Long-term evaluations 10 years after Roux-en-Y gastric bypass (RYGB) are limited. We report the development in weight and cardiovascular risk factors during 10 years after laparoscopic RYGB, with evaluation of gastrointestinal symptoms and quality of life (QoL) at 10-year follow-up.We performed a prospective longitudinal cohort study. Patients operated with laparoscopic RYGB from May 2004 to November 2006 were invited to 10-year follow-up consultations. Gastrointestinal Symptom Rating Scale (GSRS) questionnaire and two QoL questionnaires were used for analyses of gastrointestinal symptoms and QoL.A total of 203 patients were operated; nine (4.4%) died during follow-up. Of 194 eligible patients, 124 (63.9%) attended 10-year follow-up consultations. Percent excess weight loss (%EWL) and percent total weight loss (%TWL) at 10 years were 53.0% and 24.1%, respectively. %EWL > 50% was seen in 53.2%. Significant weight regain (≥15%) from 2 to 10 years was seen in 63.3%. Remission rates of type 2 diabetes, dyslipidemia, and hypertension were 56.8%, 46.0%, and 41.4%, respectively. Abdominal operations beyond 30 days after RYGB were reported in 33.9%. Internal hernia and ileus (13.7%) and gallstone-related disease (9.7%) were the most common causes. Vitamin D deficiency (<50nmol/L) was seen in 33.3%. At 10 years, bothersome abdominal pain and indigestion symptoms (GSRS scores ≥3) were reported in 42.9% and 54.0%, respectively, and were associated with low QoL.We observed significant weight loss and remission of comorbidities 10 years after RYGB. Significant weight regain occurred in a substantial subset of patients. Gastrointestinal symptoms were common and negatively impacted QoL. Roux-en-Y gastric bypass (RYGB) surgery is commonly applied for the treatment of morbid obesity. Short-term and medium-term outcomes are well documented and include health benefits such as significant weight loss, improvement in obesity-related comorbidities and improved quality of life (QoL) –5. A limLong-term evaluations on gastrointestinal symptoms after RYGB are limited. We have previously observed high rates of such symptoms 2 and 5 years after RYGB , 20. LonPending results from national and multinational databases will enlighten long-term outcomes after bariatric surgery. However, prospective cohort studies may supplement larger register-based studies, with in-depth individual evaluations. A single-center cohort study aiming to report weight development and remission of metabolic comorbidities, gastrointestinal symptoms and QoL 10 years after RYGB, were performed. We have previously reported 5-year follow-up data from this cohort .We performed a prospective longitudinal cohort study at the Department of Morbid Obesity and Bariatric Surgery, Oslo University Hospital. The institution has been a tertiary referral center for bariatric surgery since 2004, in recent years operating 250–300 patients annually. Laparoscopic RYGB was the dominating bariatric procedure performed during the inclusion period.Patients operated at our department during May 2004 to November 2006 were invited to 10-year follow-up. Preoperative and follow-up consultations were performed at the outpatient ward. Main aims were development of weight and cardiovascular risk factors during 10 years after RYGB. Postoperative complications, nutritional deficiencies, and gastrointestinal symptoms were secondary aims.The study was approved by the Regional Committee for Medical and Health Research Ethics (no. 2015/142), Health Region South-East, Norway. All participants provided written consent for study participation.Patients were advised a three-week low-calorie diet  prior to surgery. RYGB was performed laparoscopically in all patients, with a gastric pouch of about 25 ml, an antecolic alimentary limb of 150 cm, and a biliopancreatic limb of 50 cm . The gasPatients were offered follow-up consultations at 6–8 weeks, 6 months, 1, 2, 3, and 5 years after surgery. To reduce the risk of gallstone formation in patients with the gall bladder in situ, we recommend oral ursodeoxycholic acid  250 mg twice a day for six months after RYGB. Oral multivitamins (one tablet), iron (100–200 mg), vitamin D (800 IU), and calcium (1000 mg) supplementation daily, and intramuscular vitamin B12 injections (1 mg every third month) were routinely prescribed when discharged after surgery. Serum levels were evaluated at follow-up and recommendations for any adjustments were given as needed. Ten-year follow-up consultations were performed between June 2015 and June 2018.2. Percent total weight loss (%TWL) was defined as: /initial weight)) * 100. Percent weight regain (%WR) was calculated as: (current weight − weight at two years)/(preoperative weight − weight at two years) * 100, and was regarded significant if ≥ 15% [Percent excess weight loss (%EWL) was defined as: / * 100. Ideal weight corresponds to a BMI of 25 kg/mif ≥ 15% , 23.Type 2 diabetes was defined as fasting plasma glucose ≥ 7 mmol/L, glycated hemoglobin (HbA1c) ≥ 6.5% or the use of diabetic medication. Hypertension was defined by systolic blood pressure ≥ 140/90 mmHg or the use of antihypertensive medication. Dyslipidemia was defined by fasting LDL cholesterol ≥ 3.0 mmol/l, HDL cholesterol < 1.0 mmol/L (men) or < 1.3 mmol/L (women), triglycerides > 1.7 mmol/l, total cholesterol/HDL cholesterol ratio > 5 or the use of lipid-lowering medication. Metabolic syndrome was defined according to the International Diabetes Federation, and included waist circumference, type 2 diabetes, hypertension, and dyslipidemia.According to WHO’s classification, anemia was defined as hemoglobin < 12 g/dL in women and < 13 g/dL in men. Iron deficiency was defined as ferritin < 15 µg/L. Vitamin B12 and vitamin B9 deficiencies were defined as < 150 ρmol/L and < 10 nmol/L, respectively.Levels of 25-OH-vitamin D < 50 nmol/L defined vitamin D deficiency.All abdominal reoperations during the 10 years after RYGB were registered. Reoperations were defined as early (within 30 days after surgery) and late (beyond 30 days after surgery).The Gastrointestinal Symptom Rating Scale  was distributed at the 10-year follow-up consultations. GSRS yields total score and five syndrome scores  on a scale from 1 to 7  . We usedSF-36v2  was used to evaluate QoL. It provides eight health domain scores and two summary scores  from 0 to 100 . SF-36v2 was scored using Health Outcomes Scoring Software version 5.1 (Optum Inc.) .Obesity-related Problems scale  was used to measure the impact of obesity on psychosocial aspects of life and also QoL. Transformed scale scores ranged from 0 (no impairment) to 100 (maximum impairment), and scores < 40 indicated mild impairment in psychosocial functioning, scores 40–59 indicated moderate impairment and scores ≥ 60 indicate severe impairment .t test. McNemar's test or Chi-Square/χ2 test was used for comparison of categorical variables. Continuous variables are presented as means with standard deviations (SD) or median with 25th and 75th quartile, and categorical variables as number . Statistical analyses were performed using IBM SPSS® Statistics version 25 .Continuous data were compared using Student’s There was no 30-day mortality after RYGB. Nine of 203 (4.4%) patients operated died during follow-up. Among 194 patients eligible for 10-year consultations, 124 (63.9%) patients attended 10-year follow-up. Of these, 121 (97.6%) patients had also met to 5-year consultations. Median duration of follow-up was 10.8 (10.7–11.0) years. Mean age at 10 year was 50.9 (9.1) years, and 95/124 (76.6%) were females.2and 10.7 (6.1) kg/m2, respectively. In the entire cohort, %TWL > 20% was seen in 86/120 (71.7%) at 5 and in 78/124 (62.9%) at 10-year follow-up. Development in BMI during 10 years after RYGB is illustrated in Fig. Weight development is given in Table 2, weight loss and BMI change 5 and 10 years after RYGB was 33.6 (14.1) kg and 30.2 (16.6) kg, and 11.2 (4.8) kg/m2 and 9.6 (5.5) kg/m2, respectively. The corresponding numbers in 41 (33.1%) patients with preoperative BMI ≥ 50 kg/m2 was significantly higher: 44.2 (18.2) kg and 38.4 (17.7) kg, and 14.9 (6.4) kg/m2 and 12.8 (6.7) kg/m2, respectively. Patients with preoperative BMI < 50 kg/m2 had significantly higher weight and BMI loss (P < 0.005). The differences in all other parameters between the two BMI groups in Table In 83 (66.9%) patients with preoperative BMI < 50 kg/m2 and BMI ≥ 50 kg/m2. From 5 to 10 years after RYGB, we found a significant increase in the prevalence of metabolic syndrome for the entire population as well as for patients with preoperative BMI < 50 kg/m2. Lipid profiles are illustrated in Fig. P < 0.001) and to 5.5 (5.2–5.8) at 10-year follow-up (P < 0.001). The decrease from 5 to 10 year follow-up was significant (P < 0.001).For the entire cohort, the prevalence of type 2 diabetes, hypertension, dyslipidemia, and metabolic syndrome decreased significantly from baseline to both 5 years and 10 years after RYGB  and in 25/124 (20.2%) at 10-year follow-up (P = 0.004). Iron deficiency was seen in 5/99 (5.1%) at baseline, in 31/102 (30.4%) at 5-year follow-up (P < 0.001), and in 22/124 (17.7%) at 10-year follow-up (P = 0.004). The reduction in iron deficiency from 5 to 10-year follow-up was significant (P = 0.009). During follow-up, 17/124 (13.7%) received intravenous iron infusion treatment. The prevalence of vitamin D deficiency was stable from 26/83 (31.3%) at 5 to 41/123 (33.3%) at 10 years (P = 0.711).In the entire cohort, anemia was seen in 6/107 (5.6%) patients at baseline, in 19/100 (19.0%) at 5-year follow-up (At least one vitamin supplement were used by 114/124 (91.9%) patients at 10-year follow-up. The main supplements used were vitamin B12 (85.1%), vitamin D (64.9%) and multivitamins (64.9%), calcium (49.1%), and iron (44.7%).A total of 12/124 (9.7%) of patients had abdominal reoperation due to early complications. Late abdominal surgery was reported in 42/124 (33.9%) patients , reflux 1.0 (1.0–1.5), diarrhea 2.0 (1.3–3.3), constipation 1.3 (1.0–2.7), indigestion 3.0 (2.2–4.3), and total score 2.3 (1.8–3.2). Bothersome abdominal pain symptoms (scores ≥ 3) were reported by 51/119 (42.9%) patients. Bothersome symptoms for reflux syndrome, diarrhea syndrome, constipation syndrome ,and indigestion syndrome were seen in 11/119 (9.2%), 39/119 (32.8%), 25/117 (21.4%), and 65/119 (54.6%) patients, respectively. There was no significant difference in all the syndrome scores between patients with preoperative BMI < 50 kg/mLowest scores (= most disability) among the eight SF-36v2 health domains were seen for Bodily pain 41.0 (22.0–62.0), General health 45.0 (25.0–70.5), and Vitality 37.5 (12.5–57.8). The resulting physical summary score was 42.2 (32.1–51.8) and the mental summary score was 46.3 (36.2–55.2). SF-36v2 scores according to bothersome gastrointestinal symptoms are given in Fig. 2 and ≥ 50 kg/m2.Mean OP-score was 37.5 (4.2–55.2). Mild, moderate, and severe impairment of social activities were seen in 50/86 (58.1%), 15/86 (17.4%), and 20/86 (23.3%), respectively. There were no significant differences in SF-36v2 scores and OP-scores in patients with preoperative BMI < 50 kg/mOur study enlightens long-term outcomes after laparoscopic RYGB. Weight loss was significant and sustained, as were beneficial effects concerning cardiovascular risk factors. However, these health benefits were accompanied by health risks such as repeated abdominal surgery, weight regain, and long-term gastrointestinal symptoms that may negatively influence QoL.The %EWL and %TWL in our population 10 years after RYGB were comparable to what reported by others , 17. In 2 had significantly higher weight (kg) and BMI loss compared to patients with preoperative BMI < 50 kg/m2. We also found that patients with preoperative BMI < 50 kg/m2 had more weight regain 10 years after RYGB. Therefore, the differences in %EWL and %TWL in the two BMI groups were insignificant at 10 year, this in contrast to a previous study that found significant more %EWL in patients with preoperative BMI < 50 kg/m2 10–13 years after RYGB [At 10-year follow-up, patients with preoperative BMI ≥ 50 kg/m2. This increase reflects weight regain and increased weight circumference during follow-up in these patients. Beneficial lipid profiles, however, especially with significant increase in HDL, continued in the long-term as seen in Fig. 2 had higher remission rates for all comorbidities than patients with preoperative BMI < 50 kg/m2, both at 5- and 10-year follow-up, and their remission rates continued to improve to 10 years (data not shown). The metabolic benefits of RYGB may thus continue for a longer period after surgery in this subgroup of patients. A previous study evaluating 5-year outcome after RYGB has reported remission rates to be similar between the two BMI groups [The prevalence of type 2 diabetes, hypertension, dyslipidemia, and metabolic syndrome decreased significantly during follow-up  was lower than the 63.5% previously reported . ImportaThere are limitations to our study. Data on abdominal surgery after RYGB performed at other institutions were mostly patient reported, and such procedures may have been underreported. We did not have available preoperative data concerning QoL and gastrointestinal symptoms. The single-center design may reduce the external validity of our findings. Strengths of the study include the cohort design and the comprehensive and detailed follow-up from baseline to 10 years after RYGB, with paired data from several time points. Importantly, all patients were followed by in-office consultations at the same bariatric clinic. Comparable single-center and in-office long-term follow-up rates (> 10 years), has only been seen in few long-term studies , 17, 18.Significant and sustained weight loss and reduction in cardiovascular risk factors were observed 10 years after RYGB. Gastrointestinal symptoms were common and may influence QoL. The findings support the use of RYGB in selected patients.


Aldosterone and insulin elevate blood pressure by increasing the ENaC-mediated Na+ reabsorption. However, little information is available on the interactive action of aldosterone and insulin on the ENaC-mediated Na+ reabsorption. In the present study, we tried to clarify if insulin would modify the aldosterone action on the ENaC-mediated Na+ reabsorption from a viewpoint of intracellular ENaC trafficking. We measured the ENaC-mediated Na+ transport as short-circuit currents using a four-state mathematical ENaC trafficking model in renal A6 epithelial cells with or without aldosterone treatment under the insulin-stimulated and -unstimulated conditions. We found that: (A) under the insulin-stimulated condition, aldosterone treatment (1 µM for 20 h) significantly elevated the ENaC insertion rate to the apical membrane (Epithelial Na It is,K+-pump . This memembrane ,17,18,19Aldosterone and insulin are well known to modulate the intracellular trafficking process of ENaC ,21,22. H+ and is retrieved to a recycling state (Recycl) with the endocytotic rate, , kD see .The following four differential equations (see Equations (1)–(4)) respectively show the amounts of ENaC in (1) the insertion state (Insert), (2) the apical membrane state , (3) the recycling state (Recycl) and (4) the degradation state (Degrad):Equations (5)–(8) are respectively general solutions for SC observed in the present study would be the ENaC-mediated Na+ current, we applied 10 µM benzamil to the solution facing to the apical membrane where ENaC is localized, since benzamil is a specific inhibitor of ENaC at the concentration of 10 µM [SC to 0.03 ± 0.01 µA/cm2 from 0.55 ± 0.05 µA/cm2 ; 95% ISC was sensitive to 10 µM benzamil. After addition of 10 µM benzamil, basolateral application of 100 nM insulin did not show any significant action on ISC (0.04 ± 0.01 µA/cm2 30 min after insulin application (n = 3); 0.05 ± 0.01 µA/cm2 60 min after insulin application (n = 3)). In cells treated with 1 µM aldosterone for 20 h, apical addition of 10 µM benzamil abolished ISC to 0.12 ± 0.02 µA/cm2 from 3.74 ± 0.28 µA/cm2 ; 97% ISC was sensitive to 10 µM benzamil. After addition of 10 µM benzamil, basolateral application of 100 nM insulin did not show any significant action on ISC (0.15 ± 0.02 µA/cm2 30 min after insulin application (n = 3); 0.09 ± 0.02 µA/cm2 60 min after insulin application (n = 3)). Apical addition of 10 µM benzamil 5 h after insulin application respectively diminished the ISC to 0.01 ± 0.01 µA/cm2 from 0.39 ± 0.06 µA/cm2  in aldosterone-untreated cells and 0.06 ± 0.02 µA/cm2 from 2.20 ± 0.03 µA/cm2  in aldosterone-treated cells.To study if the Iof 10 µM ,25,26. ISC measured in the present study was mediated via ENaC under both conditions with and without insulin stimulation irrespective of aldosterone treatment. Therefore, in the present study, we analyzed the measured ISC considering that both the basal ISC and the insulin-stimulated ISC were the ENaC-mediated Na+ currents in cells with and without aldosterone treatment. Further, the insulin action on the ENaC-mediated ISC is mainly mediated through stimulation of ENaC translocation to the apical membrane [These observations clearly indicate that most of Imembrane , althougmembrane .SC to basolateral application of 100 nM insulin in cells with  9.6-fold to 4.02 ± 0.07 µA/cm2 (n = 5) from 0.42 ± 0.03 µA/cm2 . To compare the time-dependent change in the insulin-stimulated ISC in aldosterone-treated cells with that in aldosterone-untreated cells, we normalized the ISC to its peak value in each case ; 59.0 ± 1.1 min in aldosterone-untreated cells ). After reaching its peak value, the insulin-stimulated ISC in aldosterone-treated cells  in insulin-stimulated cells  in n = 4; p < 0.025; Insulin (−) in p < 0.001; compare Aldosterone treatment increased the recycling ratio of ENaC to the insertion state from the recycling state  in cells without insulin stimulation  in 2/day from 8111 ± 2548 μC/cm2/day in insulin-unstimulated cells  in + transport irrespective of aldosterone treatment (compare Insulin (+) with Insulin (−) in each case of ALDO (+) or (−); p < 0.001).We further evaluated the aldosterone action on cumulative NaSC) was increased 9.6-fold to 4.02 ± 0.07 (n = 5) from 0.42 ± 0.03 in cells used for Insulin (+) experiment  and 8.6-fold to 4.02 ± 0. 22 (n = 4) from 0.47 ± 0.03 in cells used for Insulin (−) experiment . These observations indicate that the aldosterone treatment increased the distribution of ENaC in the apical membrane even considering the elevation of ENaC production.Total amount of ENaC (ulin (−) . In cellulin (−) . Aldosteulin (−) , while a+ absorption 3.8-fold in insulin-stimulated cells and 5.9-fold in insulin-unstimulated cells, while the aldosterone treatment increased + absorption .As shown in ulin (−) . This me  depends p < 0.005; n = 4; Insulin (−) in First, we studied the effect of aldosterone treatment on the residency time how long an individual ENaC stays in the apical membrane each time after the insertion of ENaC into the apical membrane (+ absorption (see Equation (17) and We next studied the aldosterone action on the cumulative time p < 0.005; n = 5; Insulin (+) in p < 0.005; n = 4; Insulin (−) in n = 5) from 6.92 ± 0.33 in insulin-stimulated cells , which was almost identical to the aldosterone-induced increase in the cumulative Na+ absorption (n = 4) from 2.32 ± 0.63 , which was almost identical to the aldosterone-induced increase in the cumulative Na+ absorption . The effect of aldosterone treatment on the cumulative Na+ absorption in both insulin-stimulated and -unstimulated cells would be quantitatively explained by these two factors, + absorption , although we could not exclude a possibility that the estimated + entry through ENaC into the intracellular space across the apical membrane.The cumulative Na  depends n = 4; p < 0.025; We further studied the aldosterone treatment on the whole lifetime of ENaC  under the insulin-stimulated condition, aldosterone treatment (1 µM for 20 h) significantly elevated the ENaC insertion to the apical membrane  the ISC, 24.8 µA/cm2, in cells treated with 1 µM aldosterone for 18 h, 5-fold larger than that in the present study; and (3) the ISC, 12.1 µA/cm2, in cells treated with insulin of 100 mU/mL (≈ 600 nM) for 30 min, 10-fold larger than that in the present study. The much larger ISC reported in the study by Weisz et al. [Weisz et al.  have alsz et al.  from thaz et al.  have repz et al.  would bez et al.  and the z et al. , and TarWe also consider another possible explanation on these contradictory results as follows: (1) ENaC has very low and high open probabilities (Po) ,32; (2) SC 2.8 fold, while insulin (100 nM) has been applied for 5 h in the present study and insulin increases ISC 4.7-fold about 60 min after insulin application. These observations mean that insulin application only for 30 min would not show its maximal effect on ISC , and this short period of insulin application might be a reason why Weisz et al. [SC mainly via an increase in Po of ENaC in the apical membrane, but some part of its action would be mediated via an increase in the number of ENaC in the apical membrane, although further studies are required to clarify the contradictory results between observations reported in Weisz et al. [We should further take into consideration another possibility causing the contradictory results. Weisz et al.  have repz et al.  observe z et al.  used forz et al.  and the Gonzalez-Montelongo et al.  have repAldosterone has been well known to increase: (1) ENaC expression via elevation of ENaC mRNA expression, and (2) the residency time of ENaC in the apical membrane ,35. The After endocytosis of ENaC with ubiquitination by Nedd4-2, the ubiquitinated ENaC is trafficked to the lysosome, although some other recycling pathways are also known in the intracellular ENaC trafficking . The det of ENaC . Rab sma of ENaC . One of  of ENaC ,39. Aldo of ENaC . Thus, t+ transport by activating SGK1 via phosphatidylinositide 3’-kinase (PI3K) [Insulin is also known to elevate the ENaC-mediated transepithelial Nae (PI3K) ,41,42. Be (PI3K) ,44. Insue (PI3K) . Thus, iThese reports mentioned above indicate that both insulin and aldosterone elevate the number of ENaC in the apical membrane. However, it was unclear how aldosterone acts on the intracellular trafficking of ENaC by interacting with insulin. Aldosterone is generally known to increase the number of ENaC in the apical membrane via inhibition of ENaC endocytosis by inhibiting Nedd4-2 activity ,47, althNCTC-109 medium and fetal bovine serum were obtained from GIBCO . All other chemicals from Sigma .2, 1 mM MgCl2, 10 mM HEPES, and 5 mM glucose. The pH of both solutions was adjusted to 7.4 with NaOH.The bathing solution contained 120 mM NaCl, 3.5 mM KCl, 1 mM CaClXenopus laevis, from American Type Culture Collection  at passage 68, and were cultured on plastic flasks in a humidified incubator in a culture medium at passages 76–84. The composition of the culture media was 75% (v/v) NCTC-109 , 15% (v/v) distilled water, and 10% (v/v) fetal bovine serum at 27 °C and 1.0% CO2 in air [SC from A6 cells seeded at a density of 5 × 104 cells/well onto tissue culture-treated Transwell filter cups  and cultured for 13–15 days forming a monolayer.We obtained A6 cells, renal epithelial cells derived from the kidney of 2 in air ,25. We mSC from cultured A6 cells monolayer on the Transwell filter cup transferred to a modified Ussing chamber  by clamping the transepithelial electrical-potential-difference to 0 mV using a high-impedance millivoltmeter  [+ absorption is represented as a positive current (ISC). The bathing solution was stirred by bubbling with 21% O2/79% N2. During the ISC measuring time period, 5 h, no amino acids or serum was supplied to the bathing solution.We measured IA, USA.) ,51,52,53Insulin (100 nM) was applied to the basolateral side in Ussing chamber.Xenopus laevis (an amphibian cell line), all experiments were performed at 22~23 °C.Since A6 cells are derived from the kidney of t-test or ANOVA appropriately, and p < 0.05 was considered significant.Results shown in Tables are expressed as the mean ± standard error (S.E.). Statistical significance was determined by Student’s The present study indicates that: (A) under the insulin (100 nM)-stimulated condition, aldosterone treatment  significantly elevated the ENaC insertion rate to the apical membrane (ate (kE) A. (B) unate (kD) B; Howeve


Huntiella species are wood-infecting, filamentous ascomycetes that occur in fresh wounds on a wide variety of tree species. These fungi are mainly known as saprobes although some have been associated with disease symptoms. Six fungal isolates with typical culture characteristics of Huntiella spp. were collected from wounds on native forest trees in Greece and South Africa. The aim of this study was to identify these isolates, using morphological characters and multigene phylogenies of the rRNA internal transcribed spacer (ITS) region, portions of the β-tubulin (BT1) and translation elongation factor 1α (TEF-1α) genes. The mating strategies of these fungi were also determined through PCR amplification of mating type genes. The study revealed two new species; one from Platanusorientalis in Greece and one from Colophospermummopane and Senegalianigrescens in South Africa. These novel taxa have been provided with the names, H.hellenicasp. nov. and H.krugerisp. nov., respectively. The former species was found to have a homothallic and the latter a heterothallic mating system. Huntiella species are members of the family Ceratocystidaceae  as defined by Ambrosiella, Berkeleyomyces, Bretziella, Catunica, Ceratocystis, Chalaropsis, Davidsoniella, Endoconidiophora, Huntiella, Meredithiella, Phialophoropsis, Solaloca, Tielaviopsis, Toshiolenlla and Wolfgangiella  in Texas, USA . Moreover, one species (H.bhutanensis) is found in association with the bark beetle Ips schmutzenhoferi  J. Léonard and Senegalianigrescens (Oliv.) P. Hurter in South Africa. These fungi displayed typical culture characteristics of Huntiella spp., including rapid growth on agar medium, white fluffy mycelia when young, as well as the production of fruity aroma. Identification was accomplished, based on morphology and multigene phylogenies for the ITS, BT1 and TEF-1α gene regions. Furthermore, we considered the mating biology of these isolates in order to complement our taxonomic studies.The objective of this study was to identify two fungal isolates collected from Huntiella isolates were collected from fresh wounds of Colophospermummopane in Kruger National Park in April 2009 and another one of the South African isolates was obtained from a broken branch of a Senegalianigrescens tree damaged by elephants in Kruger National Park in June 2010. The isolates from Greece were obtained from the stump of a Platanusorientalis tree that was cut about two months before sampling, in a natural forest along the banks of the Spercheios River in Phthiotis Regional Unit during November 2018. Isolation from wood samples was performed using a trapping technique originally described by Ceratocystisplatani  Engelbrecht & Harrington using freshly-cut twigs of P.orientalis as bait , using a sterile needle under a dissection microscope . The South African isolate was obtained by transferring mycelial strands from infected wood on to MEA. Primary isolations were incubated for 3–7 d at 25 °C. From these isolations, purified cultures from single hyphal tips were prepared for morphological characterisation, phylogenetic analyses and mating-type studies. All purified isolates were deposited in the culture collection (CMW) of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, South Africa and the living culture collection (PPRI) of the South African National Collection of Fungi (NCF), Roodeplaat, Pretoria, South Africa. The dried-down type specimens were deposited in the National Collection of Fungi (PREM), Roodeplaat, Pretoria, South Africa.Isolates from Greece were made by transferring ascospore masses from the tips of the ascomata on the surface of ITS) regions 1 and 2, including the 5.8S rRNA, a partial β-tubulin 1 gene (BT1) and a partial Translation Elongation factor-1α gene (TEF-1α), amplified using the set of primers as described by All the isolates obtained in this study were used for DNA sequence-based characterisation. Total genomic DNA was extracted from the mycelium of isolates grown on 2% MEA for 3–4 d at 25 °C, using Prepman Ultra Sample Preparation Reagent  following the manufacturer’s protocols. Three gene regions were amplified for sequencing and phylogenetic analyses. These included the Internal Transcribed Spacer  to remove excess primers and dNTPs. Amplicons were sequenced in both directions using an ABI PRISM™ 3100 DNA sequencer  at the Sequencing Facility of the Faculty of Natural and Agricultural Sciences, University of Pretoria, Pretoria, South Africa.A total volume of 25 μl PCR reaction mixture contained 1 μl of DNA template, 0.5 μl (10 pM) of each primer (Forward and Reverse), 5 μl MyTaq PCR buffer  and 0.3 μl of MyTaq DNA Polymerase . The PCR reactions were conducted using an Applied Biosystems ProFlex PCR System . The PCR programme for amplification of the Huntiella spp. (except H.decorticans) were downloaded from GenBank (http://www.ncbi.nlm.nih.gov). The sequences were aligned using MAFFT v. 7 with an online FFT-NS-i strategy , Maximum Parsimony (MP) and Bayesian Inference (BI). The appropriate substitution model for each dataset was obtained using the software package jModeltest v. 2.1.5  chains were run from a random starting tree for five million generations and trees were sampled every 100 generations. Twenty-five percent of the trees sampled were discarded as burn-in and the remaining trees were used to construct 50% majority rule consensus trees. Ceratocystiscercfabiensis (isolate CMW 43029) was used as the outgroup taxon for all the phylogenetic analyses. The resulting trees were visualised using MEGA v. 7.Single gene sequence datasets of the v. 2.1.5 . The ML L v. 3.0 . Confide. 4.0b10 . Gaps we. 4.0b10  on the Ci, SMZ 18, Nikon, Tokyo, Japan) mounted with a camera (Nikon DS Ri-2) were used for all observations. Fifty measurements of each relevant microscopic structure were made when available and these are presented as minimum–maximum and average ± standard deviation.Morphological features were studied on the isolates grown on 2% MEA. The fruiting structures were initially mounted in water and this was later replaced with 85% lactic acid and in which measurements were made and images captured. Nikon microscopes  taken from an actively-growing colony was placed at the centres of Petri dishes. Four replicates per isolate were used to study growth rate and the experiment was repeated once. Colony diameters were assessed by taking two measurements perpendicular to each other for all isolates daily and growth rates were calculated. Colony characteristics were described on the same medium used for the growth studies and colours were assessed using the colour charts of MAT) of the studied Huntiella spp. was determined, based on the results of the mating type PCR reactions .All six isolates, included in this study, were successfully sequenced at all three selected gene regions for phylogenetic analyses, resulting in DNA sequence data of approximately 614, 574 and 830 bp for the ML, MP and BI were concordant and showed similar phylogenetic relationships amongst taxa , TEF-1α , 157–493 µm wide (avg. 218.2 µm), ornamented with spine-like structures, dark brown, conical, 12–29 µm long, 4–9 µm wide at base becoming attenuated; ostiolar necks upright, straight, occasionally situated at off-centre of base, darker than base when young, 344–616 µm long (avg. 515.5 µm), 34–60 µm wide (avg. 46.6 µm) at base, gradually tapering towards apex; ostiolar hyphae hyaline, straight to divergent, 15–39 µm long, 1–3 µm wide, tapering towards apex. Asci evanescent. Ascospores hyaline, subglobose, aseptate, covered with sheath giving a hat-like feature in side view, 4–5.5 × 3–4.5 µm (5 ± 0.23 × 4 ± 0.28 µm) excluding sheath.Asexual state.Thielaviopsis-like Conidiophores macronematous, simple or branched; when branched radiating from basal cell once, often reduced to conidiogenous cells. Conidiogenuscells endoblastic, hyaline, varying from lageniform to cylindrical depending spore shape; in case of thick barrel-shaped conidia, apex often becoming wider than base. Conidia hyaline, 1-celled, in two recognisable shapes; majority ellipsoidal to barrel-shaped , typical fat barrel-shaped 5–8 × 4.5–7.5 µm (5.9 ± 0.61 × 5.3 ± 0.55 µm), width of some barrel-shaped ranging 2.5–4 µm wide; rectangular-shaped , not commonly found, 5–9 × 1–3 µm (6.9 ± 1.18 × 2.3 ± 0.38 µm). Aleurioconidia not observed.Cultures on 2% MEA in dark in 8 d showing circular growth with even edge, mycelium flat, superficial, medium dense and texture becoming pelt-like with age, colour above not uniform, salmon (11f’) to ochreous (15b’) with inner half irregularly umber (13m), below ochreous (15b’) with inner half irregularly umber (13i’) at centre. Optimum growth temperatures at 30 °C at 9.6 mm/d, followed by at 25 °C (7.6 mm/d), 35 °C (7.2 mm/d), 20 °C (4.7 mm/d), 15 °C (3.2 mm/d), 10 °C (1.1 mm/d) and 5 °C (0.2 mm/d).Platanusorientalis in a natural forest along the banks of the Spercheios River, Nov. 2018, P. Tsopelas & N. Soulioti, PREM 62889, holotype (dried culture of CMW 54800), culture ex-holotype CMW 54800 = PPRI 27982, other cultures CMW 54801 = PPRI 27983.Greece, Phthiotis, near the village Kastri, occurring on freshly-cut stumps of H.hellenica developed at temperatures over 25 °C. Cultures incubated at 20 °C and below produced only the asexual state. Huntiellahellenica is closely related to H.savannae  ascomatal necks than H.savannae (average 579 μm long) and H.pycnanthi (average 673 μm long). Huntiellahellenica had larger (average 6.9 × 2.3 μm) barrel-shaped conidia than H.savannae (average 4.8 × 3 μm) and H.pycnanthi (average 6 × 3 μm). Optimal temperature for growth of H.hellenica was 30 °C, similar to H.savannae and H.pycnanthi, but H.hellenica differed from H.pycnanthi in growing minimally at 10 °C and below.The sexual state of savannae , H.pycnycnanthi  and H.kTaxon classificationFungiMicroascalesCeratocystidaceaeF.F. Liu. Marinc. & M.J. Wingf.sp. nov.60B4330B-1F8B-575A-AAA0-A8F5AE420CACMycoBank No: 835638The name refers to the Kruger National Park in South Africa, where this fungus was collected.Heterothallic with isolates having either a MAT1-1-1 gene or a MAT1-2-1 gene.Sexual state. Not observed.Asexual state. Produced on 2% MEA in 3 weeks. Thielaviopsis-like. Conidiophores macronematous, upright, simple or branched in one tier, 29–37 µm in length, often reduced to conidiogenous cells; cellsConidiogenous  enteroblastic, lageniform, 10–20 µm long, 1.5–3 µm wide, tapering towards apex. Conidia hyaline, rectangular-shaped, usually straight, with top-end conidium often club-shaped, 4–11 × 1–2 µm (avg. 6.2 × 1.7 µm). Aleurioconidia hyaline, holoblastic, mostly terminal, ellipsoidal to subglobose with an extended tube-like base, club-shaped, 4–7 × 2–3 µm (5.6 ± 0.76 × 2.5 ± 0.24 µm).Cultures on 2% MEA in dark in 8 d showing circular growth with even edge, mycelium superficial, flat, dense, colour above uniformly white, below luteous (19). Optimum growth temperatures were at 30 °C at 9 mm/d, followed by at 25 °C (8.2 mm/d), 35 °C (6.2 mm/d), 20 °C (6 mm/d), 15 °C (3.4 mm/d), 10 °C (0.9 mm/d) and 5 °C (0.3 mm/d).Senegalianigrescens, June 2010, M. Mbenoun, PREM 62883, holotype (dried culture of CMW 36849), culture ex-holotype CMW 36849 = CBS 131676 = PPRI 27952.South Africa, Mpumalanga, Kruger National Park, Satara rest camp, Colophospermummopane, April 2009, M. Mbenoun, CMW 55933, CMW 55934, CMW 55935.South Africa, Mpumalanga, Kruger National Park, Punda Maria, Huntiellakrugeri is closely related to H.hellenica described in the present study, H.cryptoformis  were longer than those of H.hellenica (average 5.9 × 5.3 μm) and H.cryptoformis (average 5.5 × 2.5 μm). In addition, H.krugeri produced hyaline aleurioconidia, which are absent in other closely-related species in the genus.toformis  and H.ssavannae . Due to Huntiella species isolated from Platanusorientalis in Greece, Colophospermummopane and Senegalianigrescens in the Kruger National Park of South Africa. These two species, provided with the name H.hellenica and H.krugeri, respectively, were shown to reside in the African Clade of Huntiella , as well as their distinct morphological characteristics. Mating studies showed that Huntiellahellenica and H.krugeri were homothallic and heterothallic, respectively. All indications were that these two species are saprobes that grow on the freshly-exposed surfaces of trees.This study led to the discovery of two novel untiella . The ideP.orientalis, from which H.hellenica emerged, was sampled approximately two months after tree felling and it was also infected by the pathogen Ceratocystisplatani, which causes a devastating disease in natural stands of P.orientalis in Greece. Colonszation of the stump with H.hellenica could have occurred on the freshly-cut surface with a contaminated tool as occurs for C.platani (The stump of platani  or was tHuntiella spp. They grew rapidly in culture; their mycelium was white when young and turned dark with age. The one species that displayed a sexual state - H.hellenica, produced hat-shaped ascospores and had short conical spines on the ascomatal bases. Temperature is known to influence the ability of Huntiella spp. to produce a sexual state (H.hellenica, which did not produce acomata below 25 °C.The novel species described in this study showed typical characteristics of al state  and thisHuntiella (BT1 and TEF-1α, provided the best resolution for species identification of Huntiella, while ITS sequences provided little information or no support.Comparison of DNA sequence data for multiple gene regions is essential when seeking to identify species in untiella . The thruntiella . Howeveruntiella . Thus prHuntiella spp. (H.hellenica has both mating-type idiomorphs and this explains the presence of sexual structures in all isolates derived from single hyphal tips. In contrast, the isolate of H.krugeri contained one mating gene and is clearly a heterothallic species of Huntiella, also consistent with the fact that the isolate produced only an asexual state. This result is also consistent with those of Huntiella spp. can have different mating strategies. Collectively, Huntiella spp. have a remarkable range of mating strategies, including homothallic and heterothallic species, as well as those exhibiting unisexuality (Huntiella.Primers, developed to identify the mating type idiomorphs in lla spp. , were efexuality . The newHuntiella, discovered in this study, bring the total number of species in the genus to 31. These are found in many different regions of the world and on a wide variety of woody substrates (Huntiella species in the future.The two new species of bstrates . The renbstrates  and reprbstrates . The desbstrates , should


The discovery of Asgard archaea has changed the existing ideas on the origins of eukaryotes. Researchers propose that eukaryotic cells evolved from Asgard archaea. This hypothesis partly stems from the presence of multiple eukaryotic signature proteins in Asgard archaea, including homologs of ESCRT proteins that are essential components of the endomembrane system in eukaryotes. However, structural and functional features of Asgard ESCRT remain unknown. Our study provides evidence that Asgard ESCRT is functionally comparable to the eukaryotic counterparts, suggesting that despite the apparent absence of endomembranes in archaea, eukaryotic ESCRT was inherited from an Asgard archaeal ancestor, alongside the emergence of endomembrane system during eukaryogenesis. vps4 null mutant of Saccharomyces cerevisiae, further supporting the functional similarity between the membrane remodeling machineries of Asgard archaea and eukaryotes. Thus, this work provides evidence that the ESCRT complexes from Asgard archaea and eukaryotes are evolutionarily related and functionally similar. Thus, despite the apparent absence of endomembranes in Asgard archaea, the eukaryotic ESCRT seems to have been directly inherited from an Asgard ancestor, to become a key component of the emerging endomembrane system.The emergence of the endomembrane system is a key step in the evolution of cellular complexity during eukaryogenesis. The endosomal sorting complex required for transport (ESCRT) machinery is essential and required for the endomembrane system functions in eukaryotic cells. Recently, genes encoding eukaryote-like ESCRT protein components have been identified in the genomes of Asgard archaea, a newly proposed archaeal superphylum that is thought to include the closest extant prokaryotic relatives of eukaryotes. However, structural and functional features of Asgard ESCRT remain uncharacterized. Here, we show that Vps4, Vps2/24/46, and Vps20/32/60, the core functional components of the Asgard ESCRT, coevolved eukaryote-like structural and functional features. Phylogenetic analysis shows that Asgard Vps4, Vps2/24/46, and Vps20/32/60 are closely related to their eukaryotic counterparts. Molecular dynamics simulation and biochemical assays indicate that Asgard Vps4 contains a eukaryote-like microtubule-interacting and transport (MIT) domain that binds the distinct type 1 MIT-interacting motif and type 2 MIT-interacting motif in Vps2/24/46 and Vps20/32/60, respectively. The Asgard Vps4 partly, but much more efficiently than homologs from other archaea, complements the  Saccharomyces cerevisiae ESCRT consists of five main subcomplexes: ESCRT-0, -I, -II, -III, and Vps4  superphylum include a homolog of eukaryotic Vps4 (CdvC) and several homologs of eukaryotic ESCRT-III subunits (CdvBs) (The cell division (Cdv) systems discovered in some archaeal orders, such as  (CdvBs) , 13. Giv (CdvBs) , 14, 15, (CdvBs) . However (CdvBs) , 17. SucThe recently discovered Asgard archaea  have been proposed to include the closest archaeal relatives of eukaryotes. This proposition stems, partly, from the findings that the Asgard genomes encode a broad repertoire of eukaryotic signature proteins (ESPs) that are far more prevalent in Asgard than they are in other archaea –21. Amon–S. cerevisiae endomembrane system. The combined phylogenetic, genetic, and biochemical analyses reveal close relationships between the ESCRT-III subunits and Vps4 of Asgard archaea and eukaryotes, to the exclusion of other archaea.Here, we explore the phylogenetic relationships among the ESCRT-III components, reconstitute and biochemically characterize the Asgard Vps4, and test its potential biological function in the heterologous Given that the ESCRT-III subunits are tightly linked to the functional complexity of ESCRT , we firs10.1128/mBio.00417-20.1FIG S1FIG S1, PDF file, 0.3 MB.Phylogenetic analysis of ESCRT-III-related proteins in the eukarya and archaea. The tree was reconstructed by maximum likelihood analysis using 156 representative amino acid sequences based on the LG+G4 model (recommended by the “TESTONLY”), with the “-bb 1000” option, and the bootstrap values are shown on nodes. Download Copyright © 2020 Lu et al.2020Lu et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.00417-20.7TABLE S1Table S1, DOCX file, 0.02 MB.Summary of proteins used in this study. Download Copyright © 2020 Lu et al.2020Lu et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the In addition to the phylogenetic results, we found that the Asgard Vps2/24/46 contained leucine-rich motifs located in the C-terminal helix and resembling the C-terminal MIM1 that are conserved in eukaryotes although some leucine residues were substituted by isoleucine in the Asgard homologs  9). The. The9). It appears likely that Vps4 structurally and functionally coevolved with ESCRT-III subunits in Asgard archaea. To explore the evolution of Vps4, an unrooted maximum likelihood phylogenetic tree was constructed for the group of ATPases, including CdvC from the TACK superphylum, Asgard Vps4, and the so-called eukaryotic “meiotic clade” comprised of Vps4, katanin 60, and spastin . As in t10.1128/mBio.00417-20.2FIG S2FIG S2, PDF file, 0.2 MB.Phylogenetic analysis of Vps4-related proteins in eukarya and archaea. The tree was reconstructed by maximum likelihood analysis using 76 representative amino acid sequences based on the LG+I+G4 model (recommended by the “TESTONLY”), with the “-bb 1000” option, and the bootstrap values are shown on nodes. Download Copyright © 2020 Lu et al.2020Lu et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Despite their high divergence demonstrated by the lack of monophyly in the phylogenetic tree , all Asg10.1128/mBio.00417-20.3FIG S3FIG S3, TIF file, 2.6 MB.Predicted “arginine collar” in Vps4 of Asgard archaea and eukarya. (A) The Walker A, Walker B, Sensor I, ARG finger, and Sensor II are conserved across all the indicated sequences and are shown to confirm the location of the “arginine collar”. Conserved arginine residues of the “arginine collar” are highlighted . The information of proteins used here can be found in Copyright © 2020 Lu et al.2020Lu et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Because Vps4 recognizes ESCRT-III subunits via the MIT domain, we specifically analyzed the phylogeny of the MIT domains of the Vps4 proteins from Asgard archaea, eukaryotes, and TACK archaea. The tree demonstrates a clear affiliation of Asgard archaea with eukaryotes that, in this case, form a clade with one of the MIT domains from Heimdallarchaeota . Affilia10.1128/mBio.00417-20.4FIG S4FIG S4, PDF file, 0.2 MB.Phylogenetic analysis of the MIT domain in Vps4-related proteins. The tree was reconstructed by maximum likelihood analysis using 48 amino acid sequences based on theLG+I+G4 model (recommended by the “TESTONLY”), with the “-bb 1000” option, and the bootstrap values are shown on nodes. Download Copyright © 2020 Lu et al.2020Lu et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the S. cerevisiae Vps4 structure. For a control for the Asgard Vps4, we include CdvC from Sulfolobus solfataricus_P2 , Odinarchaeota_LCB_4 (Odin_LCB_4), Thorarchaeota_AB_25 (Thor_AB_25), and Lokiarchaeum_GC14_75 (Loki_GC14_75) using homology modeling and molecular dynamics simulation, and compared these with the tructure .10.1128/mBio.00417-20.5FIG S5Saccharomyces cerevisiae, and TACK archaea during molecular dynamics simulations. The curves of the numbers of Vps4 MIT domain amino acid residues in the alpha conformation of Asgard and S. cerevisiae, which were calculated from the last 200-ns MD simulation trajectories, were plotted against simulation time. Download FIG S5, TIF file, 2.8 MB.The number of Vps4 and CdvC MIT domain amino acid residues in alpha conformation of Asgard archaea, Copyright © 2020 Lu et al.2020Lu et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Taken together, the above data suggest that the evolution of Asgard Vps4, especially their MIT domain, was accompanied by the functional divergence of the ESCRT-III subunits. Thus, although the Asgard Vps4 proteins are highly diverged, the results of sequence comparison, phylogenetic analysis, and structural modeling are compatible with coevolution of Vps4 with ESCRT-III subunits and an ancestral relationship between the membrane remodeling machineries of Asgard and eukaryotes. Furthermore, it can be predicted that Asgard Vps2/24/46 and Vps20/32/60 form ESCRT-III-like filaments similar to those in eukaryotes.S. cerevisiae, suggesting that the affinity of Asgard Vps4 for Vps2/24/46 is weaker than that of S. cerevisiae Vps4 for Vps2. The binding free energies for Asgard Vps4-Vps20/32/60 differed to a greater degree, indicating variation in the affinities , the values for Heimdall_LC_3  and Loki_GC14_75  were comparable to those for S. cerevisiae, and the value for Odin_LCB_4  was much lower than in S. cerevisiae.As previously described, unlike the CdvBs, Asgard Vps2/24/46 and Vps20/32/60 share the same eukaryotic ESCRT-III secondary structure, and these structures are probably responsible for their ability to bind to Vps4 like their eukaryotic counterparts . Isotherfinities . The Tho10.1128/mBio.00417-20.6FIG S61 = −4.73 × 105 cal mol−1 and ΔH2 = 9.31 × 105 cal mol−1. The curve of Heimdall_LC_3-Vps4-MIT titrated with Heimdall_LC_3 TF-Vps20/32/60 was fit to Sequential Binding Sites with ΔH1 = 2.78 × 106 cal mol−1 and ΔH2 = −2.31 × 106 cal mol−1. (B) The curve of Odin_LCB_4 TF-Vps4-MIT titrated with Odin_LCB_4 Vps2/24/46 was fit to One Set of Sites with ΔH  = 3.92 × 105 cal mol−1. The curve of Odin_LCB_4 TF-Vps4-MIT titrated with Odin_LCB_4 TF-Vps20/32/60 was fit to One Set of Sites with ΔH  = 3.31 × 105 cal mol−1. (C) The curve of Thor_AB_25 TF-Vps4-MIT titrated with Thor_AB_25 TF-Vps2/24/46 was fit to Sequential Binding Sites with ΔH1 = 3.25 × 108 cal mol−1 and ΔH2 = 1.29 × 106 cal mol−1. The curve of Thor_AB_25 TF-Vps4-MIT titrated with Thor_AB_25 TF-Vps20/32/60 was fit to One Set of Sites with ΔH  = 1.62 × 106 cal mol−1. (D) The curve of Loki_GC14_75 TF-Vps4-MIT titrated with Loki_GC14_75 TF-Vps2/24/46 was fit to Sequential Binding Sites with ΔH1 = −1.21 × 105 cal mol−1 and ΔH2 = 4.22 × 105 cal mol−1. The curve of Loki_GC14_75 TF-Vps4-MIT titrated with Loki_GC14_75 TF-Vps20/32/60 was fit to One Set of Sites with ΔH  = 1.42 × 106 cal mol−1. Binding to a transcription factor (TF) control surface was negligible (not shown). Download FIG S6, TIF file, 2.9 MB.ITC binding profiles of Asgard Vps4 MIT domain titrated with Asgard Vps2/24/46 and Vps20/32/60. (A) The curve of Heimdall_LC_3 TF-Vps4-MIT titrated with Heimdall_LC_3 TF-Vps2/24/46 was fit to Sequential Binding Sites with ΔHCopyright © 2020 Lu et al.2020Lu et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.00417-20.8TABLE S2Table S2, DOCX file, 0.02 MB.Predicted binding free energies between Vps4 and ESCRT-III subunits (Vps2/24/46 [A] and Vps20/32/60 [B]). Download Copyright © 2020 Lu et al.2020Lu et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the S. cerevisiae Vps4  and an S. cerevisiae cytochrome c isoform 1 (CYC1) terminator using the YeastFab Assembly method (S. cerevisiaevps4Δ strain by the lithium acetate (LiAc)/polyethylene glycol (PEG) method , respectively. After expression in Escherichia coli BL21, proteins were purified by Mag-Beads His-Tag Protein purification kit . The biochemical experiments in vitro show that these purified proteins are active ATPases both at 30°C and 39°C  method . As prev at 39°C , 34. We  at 39°C . Remarkaobserved . Neverthand 39°C . This obvps4 null mutation could induce formation of an aberrant prevacuolar compartment adjacent to the vacuoles, known as class E compartment, due to the block of intracellular protein trafficking  archaea possess intracellular membranes , so the https://www.ebi.ac.uk/interpro/) and NCBI’s conserved domain database.All the protein sequences were obtained either by NCBI accession number or by BLAST search  of the nS. cerevisiae, Lokiarchaeum_GC14_75, Thorarchaeota_AB_25, Heimdallarchaeota_LC_3, and Odinarchaeota_LCB_4 from the NCBI database and CdvC sequences belonging to Sulfolobus solfataricus_P2, and Bathyarchaeota  to build the homology model. The cryo-electron microscopy (cryo-EM) structure of Vps4 (E233Q) hexamer belonging to S. cerevisiae was obtained from the Protein Data Bank (PDB accession number or code 5XMI) (http://zhanglab.ccmb.med.umich.edu/I-TASSER). Sequence alignments and homology modelings of Vps4 for Lokiarchaeota, Thorarchaeota, Heimdallarchaeota, and Odinarchaeota with unknown structures were conducted using the MODELLER program . The three-dimensional structures of Vps2/24/46 and Vps20/32/60 for S. cerevisiae and four Asgard archaea were also built at the I-TASSER server. Among several three-dimensional models generated using homology modeling and ab initio method, the best model was selected after a series of refining and minimization and molecular dynamics simulation employing ff14SB force fields parameters by AMBER 16.0 package  ; subunit+ or Cl− counterions were added in sufficient number to neutralize any net charges of the structures above. All of the chemical bond lengths of hydrogen-heavy atoms were restrained by the SHAKE algorithm  simulations employing ff14SB force field parameters. The ionizable residue default protonation states in AMBER 16.0 were assigned. All MD simulations were conducted by applying cubic periodic boundary conditions (PBC) and in an explicit water box of TIP3P  water molecules  with a mlgorithm . A cutoflgorithm . Langevi2) restraints and (ii) reducing to 10 kcal/(mol·Å2), and (iii) last, for the entire system without any restraining force. Each stage was conducted with 10,000 steps of steepest descent algorithm, followed by 1,000 steps of conjugate gradient minimization to get rid of any unfavorable steric contacts for both solvent and protein molecules. Then, a NVT  simulation was conducted to slowly heat the systems temperature from 0 K to 300 K over a period of 500 ps, and density was equilibrated for 2,000 ps with a weak restraint applied to the whole protein at 1 atm and 300 K. Finally, all restraints were removed, and production MD simulations were conducted at constant pressure (1 atm) and temperature (300 K) in the NPT  ensemble. For each system, MD simulation was performed for 500 ns and repeated thrice with different random number, and a total of 1.5-μs trajectory was analyzed by using a CPPTRAJ module (The solvated systems were minimized using the PMEMD.CUDA module enabled with NVIDIA graphics processing units (GPUs) , 47 in tJ module .The binding free energies of Vps2/24/46 and Vps20/32/60 against Vps4 were calculated by molecular mechanics-generalized Born surface area (MM-GBSA) method , 29. AllEele, ΔEvdw, ΔGnp, and ΔGele represented electrostatic energy in the gas phase, van der Waals energy in the gas phase, nonpolar solvation energy, and polar solvation energy, respectively. All energy terms were calculated using MM-GBSA calculations, and the ΔGele is estimated by the generalized Born (GB) model  of the molecules by molsur with the 0.00542 and 0.92 values for SURFTEN and SURFOFF, respectively (The ΔB) model , and theectively . The decectively , 51 implS. cerevisiae, Lokiarchaeum_GC14_75, Thorarchaeota_AB_25, Heimdallarchaeota_LC_3, and Odinarchaeota_LCB_4, and CdvC coding sequences belonging to Sulfolobus solfataricus_P2, and Bathyarchaeota from NCBI database  were codon optimized by GeneDesign (http://54.235.254.95/gd/) for expression in E. coli BL21, synthesized , and respectively, cloned into a pCold-TF vector  that includes an N-terminal His tag and a soluble trigger factor chaperone tag. The E. coli BL21  bearing the recombinant vectors were inoculated in LB medium containing 100 μg/ml carbenicillin, and incubated at 37°C until the optical density at 600 nm (OD600) reached 0.6 to 0.8, and then isopropyl-d-1-thiogalactopyranodside was added at the final concentration of 0.5 mM, followed by incubation at 15°C for 18 to 24 h. The cell pellets were collected and resuspended in 20 ml binding buffer , followed by ultrasonic decomposition. Next, the target proteins were purified by Mag-Beads His-Tag Protein purification kit  with wash buffer  and elution buffer . Finally, the purified proteins were concentrated to 1 to 2 ml in phosphate-buffered saline (PBS) (pH 7.4) by 30K Amicon Ultra-15 . The concentrations of these proteins were determined by Bradford Protein assay kit . The purified Vps4, Vps2/24/46, and Vps20/32/60 belonging to S. cerevisiae, Lokiarchaeum_GC14_75, Thorarchaeota_AB_25, Heimdallarchaeota_LC_3, and Odinarchaeota_LCB_4 were used for isothermal titration calorimetry assay. The purified Vps4 belonging to S. cerevisiae, Lokiarchaeum_GC14_75, Thorarchaeota_AB_25, Heimdallarchaeota_LC_3, and Odinarchaeota_LCB_4, and Cdv belonging to Sulfolobus solfataricus_P2, and Bathyarchaeota were used for ATPase activity assay.The Vps4, Vps2/24/46, and Vps20/32/60 coding sequences belonging to The isothermal titration calorimetry (ITC) assay was conducted at 25°C using an ITC200 system . The Vps4 MIT domain (3 μM in PBS buffer) was placed in the cell and titrated with 19 injections of 10 μl of Vps2/24/46 or Vps20/32/60 (33 μM in PBS buffer) at 2-min intervals. The heat of ligand dilution into buffer was subtracted from the reaction heat, after removing the data of first injection. Data analysis was conducted using Origin 7.0 .2, 1 mM dithiothreitol [DTT]) in a total volume of 50 μl at the indicated temperature for 90 min, and was immediately stopped by liquid nitrogen. Then, the reaction mixture was added with 100 μl of malachite green color buffer  and 50 μl of 21% (wt/vol) citric acid, followed by incubation at room temperature for 30 min. Finally, the reaction mixture that turned green was attributed to the free phosphate released by Vps4 ATP hydrolysis. Additionally, the control experiments were identical to the treatment group, except that the mixture of Vps4 and reaction buffer was immediately treated with liquid nitrogen before the addition of malachite green color buffer and citric acid, and these experiments were performed to eliminate the interference of irrelevant free phosphate. Also, the empty vector was used to prove that the ATP hydrolysis is due to Vps4.The ATPase activity was determined by a slightly modified malachite green assay . In shorS. cerevisiae strain BY4741 (MATa leu2Δ0 met15Δ0 ura3Δ0 his3Δ1) and its derivative vps4 null mutant strain YPR173Ca (designated S. cerevisiaevps4Δ in this study) were from S. cerevisiae deletion mutant library  or SC-Ura medium (synthetic complete medium lacking uracil)  at 30°C unless otherwise noted. The solid media were identical to those of YPD or SC-Ura except that agar was present.The  library . S. cereSulfolobus solfataricus_P2, and Bathyarchaeota  were codon optimized by GeneDesign (http://54.235.254.95/gd/) for expression in S. cerevisiae, before synthesis by BGI Genomics Co., Ltd. (S. cerevisiae BY4741 vps4 (a 500-bp DNA sequence region upstream from the ATG start codon of this gene) was used to drive the coding sequences. Then, we assembled the coding sequences, the S. cerevisiaevps4 native promoter, and an S. cerevisiae CYC1 (cytochrome c isoform 1) terminator into a pPOT-RFP vector according to a developed YeastFab Assembly protocol  at 30°C for 20 min, and next cultured for 120 min after washes with medium. Finally, the S. cerevisiae cells were examined under an N-STORM fluorescence microscope .


This work shows that the coordination network formed by the trioxotriangulene-based ligand was effective in the improvement of cycle performance of the organic rechargeable battery.A three-fold symmetric trioxotriangulene derivative with three pyridyl groups as coordinating sites was designed and synthesized. In a cyclic voltammetry measurement, the trioxotriangulene skeleton exhibited a multi-stage redox ability from neutral radical to radical tetra-anion species. In the zinc complex of monoanion species, three pyridyl groups coordinated to the zinc ion to build up a two-dimensional coordination network with a cavity larger than 12 Å in diameter. This complex was utilized as a cathode active material of a lithium ion battery, and it exhibited a capacity of ca. 60 mAh g Organic electrode-active materials can be engineered at the molecular level, and recent investigations have improved electrochemical performances of ORBs. On the other hand, some problems specific to organic materials remain. One of the most significant issues is the decrease in capacity during the charge–discharge cycle, mainly due to the decomposition of organic materials and their dissolution into electrolyte solution (−4). The 4 neutral radical was obtained as an insoluble black solid by chemical oxidation of [(n-Hex)4N+](−4) in CH2Cl2 with 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ). This solid was stable at room temperature under air for a long period of time. 1H NMR spectrum of the monoanion −4 salt in dimethyl sulfoxide (DMSO)-d6 shows a simple spectrum with three signals at 8.90, 8.67 and 7.82 ppm 4N+](−4) salt, the asymmetric unit includes crystallographically two kinds of −4 molecules. The crystal also included crystallographically five independent water molecules and one dimethoxyethane (DME) molecules (n-Hex)4N+](−4):H2O:DME = 2:5:1. The TOT skeletons of both −4 molecules possessed nearly planar structures with C–C and C–O bond lengths of 1.36–1.48 Å and 1.23–1.26 Å  of [(n-Hex)4N+)](−4) in N,N-dimethylformamide (DMF)  and three reduction (E2–4) waves. The potentials of E2–E4 of −4 showed positive shifts by 0.2–0.3 V from those of pristine 1 due to the electron-deficient feature of the pyridine group. n-Hex)4N+)](−4) under the electrochemical oxidative condition at +1.2 V (vs. Ag/Ag+) in CH2Cl2. At the initial state, the monoanion −4 shows absorption bands of λmax = 440 and 680 nm−1, the latter of which corresponds to the highest occupied molecular orbital–lowest unoccupied molecular orbital (HOMO–LUMO) transition. As the electrochemical oxidation proceeded, these absorption bands gradually disappeared, and a new broad absorption band with λmax of ~530 nm appeared. The feature of the oxidized state is in good agreement with the 4 neutral radical synthesized by the chemical oxidation (The electronic effect of pyridyl groups on the redox ability of de (DMF) . The 4− xidation b.−4 was prepared by diffusion of solutions of Li+−4 salt in 1:1 DMSO/nitrobenzene and zinc nitrate in EtOH to give a [Zn2+(−4)(DMSO)2](NO3−)(solvent)x complex 5 as green crystals 4N+](−4)  ion. Thus, the −4 molecule can be regarded as a three- connected node constructing a 2D coordination sheet with a 63-honeycomb-type topology  ion even after the crystal structure is deformed.A zinc(II) complex of the monoanion species crystals . Complexependent . The int4N+](4−) . The solon state b. The zion state a. Bond ltopology b. The shf 3.40 Å a. A smalsordered b. In orderformed . The obs5 in the solid state was measured by the coin-type cell (vide infra) and showed several redox waves 4N+)](−4) in the DMF solution  complex. The slightly higher potential shift might be derived from the coordination to the zinc(II) ion. At a lower potential, several reduction peaks were observed at 1.5–2.2 V vs. Li/Li+, which correspond to −2.2 to −1.5 V vs. Fc/Fc+, respectively. These peaks were assignable to changes to the polyanionic species (E2–E4 in Cyclic voltammogram (CV) of complex ox waves . The firE2–E4 in , respect5 due to the strong coordination interaction is considered to be advantageous for the improvement of cycle performance as a cathode-active material of ORBs, even after deformation of the cavity shown in the single crystal structure  and acetylene black . The charge–discharge measurement of the cell was performed at 1 C in the voltage range of 1.4 to 4.0 V vs. Li/Li+. In the discharge curve, broad plateaus were observed around 3.5 V and 2.0–1.5 V vs. Li/Li+  at the first cycle was of 9.6 mA h g−1, from which the capacity of 5 itself (CTOT) was calculated as 96 mA h g−1, assuming that PVDF and AB do not contribute to charge and discharge. The initial CTOT value was ~75% of the theoretical value of 5, 128 mA h g−1, which was calculated assuming the four-electron redox of the TOT skeleton. The discharge capacity Ccathode drastically decreased to ~6 mA h g−1 (~60 mA h g−1 of CTOT) within initial several cycles, and the capacity was almost maintained up to the 1000th cycle    as a green solid. Recrystallization from DME/hexane resulted in a fine crystal suitable for single crystal X-ray diffraction analysis. melting point (mp). 154–156 °C; 1H NMR : δ 0.86 , 1.27 , 1.54 , 3.13 , 7.82 , 8.67 , 8.90  ppm; UV-Vis (CH2Cl2): λmax (ε/M−1 cm−1) 333 , 440 , 602 , 624 , 664 , 681  nm; IR (KBr): ν1242, 1292, 1486, 1598, 1614, 2859, 2953 cm−1; FAB-MS (negative mode) (%intensity): C37H18N3O3: Calcd: 552.1348. Found: 552.1354 (100%); Anal. Calcd for (C61H70N4O3)(H2O)2: C, 77.67; H, 7.91; N, 5.92%. Found: C, 77.49; H, 7.72; N. 5.82%.To a three-necked flask, n-Hex)4N+](−4)  was placed in a round bottled flask and dissolved with methanol (60 mL). To this solution, 2 M HCl aq (60 mL) was added at room temperature and stirred for 3 h. The green solution was changed to a purple suspension. The precipitate was collected by filtration and washed with water and methanol to give a brown solid (103 mg). The resulting solid was suspended in methanol in round bottled flask and LiOH•H2O  was added to the mixture and stirred for 3 h under Ar. The mixture changed from a purple suspension to a green solution. The reaction mixture was evaporated under reduced pressure to dryness. The residue was washed with water and collected by filtration. After washing with a small amount of methanol, the product was dried under reduced pressure at 50 °C for 2 h. . 1H NMR : δ 7.93 , 8.71 , 9.10  ppm; UV (KBr): λmax 268, 429, 632 nm; IR (KBr): ν1247, 1295, 1487, 1542, 1561, 1600 cm−1; Anal. Calcd for (LiC37H18N3O3)(H2O)6: C, 66.59; H, 4.53; N, 6.29%. Found: C, 66.54; H, 4.59; N, 6.16%.(−4)  in CH2Cl2 (10 mL) was added slowly. The mixture was stirred at room temperature for 2 h. The resulting precipitate was collected by filtration, and then washed with CH2Cl2, to give the neutral radical 4  as a black powder. decomposition point (dp). > 300 K (in air); UV (KBr): λmax 259, 527 nm; IR (KBr): ν1222, 1311, 1482, 1612, 1712 cm−1.In a 200-mL round bottomed flask, DDQ  was dissolved in CH5 were grown from a double-layered solution consisting of an ethanol solution of Zn(NO3)•6H2O (5.0 mM) as the top layer, and a DMSO/nitrobenzene solution (1:1) of Li+−4 (1.0 mM) as the bottom layer in ϕ 8 mm × 20 mm length glass tubes. After slow diffusion of the solution at room temperature for 2 weeks, green crystals suitable for X-ray crystal structure analysis appeared. During the diffusion, not only crystals of complex 5 but also a small amount of insoluble powder, which may be the polymorphs, appeared. This fine powder could be removed by dispersing the crude product in a solvent and the decantation, and the pure crystals were obtained by filtration followed by the drying in vacuo. UV (KBr): λmax 270, 447, 620(sh) nm; IR (KBr): ν1248, 1296, 1385, 1487, 1561, 1601 cm−1; Anal. Calcd for C41H30N4O8S2Zn: C, 58.89; H, 3.62; N, 6.70%. Found: C, 59.18; H, 4.09; N, 6.25%.The single crystals of F2 using SHELXL-2014 , data corrected for Lorentzian and polarization effects; an empirical absorption correction resulted in transmission factors ranging from 0.925 to 0.984. The final values were R1 = 0.1045, wR2 = 0.3554, and GOF (Goodness-of-Fit) = 0.912, and the maximum positive and negative peaks in the ∆F map were ρmax = 0.564 e Å−3 and ρmin = −0.312 e Å−3. The disorder of the DME molecules and flexible n-hexyl groups caused the large wR2 value.C41H24N3O14S2Zn, Mr = 912.14, monoclinic, C2/c (#15), a = 15.0694(4) Å, b = 31.7869(9) Å, c = 9.9300(3) Å, β = 118.824(2)°, V = 4167.2(2) Å3, T = 93 K, Z = 4, dcalc = 1.454 g cm−3, µ = 2.371 mm−1, 2θmax = 136.68°, λ(CuKα) = 1.54178 Å, ω scan mode, 21,303 reflections, of which 3746 were unique and 3237 were included in the refinement [I > 2.00 σ(I)], data corrected for Lorentzian and polarization effects; an empirical absorption correction resulted in transmission factors ranging from 0.9198 to 0.9944. The final values were R1 = 0.0990, wR2 = 0.2886 and GOF = 1.046, and the maximum positive and negative peaks in the ∆F map were ρmax = 2.647 e Å−3 and ρmin = −1.385 e Å−3. Due to the severe disorder of NO3− and solvent molecules within the cavity, these atoms could not be determined, and large residual densities remained in the refinement.C3 electrode in acetonitrile served as reference electrode, a 3 mm diameter glassy carbon electrode as counter electrode, and a Pt wire as working electrode. CV and DPV measurements were performed at room temperature under Ar in dry DMF containing 0.1 M (n-Bu)4N+ClO4− as a supporting electrolyte. The redox potentials were finally calibrated against the ferrocene/ferrocenium couple.Electrochemical measurements in the solution state were conducted on an ALS electrochemical analyzer model 630A . An Ag/AgNO3 as a reference electrode at suitable external potentials that could generate the oxidized species in CH2Cl2 in the presence of 0.1 M (n-Bu)4N+PF6−.UV-Vis/near-IR absorption spectra during electrochemical oxidation were measured at room temperature using an ultra-high resolution spectrometer HR4000  with a DH-2000-BAL UV-Vis-NIR light source , a 1 mm wide cell equipped with fine-mesh platinum as a working electrode, Pt wire as a counter electrode, and an Ag/AgNO5 and AB. The materials were thoroughly ground in a mortar and dispersed in N-methyl-2-pyrrolidone (NMP) containing PVDF (10 wt%), and then kneaded with an ARE-310 centrifugal mixer . A cathode sheet was prepared by pressing the composite using a PI-1201 filmcoater  on an Al sheet, followed by drying in vacuo at 120 °C for 1 h. The sheet was cut out to a diameter of 12 mm and used as a cathode disk. In a coin-type cell, a Li metal plate, a porous polymer film separator and the cathode disk were stacked, and the resulting cell was filled with the electrolyte solution in an argon-filled glove box. The electrolyte solution was a 1 M LiPF6 solution in a mixture of ethylene carbonate (EC) and diethyl carbonate (DEC) in a 3:7 ratio (vol/vol%). A VMP3 multichannel potentiostat  was used for the solid-state CV and galvanostatic discharge–charge experiment.Cathode composites of coin-type cells containing 10 wt% active cathode material were fabricated by mixing 10:80 (wt%) of zinc(II) complex TOT 4 as a tridentate ligand with three-fold symmetry successfully formed a new redox-active MOF with a zinc(II) ion. Complex 5 formed a 2D coordination network with a cavity and exhibited a multistage redox ability in the solid state. Although the ORB using 5 as a cathode active material had a small capacity due to the collapse of the crystal structure, the robust coordination bond of 4 demonstrated the improvement of the cycle performance. Although the 2D coordination network and cavity of complex 5 did not show sufficient strength, these experimental results indicate that the combination of TOT and a porous structure will highly contribute to the development of various electronic materials such as energy storage by providing an intriguing strategy for the material design. The most striking feature of TOT is the extremely high stability of the neutral radical species and its strong intermolecular singly occupied MO (SOMO)–SOMO interaction [5, by controlling the arrangement of the TOT skeletons by coordination bonds, new electrical and chemical functions can be explored, where the magnetic and/or electrical properties are deeply linked with inclusion behavior of the cavity. Studies using a redox-active porous coordination polymer as an electrochemical catalyst for oxygen reduction and oxygen evolution reactions, for example, have recently attracted much attention [TOT possess a high potential to exhibit intriguing electrical/magnetic properties [Tripyridyl-eraction . As showttention ,67. In aoperties . We are operties .


In this preclinical investigation, the feasibility of using diffusion tensor imaging (DTI) to study contrast-induced acute kidney injury (CIAKI) is explored, comparing radiographic outcomes with histopathologic and immunohistochemical findings after repeated animal exposures to iodinated contrast agent.n = 15 each), each receiving two separate injections 1 day apart: group 1 ; group 2 (iodixanol twice); and control group . Five rats were then randomly selected from each group at three separate time points  for magnetic resonance imaging (MRI). Upon MRI completion, the animals were sacrificed, examining renal tissue and serum creatinine level. DTI data served to calculate fractional anisotropy (FA) and apparent diffusion coefficient (ADC).Forty-five male wistar rats were allocated to three groups  or ADC  and tubular injury and between FA  or ADC  and hypoxia-inducible factor-1α.FA values were significantly lower in group 2 than in the others. Compared with controls, FA assessments at 1 h, 24 h, and 120 h after injections commenced were significantly lower in group 2; and ADC was significantly more pronounced at 24 h. Serum creatinine levels at 24 h were markedly elevated in both groups 1 and 2. Pearson correlation analysis revealed significant negative correlations between FA (Analytic approaches to DTI with better reproducibility should aid in monitoring the early pathophysiologic derangements of CIAKI, thus facilitating timely reversal of the detrimental effects. Nonionic iodinated contrast agents (CAs) are frequently used in medical practice, especially in computed tomography studies and interventional procedures . AlthougAccording to established guidelines, CIAKI is defined as an increase in serum creatinine (SCr), in the absence of overt kidney damage . HoweverRenal hypoxia and direct nephrotoxicity of CA are the universally acknowledged causes of CIAKI. Given the well-known fact that tubular vacuolar, tubulointerstitial fibrosis, and tubular damage occur in CIAKI, the hypoxia-inducible transcription factor-1α (HIF-1α) is also highly expressed in CIAKI . We hypoThe goal of this pilot imaging study was to explore the feasibility of DTI use for visualizing/quantifying diffusion properties and directions in the setting of CIAKI. A rat model was engaged for this purpose, delivering a single dose of CA to one group of animals and two doses to another, 1 day apart. We also measured expression levels of marker genes signaling renal damage and compared DTI-derived parameters with scored histopathologic changes.n = 15 each) for consecutive injections (1 day apart) as follows: group 1: CA then saline; group 2: CA both days; and control group: saline both days. All iodixanol  injections were intravenous (IV), administered via tail vein at a dosage of 4 g iodine/kg body weight (BW)  and formulated in accordance with the National Institutes of Health Guide for the Care & Use of Laboratory Animals. A total of 45 male wistar rats  were obtained and randomly allocated to three groups (ght (BW) . The rat2 and respiratory triggering stipulated as follows: repetition time/echo time, 3,175/126 ms; field of view (FOV), 100 × 65 mm2; slice thickness, 2.4 mm; matrix, 160 × 160; bandwidth, 125 kHz; number of signal averages (NEX), 4; number of slices, 3; acquisition time, 4 min and 4 s.All rats were fasted for 8 h before MRI examinations. In each group, 5 rats selected at random were subjected to magnetic resonance imaging (MRI) at three separate time points  post injection (PI) . MRI acq2)) on DTI images . The diffusion measures along the six axis are fitted to a 3 × 3 symmetric matrix to form a three-dimensional ellipsoid model for DTI analysis. Characteristics of this model are defined by Eigenvectors  and Eigenvalues , so as that analysis of the diffusion along the main axis can be conducted . The extI images . Mean FAAll rats were sacrificed at specified time points after MRI examinations. Blood was extracted from inferior vena cava (0.5–1.0 mL) standing in room temperature (20–25 °C) for 30 min and centrifuged at 3,500 rmp for 10 min, collecting serum for creatinine analysis serum creatinine concentration at a local clinical laboratory using an automatic biochemical analyzer according to the manufacturer’s instructions.Sections of right kidney were subjected to routine tissue processing, then embedded in paraffin and sectioned (5 μm) for hematoxylin and eosin (H&E) staining. The slides prepared were examined in blinded manner, selecting five fields of renal cortex and medulla (200× magnification) at random to gauge the extent of morphologic changes. Renal injury  was scored on a scale of 0–4 as follows: 0 ; 1 ; 2 ; 3 ; and 4  .Formalin-fixed, paraffin-embedded sections of kidney were used for immunostaining of hypoxia-inducible factor-1α (HIF-1α). After 20-min Tris-EDTA (pH 9.0) incubation at 95 °C for antigen retrieval, the slides were then blocked in 2% normal horse serum for 30 min, followed by 20 min in 3% hydrogen peroxide. Primary antibody HIF-1α  was applied at room temperature for 120 min. Biotin-labeled goat anti-mouse IgG served as secondary antibody, using diaminobenzidine (DAB) as chromogenic substrate. The brownish precipitates at points of peroxidase localization were interpreted using the smart segmentation tool of ImageJ  open-source software .p < 0.05; and all data were tested for normality. In group-wise assessments of FA or ADC, one-way analysis of variance (ANOVA) was used, conducting post-hoc comparisons via Fisher’s Least Significant Difference  or Kruskal–Wallis test . Pearson’s analysis was applied to examine correlations between DTI-derived variables  and histopathologic changes.Data collected were expressed as mean ± standard deviation values. All computations were driven by standard software , setting significance at p = 0.095 and p = 0.001, respectively). Group 2 significantly surpassed group 1 (p = 0.033) and the control group (p = 0.001) in this regard. At 120 h, mean SCr levels of groups 1 and 2 were 31.64 ± 2.09 µmol/L, and 36.74 ± 2.97 µmol/L, respectively, group 2 again significantly surpassing group 1 (p = 0.032) and the control group .Serum creatinine determinations are listed in FA and ADC values of group 1, group 2 and the control group are provided in p = 0.09); group 2: 0.236 ± 0.0140 (p < 0.0001)) or at OM (group 1: 0.433 ± 0.115 (p < 0.0001); group 2: 0.394 ± 0.00426 (p < 0.0001)), compared with controls . FA values in group 2 also significantly exceeded those in group 1, whether at CO (p = 0.003) or at OM (p < 0.0001). At 24 h, FA determinations again were significantly lower in both test groups at CO (group 1: 0.290 ± 0.0743 (p = 0.141); group 2: 0.259 ± 0.0158 (p < 0.0001)) and at OM (group 1: 0.522 ± 0.140 (p < 0.0001); group 2: 0.483 ± 0.00377 (p < 0.0001)), compared with controls ; and they were significantly greater in group 2 than in group 1 at CO (p = 0.002) and at OM (p < 0.0001). At 120 h, FA values once more were significantly lower in both test groups at CO (group 1: 0.294 ± 0.0359 (p = 1.000); group 2: 0.259 ± 0.0158 (p = 0.558)) and at OM (group 1: 0.571 ± 0.00259 (p < 0.0001); group 2: 0.510 ± 0.0342 (p < 0.0001)), compared with controls . However, only FA values at OM differed significantly in the test groups, group 2 surpassing group 1 (p < 0.0001)  .−3mm2/s) also showed similar trends in all groups (p < 0.0001); group 2: 1.968 ± 0.156 (p = 0.025)) and at OM (group 1: 2.094 ± 0.230 (p = 0.0001); group 2: 1.780 ± 0.181 (p = 0.071)), compared with controls ; and ADC values in group 2 were higher than those in group 1 at CO (p = 0.022) or at OM (p = 0.046). At 24 h, the ADC values at OM was significantly lower in group 2 (2.096 ± 0.184) than in controls . At 120 h, ADC values declined slightly at CO and at OM in all three groups, but observed differences did not reach statistical significance.Renal ADC values . Scored renal histopathology at 1 h in group 2 surpassed the 1 h scores in group 1 but failed to show significance (p = 0.111). At 24 h, significant renal injury (p = 0.001) was evident in group 2, compared with controls, whereas groups 1 and 2 did not differ significantly (p = 0.113); The same was true at 120 h, significant tubular vacuolar degeneration (p = 0.000) appearing in group 2 (vs controls), groups 1 and 2 showing similarities (p = 0.623) (p = 0.035), 24 h (p = 0.028), and 120 h (p = 0.025)  . Within = 0.025) .p = 0.008, p = 0.002, and p = 0.005, respectively).HIF-1α expression was confined to OM, as was our analysis of scored expression levels presented in r = −0.730; p < 0.001) in all animals, as did HIF-1α expression levels and FA values at OM . In all animals, tubular injury and ADC values at OM correlated moderately , whereas a strong positive correlation was shown between HIF-1α expression levels and ADC values at OM .As illustrated in MR diffusion imaging is a sensitive tool for characterizing the random microscopic motion of water molecules and has now prevailed in routine MRI protocols applied to human and animal brain studies . The conOur data indicate greater depression of FA in test groups 1 and 2, compared with control animals, although the pathologic mechanisms implicated in such shifts are uncertain . Still, r = −0.730) has indeed emerged in the present study; and as our tissue sections (H&E) attest, parenchymal damage caused by interstitial fibrosis may also alter diffusivity and impair renal function. A relation between faltering and tissue fibrosis has actually been demonstrated in a prior rat model of diabetic nephropathy  and impose long-term adverse results. However, the reason that multiple episodes of AKI accelerate long-term kidney injury is obscure. First, renal elimination of iodine was delayed after administering iodixanol, chiefly due to viscosity  , which lIncomplete clinical recovery from AKI may promote a long-term decline in kidney function. The biochemical pathways related to such progression have attracted a great deal of attention . AccordiAlthough iodixanol was injected at a dose of 4 g iodine/kg body weight, just as in previous study . This doForthcoming technologic innovations, such as arterial spin labeling, intravoxel incoherent motion, and blood-oxygen-level-dependent imaging, are awaited to clarify how changes in renal microstructure and function influence DTI parameters  and should enable acquisition of true DTI-based metrics. Unlike the intuitively gauged diffusion properties recorded in our animal groups, these may be critical when investigating potential renal injury. Partial volume effect also limited our ability to optimize DTI parameters and should be a focus of research going forward.In conclusion, we found DTI useful for assessing temporal changes of CIAKI in various animal groups. As a DTI-derived parameter, FA helped with early detection and monitoring of renal damage after repeated CA injection of rats. Ultimately, our understanding of functional recovery from AKI is pivotal in preventing AKI-to-CKD transition, unleashing substantial therapeutic and economic benefits.10.7717/peerj.10620/supp-1Supplemental Information 1FA: fractional anisotropy; CO: cortex; OM: outer medulla.Click here for additional data file.10.7717/peerj.10620/supp-2Supplemental Information 2ADC: apparent diffusion coefficient; CO: cortex; OM: outer medulla.Click here for additional data file.


Little is known about the jejunal insulin signalling pathways in insulin resistance/diabetes states and their possible regulation by insulin/leptin. We study in jejunum the relation between insulin signalling and insulin resistance in morbidly obese subjects with low (MO-low-IR) or with high insulin resistance (MO-high-IR), and with type 2 diabetes treated with metformin (MO-metf-T2DM), and the effect of insulin/leptin on intestinal epithelial cells (IEC). Insulin receptor substrate-1 (IRS1) and the catalytic p110β subunit (p110β) of phosphatidylinositol 3-kinase (PI3K) were higher in MO-high-IR than in MO-low-IR. The regulatory p85α subunit of PI3K (p85α)/p110β ratio was lower in MO-high-IR and MO-metf-T2DM than in MO-low-IR. Akt-phosphorylation in Ser473 was reduced in MO-high-IR compared with MO-low-IR. IRS1 and p110-β were associated with insulin and leptin levels. The improvement of body mass index (BMI) and HOMA-IR (homeostasis model assessment of insulin resistance index) after bariatric surgery was associated with a higher IRS1 and a lower p85α/p110β ratio. IEC  incubation with a high glucose + insulin dose produced an increase of p85α and p110β. High dose of leptin produced an increase of IRS1, p85α and p110β. In conclusion, despite the existence of insulin resistance, the jejunal expression of genes involved in insulin signalling was increased in MO-high-IR. Their expressions were regulated mainly by leptin. IRS1 and p85α/p110β ratio was associated with the evolution of insulin resistance after bariatric surgery. Intestine may play an important role in glucose homeostasis ,2,3. PreInsulin sensitivity is mediated by insulin receptor substrate-1 (IRS1) and phosphatidylinositol 3-kinase (PI3K). A reduction in IRS protein expression has been found in obese, insulin resistant and diabetic patients in skeletal muscle and adipocytes ,15. On tInsulin resistance has been widely documented in muscle and adipose tissue ,19. HoweTherefore, the aim of the present study was (a) to analyse the jejunal expression of genes involved in insulin signalling and glucose metabolism pathway in morbidly obese subjects according to the insulin resistance grade and in those treated with metformin, (b) to analyse the association with the change of body mass index (BMI) and insulin resistance of those subjects after bariatric surgery, and (c) to study the effect of insulin, glucose and leptin on the expression of relevant genes.n = 15) and with high HOMA-IR value (>4.7)  (both groups without treatment for T2DM) and a third group (n = 15) with T2DM who were only receiving metformin treatment (MO-metf-T2DM) [The study was undertaken in 45 morbidly obese subjects who underwent Roux-en-Y gastric bypass (RYGB) at baseline and 1, 3, 6 and 12 months after RYGB  Table 1Table 1. tf-T2DM) . Subjectbaseline − variable1, 3, 6 or 12 months) × 100/variablebaseline [Blood samples were collected after 10–12 h fasting at baseline and 1, 3, 6 and 12 months after RYGB. Serum was separated and immediately frozen at −80 °C until analysis. Serum biochemical parameters were measured in duplicate, as previously described ,6. Changbaseline .n = 15 per group) were obtained during bariatric surgery, 40 cm from the ligament of Treitz [Jejunal biopsy samples  was performed in triplicate using the CellTiter-Glo® Luminescent Cell Viability Assay  according to the manufacturer’s instructions [A cell viability assay in jejunal biopsy samples (ructions .n = 6) during RYGB since this group was the one that had less metabolic alterations and could be considered as the control group of the three groups of subjects studied. IEC were isolated as previously described [l-glutamine, 1% penicillin and streptomycin at 37 °C and 5% CO2 for 3 h. Tests were performed in different conditions: 5.5 mM glucose, 5.5 mM glucose + 100 nM insulin, 25 mM glucose, 25 mM glucose + 100 nM insulin, leptin 50 mg/mL and leptin 150 mg/mL. Each treatment was performed in triplicate. For this experiment, jejunal biopsy samples were only obtained from MO-low-IR subjects  were extracted with RIPA buffer  and protease and phosphatase inhibitor cocktails . Homogenates were centrifuged at 14,000× g rpm for 10 min at 4 °C, and supernatants were aliquoted and immediately frozen at −80 °C until analysis. Protein levels were analysed using the bicinchoninic acid (BCA) method . Proteins (50 mg) were denatured in 0.125 M Tris-HCl (pH 6.8), 20% glycerol, 4% SDS and 10% β-mercaptoethanol, and subjected to SDS-PAGE on polyacrylamide 4%–20% Mini-PROTEAN® TGX™ Precast Protein Gels  and electrotransferred on a polyvinylidene fluoride membrane (Trans-Blot® Turbo™ Midi PVDF) . Membranes were blocked in TBS-Tween-20 (50 mmol/L Tris-HCl (pH 7.5), 0.15 mol/L NaCl and 0.1% Tween-20) containing 5% skimmed milk for 1 h at room temperature. Afterwards, membranes were incubated with phospho-Akt serine/threonine kinase 1 (phospho-Akt) (Ser473) monoclonal antibody at 1:1000 dilution  and human/mouse/rat Akt pan specific antibody at 0.2 µg/mL  overnight at 4 °C. Membranes were washed , 0.15 mol/L NaCl and 0.1% Tween-20) and incubated with VeriBlot for IP Detection Reagent (HRP)  at 1:4000 for 2 h at room temperature. We used Clarity Max™ Western ECL Blotting Substrates  to easily and quickly visualize proteins with shorter acquisition times. Proteins were visualized using ImageQuant LAS 4000 , and quantified with the image acquisition analysis software ImageJ .Jejunal proteins (n = 45) and IEC incubations were obtained using RNeasy Mini Kit  [Total RNA from the jejunal biopsy samples (Germany) ,5,25. cDPrimers for the PCR reaction were designed based on NCBI database sequences and obtained from Proligo :18S , IRS1 , p85α , p110β , G6Pase , HK1 , PKLR ,GLUT1 ,PEPCK . Ct−Δ), and expression results are given as the expression ratio relative to the 18S gene expression.Relative expression was calculated using the comparative Ct method (2n = 5 per group) was incubated with 100 mM BIS-TRIS buffer, pH 6.5, and 200 mM glucose-6-phosphate at 37 °C for 5 min, and afterwards, 20% trichloroacetic acid was added. Samples were mixed, incubated for 5 min at 25 °C and centrifuged at 4000 rpm for 10 min. The supernatant was incubated at 25 °C for 5 min for the colour development step with the Taussky–Shorr colour reagent, and absorbance readings at 660 nm were obtained. G6Pase was analysed in triplicate. Blanks for each sample and phosphorus standard curve were included. Results were adjusted by jejunal protein concentration, which was determined according to the bicinchoninic acid method .Enzymatic activity of G6Pase was analysed by a procedure described by Sigma-Aldrich  (Sigma-Ahttp://www.r-project.org/). The sample size was calculated based on a preliminary study. A one-way analysis of variance was used for mRNA expressions and G6Pase activity, and a repeated measurements analysis for incubated IEC, accepting an alpha risk of 0.05 and a beta risk of 0.2 in a two-sided test. Differences between groups were made with Kruskal–Wallis test. The statistical differences between before and after RYGB were made with Wilcoxon test [p ≤ 0.05. The results are given as the mean ± standard deviation (SD) or mean ± standard error of the mean (SEM) in figures.All analyses were performed using R statistical software, version 2.8.1 . No significant differences were found between groups.First, we analysed the cell viability in jejunal samples by the CellTiter-Glop = 0.037) and p110β (p = 0.041) expressions were higher than in the MO-low-IR group, while the p85α/p110β ratio was lower (p < 0.001) (p = 0.010) was lower in the MO-metf-T2DM group  . CompareDM group .p = 0.005)   comparedp = 0.024) and PEPCK (p = 0.027), the expression of glycolytic enzymes HK1 (p = 0.021) and PKLR (p = 0.048), and GLUT1 (p = 0.048) were higher than in the MO-low-IR group (p = 0.041) was lower and G6Pase (p = 0.014) was higher in the MO-metf-T2DM group  and MO-metf-T2DM (p = 0.020) groups than in MO-low-IR group . p85α/p110β ratio correlated negatively with IRS1  and p110β  expressions. Also, we found different significant correlations between mRNA expression levels with anthropometric and biochemical variables, mainly insulin, HOMA-IR, BMI and waist circumference . The incubation with high levels of glucose (25 mM glucose) produced an increase of p110β (p = 0.046).Regarding the IEC treated with 5.5 mM glucose , the incp = 0.018) was higher than in the IEC incubated with a high dose of insulin (5.5 mM glucose + 100 nM insulin). p85α (p = 0.018) and p110β (p = 0.028) expressions were higher and the p85α/p110β ratio (p = 0.043) lower than in the IEC incubated with a high dose of glucose (25 mM glucose). Also, p85α (p = 0.030) and p110β (p = 0.003) expressions were higher than in the IEC incubated with 5.5 mM glucose.In the IEC incubated with a high dose of glucose and insulin (25 mM glucose + 100 nM insulin) , p110β ep = 0.033) expression, and a decrease of the glycolytic enzymes PKLR (p = 0.05) and HK1 (p = 0.01). The incubation with high levels of glucose (25 mM glucose) produced an increase of PEPCK (p = 0.035), and a decrease of GLUT1 (p = 0.023) and the glycolytic enzymes PKLR (p = 0.001) and HK1 (p = 0.002) when compared with IEC treated with 5.5 mM glucose. Regarding the IEC treated with 5.5 mM glucose , the incp = 0.035) was higher and GLUT1 (p = 0.028) and HK1 (p = 0.028) lower than in the IEC incubated with a high dose of insulin (5.5 mM glucose + 100 nM insulin). GLUT1 expression (p = 0.046) was lower than in the IEC incubated with a high dose of glucose (25 mM glucose). Also, PEPCK expression (p = 0.025) was higher and the glycolytic enzymes HK1 (p = 0.02) and PKLR (p = 0.015) lower than in the IEC incubated with 5.5 mM glucose.In the IEC incubated with a high dose of glucose and insulin (25 mM glucose + 100 nM insulin) , PEPCK ep = 0.043), p85α (p = 0.043), p110β (p = 0.043), GLUT1 (p = 0.018) and G6Pase (p = 0.020) expressions were significantly higher than in IEC incubated with 50 ng/mL of leptin  and gluconeogenic enzymes (PEPCK and G6Pase), and a decrease in phospho-Akt levels. This altered profile is slightly attenuated in MO-metf-T2DM. The IRS1, p85α/p110β ratio, HK1 and G6Pase expression are also associated with the changes in BMI and HOMA-IR after RYGB. The expression of most of the studied genes involved in the insulin signalling pathway in IEC is regulated by glucose + insulin and/or leptin.One of the main finding of this study is the increase in IRS1 and the decrease in the p85α/p110β ratio in MO-high-IR. In human adipose tissue and skeletal muscle, a decrease in IRS1 levels has been described in a state of insulin resistance ,28, althOur data could also suggest an increase in the cycle gluconeogenesis/glycolysis in MO-high-IR. We have found an increased expression of genes involved in glycolysis (HK1 and PKLR) and gluconeogenesis (PEPCK and G6Pase expression and G6Pase activity). However, the decrease found in the expression of genes involved in gluconeogenesis/glycolysis in those subjects treated with metformin was not observed for G6Pase expression and activity. This suggests that the glucose-6-phosphate continues to turn into glucose, with a possible greater release to blood. Another important finding is that IRS1 and p110β expression decreases in MO-metf-T2DM. To our knowledge, there are no studies evaluating the effect of metformin on intestinal insulin signalling.GLUT1 is involved in the insulin-independent glucose intake in the enterocyte. The increase of GLUT1 in MO-high-IR suggests that the enterocytes would have a higher glucose uptake at baseline in insulin-resistant state, slightly decreasing in those subjects in metformin treatment. This could be the result of the combination of its stimulation by leptin and insulin, as we found in the in vitro incubations and in other studies ,35. AlsoThe correlations found in this study suggest that IRS1, p85α and/or p110β expression may be regulated by molecules such as insulin and leptin. It is known that insulin and leptin share signalling pathways. Leptin may be involved in the regulation of glucose metabolism . Our in Our results also suggest that the cycle gluconeogenesis/glycolysis is regulated by insulin/leptin. High leptin levels increased G6Pase in IEC, which could contribute to the higher expression of gluconeogenic enzymes found in MO-high-IR. Interestingly, we have found a decrease in glycolytic enzymes HK1 and PKLR when IEC were incubated with high doses of glucose or insulin. However, we do not know the mechanism or the reason for the decrease of the expression of these enzymes. We could hypothesize that IECs in a state of non-insulin resistance might not metabolize a large amount of glucose under normal conditions so as not to increase hexosamine levels, since they may lead to insulin resistance . So, theHowever, what are the clinical implications of our results? Abnormalities in intestinal insulin signalling could be present in several diseases, including obesity, diabetes and cancer and could contribute to/prevent the comorbidities associated with these diseases. We found that a high IRS1, HK1 and GLUT1 expression and a low p85α/p110β ratio are associated with the changes of BMI and HOMA-IR after RYGB. However, this relation between leptin, the PI3K signal pathway and insulin resistance can be involved in other pathologies, such as colorectal cancer. A meta-analysis reported a relationship between BMI and colorectal cancer . ConversThis study has several limitations. We think that the studied proteins may be the most interesting ones involved in insulin signalling and glycolysis. However, other proteins are also involved, as those of the leptin pathway. We did not obtain enough tissue to measure all the protein and phosphorylation levels and the enzymatic activity of the studied genes. Also, it would have been interesting to introduce a control group of lean patients. However, we could not obtain jejunal biopsies from the same location of healthy non-obese patients.Our results show that in the jejunum of MO-high-IR, the IRS1, p110β, HK1 and G6Pase expression, the p85α/p110β ratio and the level of Akt phosphorylation are altered. These molecules are involved in the regulation of the insulin resistance and glucose metabolism pathways. On the other hand, metformin could be slightly counterbalancing this situation. Moreover, these genes involved in insulin signalling are associated with the changes of HOMA-IR after RYGB. We also suggest that leptin could play an important role in the increased IRS1 and p110β mRNA levels found in MO-high-IR. Together, our results could imply a great involvement of jejunum in the regulation of serum glucose levels.