despite the increasingly widespread use of video consultations, there are very few documented descriptions of how to set up and implement video consultations in real-time practice. This step-by-step guide will describe the set-up process based on the authors’ experience of two real-time National Health Service (NHS) examples: a single health board use , and an All-Wales National Video Consultation Service roll-out (delivered during an emergency pandemic as part of the COVID-19 response). This paper provides a simple visual step-by-step guide for using telepsychiatry via the remote use of video consultations in mental health services, and outlines the mandatory steps to achieving a safe, successful and sustainable use of video consultations in the NHS by ensuring that video consultations fit into existing and new NHS workflow systems and adhere to legal and ethical guidelines. This is because many treatments are based on interpersonal ‘talking therapies’ and medication management, which typically do not require any other medical devices for clinical use, perhaps in contrast to other specialties.21 Video consultations have the potential to offer many additional benefits to patients, families and clinicians besides treatment. They are reported to improve and widen patient and family access to healthcare, support co-production and self-management, increase efficiency and improve clinical outcomes, as well as significantly reducing clinical time and patient and family travel.23There is a large and growing evidence base of published data that demonstrates an overall consensus of suitability, acceptability and satisfaction regarding the use of digital technology,24 funded Aneurin Bevan University Health Board (ABUHB) for 1 year to establish a telepsychiatry programme with the objective of providing mental health appointments to children and adolescents within Gwent mental health services. This ABUHB programme is called Connecting with Telehealth to Children in Hospital and Healthcare (CWTCH). It is a National Health Service (NHS) quality improvement project that provides faster and more efficient appointments in child and adolescent mental health services (CAMHS) using a communication platform called Attend Anywhere (https://www.attendanywhere.com). The programme tested the suitability and acceptability of telepsychiatry and measured satisfaction across a wide range of settings and uses, including paediatric wards for emergency assessments, out-patient appointments, medication reviews, autism assessments, school postvention clinics for pupil suicides, virtual groups and more. It demonstrated that telepsychiatry in CAMHS is a highly suitable adjunct to routine ways of working; once people became familiar with this way of holding appointments, it was rated as acceptable and satisfactory by patients, families and clinicians.26In 2019, the Health Foundation26 have also been endorsed by the Welsh Royal College of Psychiatrists.27CWTCH has now been rebranded and is called CWTCH Cymru. It has received local and national recognition for its success and is now considered an exemplar of good practice across Wales. CWTCH Cymru and its guiding principleshttps://digitalhealth.wales/tec-cymru) and the Welsh Government to form a National Video Consultation Service.28 This service is currently rapidly scaling up the routine use of video consultations across Wales, using the Attend Anywhere communication platform, to all appropriate primary, secondary and community care services, including mental healthcare for all ages. The National Video Consultation Service has a fully resourced website, with helpful guides, videos and toolkits, which can be accessed and used in addition to this paper.29In March 2020, in response to the COVID-19 emergency, CWTCH Cymru went into partnership with Technology Enabled Care  and in two very different contexts  has demonstrated that regardless of the scale and rate of adoption, there are distinct challenges in introducing video consultations as a new way of working in the NHS for clinicians. Clinician acceptance and use of video as an accepted alternative to established ways of working were found to be rate-limiting factors with respect to adoption and spread. This challenge is defined by the authors as ‘clinician need versus clinical need’. This definition suggests that video consultations are more likely to be adopted and accepted as a feasible approach to healthcare delivery when the ‘need’ for this way of working is defined and accepted by the clinician or service, compared with when they are defined and accepted as a clinical need. In other words, video consultations are generally more successful and sustainable when a clinician or service perceives the need themselves and requests the service, rather when they are motivated by the projection of need or want from their patient population. It is therefore essential to establish and define this ‘need’ within a service, seeing clinician/service ‘pull’ for video consultation as an essential criterion for successful adoption. When the perceived need for video consultations by clinicians and services increased in the pandemic context, there was greater willingness to test new ways of working and overcome perceived or internal barriers to change. This has significant implications for how video consultations should be introduced into services and organisations, highlighting the need to focus on both the technical and the cultural aspects of the adoption process. These findings mirror the work of Greenhalgh et al and their ‘NASSS’  framework.Despite the increasingly widespread use of video consultations, there are very few visual and fully documented descriptions of how to set up and implement such consultations in real-time practice with specific attention to features such as fitting into existing systems and the legal and ethical requirements of video consultations. This step-by-step guide will describe the process based on two examples: a single health board project , and an All-Wales National Video Consultation service roll-out (delivered during the COVID-19 pandemic emergency).33 Note that the visual diagrams and infographics used in this paper are examples based on the communication platform Attend Anywhere (https://www.attendanywhere.com); therefore, descriptions and instructions may differ slightly depending on the type of platform used in your health service.This paper was designed to be a simple and visual step-by-step guide describing how to set up and use video consultations for mental health services, including a range of ‘technical’ skills and knowledge that clinicians and services may find helpful for the adoption of video consultations. This paper will describe the various steps that have been taken by the authors in their real-time experience to initiate and implement a new video consultation programme in an existing NHS systems. The visuals used on the TEC website and in this paper are adaptations and syntheses of work from other recent video consultation reports in the UK.Ethical approval was obtained from all seven Welsh research and development departments. Consent was obtained from all participants in both CWTCH and the National Video Consulting Service.This section of the paper will provide a step-by-step guide that is divided up into three distinct sections. The first section demonstrates how to set up video consultations in an existing healthcare workflow. The second section provides the appropriate steps regarding ethical and legal principles. The third section discusses how to use video consultations in a secondary care health service, with the help of an infographic diagram.Within your healthcare service, you will need to identify a lead clinician who will decide how best to set up, use and manage the video consultations . This peYour healthcare service will need to define and agree on clinical criteria for video consultations and the suitability and appropriateness of appointment types. Following this agreement, a template for triage or suitability would need to be developed and provided to the person(s) in charge of making video appointments, e.g. a receptionist.Your service will need to identify how video consultations will be made, and who will make and deliver these appointments. Your service will also need to identify how appointment links and information will be sent to patients – for example, via a letter, verbally or via an SMS text or email – and how appointment slots will be offered, documented and given to the delivering clinician. In addition, your service will need to decide how the video consultation will be managed, how clinical templates and coding will be used for video consultations, and how these will be matched to existing systems. Your service will need to have a contingency plan for possible scenarios or problems . Finally, your service will need to consider how clinical information is later documented, for example, similarly to the hand-written notes used in usual practice.Your health service will need to identify how video consultations will be set up. For example, you will need to think about factors such as the room layout, e.g. whether it is well-lit and well-positioned, confidentiality issues and clinical appropriateness. More about this can be found in the following sections.Your service will need to decide how to deal with providing additional information; for instance, if the picture definition obtained via a video consultation is not good enough to allow accurate visualisation and identification of skin lesions, your service needs to decide how to obtain this additional information. It is important that patients are able to receive patient information leaflets as they would in a standard consultation. Your service needs to decide the best mechanism for communicating this information to them. For example, you could email them. Your service would also need to consider additional plans for in-person contact, such as for collection of prescriptions.Key considerations in the use of video consultations include legal and ethical issues, such as defining and documenting patient suitability and the role of the clinician, risk assessments and contingency planning, privacy, confidentiality, security and consent . ApproprYour healthcare service will need to define and agree to clinical criteria for video consultations and the suitability and appropriateness of appointment types. This defining and documentation process would need to include the roles and responsibilities of clinical staff.All types of mental health services using video consultations, regardless of their level of risk, should consider conducting risk assessments, and abide by safeguarding principles and ethical guidelines.To start thinking about the population your service will deliver a video consultation to, and the risk exposure that may arise, it is advised to conduct an initial ‘process mapping’ of your service. Process mapping will help your team to define video consultations in the context of your service, understand who is responsible for what and where, and to flag up any potential concerns or risk exposures. Process mapping and risk assessment will help your service to start thinking about and understanding what types of risks may arise in specific situations, and possibly how to avoid them.•Would using video consultations instead of in-person care increase risk in any way?•If so, what are these risks, and can they be resolved?•Is the risk of using video consultations greater than not seeing the patient at all?•Would these risks be the same if the service was delivered in person?•What other types of risks might there be – such as the setting, environment and clinical outcome?You would need to think about the following.On completion of process mapping and initial risk assessments, the next step would be to start thinking about – and formally agreeing to and documenting – clear and concise safeguarding contingency plans for your video consultation service. This would involve a ‘what to do’ plan in the event of an emergency or concern arising during a virtual appointment. It is advised to list a wide range of scenarios, ranging from low- to high-risk possibilities. Make it as specific to your service as possible, to make potential scenarios relatable to your staff. When developing the contingency plan, think about who is best suited to develop it and who will be following it, and consider a wide range of opinions and possibilities. Develop a list of all possible scenarios and all levels of risk exposure, and make them specific, applicable and relatable to your service.Video consultations, like any other form of healthcare delivery, will need to be treated exactly the same way as in-person care with regards to ethical guidelines and procedures. However, owing to the obvious remoteness of a virtual appointment, there are additional ethical considerations which need to be considered and applied, such as confidentiality, privacy and security issues.https://www.attendanywhere.com).It is essential that a video consultation service replicates an in-person appointment or assessment as much as possible. For example, the setting of an appointment room would ideally need to be the same as an in-person appointment room, e.g. if your service would normally use a private room for an in-person appointment, then a virtual appointment would also need this. It is also important to ensure that the platform used for video consultations is safe and secure, and that it meets your existing health systems standards, including software encryption. Many popular video chat platforms such as FaceTime and WhatsApp are not compliant with healthcare standards; therefore, you would need to seek out a safe and secure platform such as Attend Anywhere (•Implied consent (or tacit consent), which is signalled by the behaviour of an informed person in agreement. This type of consent is typically used in the delivery of ‘in-person’ healthcare.•Explicit consent is when a person actively agrees, either verbally or in writing. This type of consent is highly recommended for video consultations, as signalled (implied) behaviour may be more difficult to capture remotely.Informed consent is the process of seeking agreement from a person before taking a course of action that requires consent. Informed consent is required from any person who is receiving a video consultation. There are two types of consent.To obtain consent, the person giving it would need to be considered to fully understand the process and to have full capacity to do so. A person with incapacity, such as a child or vulnerable adult, may not be able to give informed consent; therefore, parental or guardian consent (known as assent) would be required.The final step of this guide describes how to use video consultations in a secondary healthcare service .Fig. 3HAs shown in the above Depending on the type of communication platform (e.g. Attend Anywhere) you will be using, the patient will need to be offered the video appointment; they will then need to agree to it, after which they will need to be sent the patient information sheet and URL link to access the video call.Again, depending on the type of communication platform you will be using, there will need to be clear steps set out to determine how best to use video consultations in your service.To ensure that the use of video consultations is properly integrated into your health service, it is important to capture feedback on use, acceptability, suitability and satisfaction. It is therefore advisable to establish an evaluation framework to capture this. An example of this may include attaching a basic satisfaction survey to the end of the video consultation, asking a few ‘how did it go’ questions.https://digitalhealth.wales/tec-cymru), which provides a detailed step-by-step guide on setting up a video consultation (For additional information, please see the TEC website (ultation , healthcThis paper outlines a simple visual step-by-step guide to help clinicians to set up and use video consultations in mental health services. This resource has been used to support clinicians to gain the technical skills and knowledge required to routinely use video consultations in practice. In addition, we found that there is an important ‘cultural’ aspect to successful adoption of video consultations, where the rate-limiting factor for successful adoption is the ‘need’ and ‘pull’ for this way of working to be defined and accepted by the clinician or ser vice.When the perceived need for video consultations by clinicians and services increased in the pandemic context, there was greater willingness to test new ways of working and overcome perceived or internal barriers to change. It will be interesting to see whether having made the change to this new way working in the pandemic context, clinicians and services actively choose to maintain these newly gained technical skills – and also whether patients and carers, having experienced video consultations for the first time, increase their demand for this to become a new ‘routine’ way of working. As use of video consultations increases, we will also undoubtedly learn how to titrate this offer according to need, circumstance and demographics, and discover which healthcare treatments can best be deployed or augmented through the use of video consultations.This paper provides a guide to using video consultations in the NHS, based on personal experience of the authors and feedback from their evaluation. However, it is still early days for video consultations in Wales, and more research is needed to understand more about their use, particularly what can and can't be done using video consultation, as this is still unspeculative and unproven.