Previous research has suggested that videos can be used to experimentally manipulate social stimuli. In the present study, we used the California scrub-jays’ cache protection strategies to assess whether video playback can be used to simulate conspecifics in a social context. In both the lab and the field, scrub-jays are known to exhibit a range of behaviours to protect their caches from potential pilferage by a conspecific, for example by hiding food in locations out of the observer’s view or by re-caching previously made caches once the observer has left. Here, we presented scrub-jays with videos of a conspecific observer as well as two non-social conditions during a caching period and assessed whether they would cache out of the observer’s “view” (Experiment 1) or would re-cache their caches once the observer was no longer present (Experiment 2). In contrast to previous studies using live observers, the scrub-jays’ caching and re-caching behaviour was not influenced by whether the observer was present or absent. These findings suggest that there might be limitations in using video playback of social agents to mimic real-life situations when investigating corvid decision making. The use of video stimuli offers a high degree of flexibility in the presentation of stimuli in animal experiments as it allows for a controlled presentation of inanimate objects as well as conspecifics and social agents in general. Furthermore, the possibility to alter certain features allows the experimenter to create well-suited control stimuli . This chto birds in different contexts. For example, in chickens, video playback of conspecifics produces audience effects  and re-caching once the observer has left (Experiment 2). If scrub-jays interpreted the conspecific observer in the video as a threat to their caches—like they do with live conspecific observers—they should selectively protect their caches, i.e., preferentially cache out-of-view (Experiment 1) and re-cache (Experiment 2) when the video showed a conspecific than when the video showed an empty cage or a rope hanging from the cage ceiling .Observer condition). In addition, we tested the birds in two non-social conditions: in one, the birds were presented with a video of an empty cage (Empty Cage condition) and in the second condition, birds were presented with a video of a lightly swinging rope hanging from the cage ceiling (Rope condition). This second non-social condition was run to ascertain that a difference between the Observer and the Empty Cage condition would not be due to movement on the screen.In Experiment 1, scrub-jays were presented with videos of an observer . When not being tested birds rested in outside aviaries.In a within-subject design, each bird participated in all of the three conditions and completed three separate test trials on three consecutive days. Hence, each bird saw each video once. Trial order was pseudo-randomised across birds.Individuals could either cache in a location hidden behind the opaque side of a Perspex barrier (“out-of-view” location) or behind a transparent side of a Perspex barrier (“in-view” location). Which side of the barrier was opaque was counterbalanced across subjects but held constant across conditions.Two hours prior to testing, individuals were separated from their cage partner, and maintenance diet was removed to ensure that birds were mildly hungry and thus motivated to cache or eat at the time of testing. Birds were tested in cage 1 see . At the In a pre-test, two trials were conducted to ensure that birds were comfortable with caching in the experimental set-up, particularly in the presence of the computer screen. In these pre-test trials, birds were given access to a bowl containing food items and a single caching tray, which was placed in one trial behind the opaque, and in another trial behind the transparent side of the Perspex barrier. Birds were allowed to cache for 15 min whilst the video on the computer screen showed an empty cage. The order in which the birds experienced the caching tray being behind the opaque or the transparent sides of the barrier was counterbalanced across birds. Trials were repeated until each bird cached in both trays, with a maximum of two trials per side.® Hero 4 Black with 60 frames per second and a resolution of 930p and presented with a Lenovo® Thinkpad Edge E330  on a 17″ portable Lenovo® LCD monitor (60 Hz refresh rate). Note that LCD pixels do not flash between frames, and hence, LCD monitors exhibit no refresh-induced flicker.The videos presented were 15-minute long, without sound, and consisted of a looped sequence of 4 to 5-second long recordings. Videos were filmed using a GoProThe screen with the video was mounted to the wall of cage 2 see . The vidBirds serving as actors in the Observer Condition were kept mildly hungry by removing all food from their cage 30 min prior to filming. Actor birds were presented with food behind the camera to ensure that they attended to the direction of the camera and thus would appear to be attending to the cacher during the test trials.Actors were chosen based on their reaction to being filmed: we choose only birds that sat calmly on a perch in front of the camera. All actor birds were familiar to the focal birds and were of the same sex as the respective focal bird. Three sets of videos were shot with three different birds to prevent any effects being caused by study-irrelevant features of the video, such as the identity of the conspecific shown.® blocks attached to one long side of the tray. A bowl containing 50 food items (the type of food depended on the cacher’s food preference) was placed equidistantly to both trays.The caching trays were plastic ice-cube trays with 2 × 7 cubes moulds filled with corn kibble. These caching trays were made visually trial-distinctive with Lego Duploout−of−view–Cachedinview) was compared across conditions. If scrub-jays represented the conspecific observer in the video as a threat to their caches, this difference should be larger in the Observer condition than in the two non-social conditions, DObserver > DEmptyCage and DObserver > DRope.Items cached in the trays during the caching period were counted and recorded by the experimenter. To obtain one independent measure for the birds’ preference for caching in one of the two locations (“in view” versus “out-of-view”) in Experiment 1, the difference of the number of caches made in the “out-of-view” tray minus the number of caches made in the “in-view tray” (D = CachedAligned rank transformed data  were subBF) using JASP Version 0.8.1.2  with a Bayesian repeated measures ANOVA with the within-subject factor Condition (Empty Cage vs. Observer vs. Rope). Here, the null hypothesis was that Condition does not influence the preference for one of the two trays. A BF10 > 3 would suggest support for H1  and a BF01 < 0.333 would suggest support for the H0 .Additionally, we analysed the data using Bayesian statistics. A failure to reject the null-hypothesis using frequentist inferences cannot be used to draw conclusions about the absence of an effect . Thus, wn = 9). F = 1.34, p = .290).One male subject (Subject No. 210) was excluded from the analysis because he failed to cache for the duration of all test trials  = 0.572, p = .575; see Z =  − 0.043, p = .523, d =  − 0.014), or the Rope condition .Critically, there was also no main effect of Condition on the difference of caches made in the “in-view” minus “out-of-view” tray  = 0.021, p = .980).In order to check whether the trial sequence had an effect on caching, we assessed the number of caches made in both trays across trials post-hoc. BF10 = 0.279 ± 0.572, suggesting that our data support the null-hypothesis, i.e., a similar preference for the two trays across all three conditions. Thus, our data suggest that the scrub-jays did not adjust their cache-protection strategies in response to the video playback presented. When comparing the Empty Cage and the Observer condition with a Bayesian t-test, we found again that our data supported the null-hypothesis, DObserver = DEmptyCage, BF10 = 0.331  ±  ∼ 0.004, whilst the comparison between the Rope and the Observer conditions showed only anecdotal evidence for the null-hypothesis, DObserver = DRope, BF10 = 0.780  ±  ∼ 1.560e−4.The Bayes factor was a conspecific compared to the non-social controls.These findings suggest that the scrub-jays’ caching behaviour was not affected by the presence of the conspecific displayed on the video screen: the birds were not more likely to employ cache protection strategies when presented with a video of Experiment 2 addressed the question of whether the jays’ re-caching behaviour was influenced by the different videos. Cachers had access to two caching trays, both “in-view”, and, after a break of 15 min, had the opportunity to re-cache ‘in private’, i.e., in the absence of the observer, any caches made in the initial caching period. As in Experiment 1, birds were presented with different videos during caching, namely of an observer , as well as of an empty cage and a rope hanging from the ceiling . The proportion of re-caches  made was investigated. If the scrub-jays were sensitive to the video presented on the video screen, they were expected to re-cache a larger proportion of initially made caches after having had cached in front of a video showing the observer (Observer condition) than after having had cached in front of a video showing an empty cage (Empty Cage condition) or a rope hanging from the ceiling (Rope condition).1One male bird, 210, was not tested because he failed to cache in Experiment 1.sexually mature scrub-jays were housed and tested under the same conditions as laid out in Experiment 1 between February and June 2015. All birds had previously participated in Experiment 1. The overall setup was the same as in Experiment 1, except that a fully transparent divider was placed between cages 1 and 2 such that there was no difference in the visual accessibility of the two caching locations, i.e., both caching trays were “in-view”.Five female and four maleEach bird completed three test trials, separated into 3 phases, as described in Experiment 1. Importantly, during the 15-minute long re-caching phase, the video screen was covered, thus re-caching was in “private”. Note that new videos were shot for this experiment to avoid habituation, and new actor birds were used, that is, the focal birds saw a different observer than in Experiment 1.To ensure that birds could re-cache items, birds had to initially have cached at least one food item in each condition. In an equivalent manner to Experiment 1, we analysed the proportion of re-cached items  with a repeated measures ANOVA (factor Condition) and one-sided permutation tests as planned contrasts.BF) using JASP Version 0.8.1.2  with a Bayesian repeated measures ANOVA with the within-subject factor Condition (Empty Cage vs. Observer vs. Rope). Here, the null hypothesis was that Condition does not influence the preference for one of the two trays. A BF10 > 3 would suggest support for H1 and a BF01 < 0.333 would suggest support for the H0.Additionally, we calculated a Bayes Factor (n = 8). F = 1.133, p = .349).Subject Nr. 215 was excluded from the analysis because she failed to cache across all test trials  = 0.29, p = .75, see Z =  − 0.041, p = .520, d = 0.00), or than in the Rope condition .Crucially, there was no effect of Condition on re-caching, that is, birds did not alter the proportion of re-cached items depending on the different stimuli presented on the video,  = 0.009, p = .991). There was also no effect of trial number on the number of cached items  = 0.404, p = .675).Similarly to Experiment 1, we assessed whether the trial sequence had an effect on caching as well as on the proportion of re-caching. BF10 = 0.282 ± 0.780, suggesting that our data support the null-hypothesis, i.e., the amount of re-caches was similar across conditions. Comparisons using Bayesian t-tests showed that there was no difference in re-caches between the Observer and the Empty Cage conditions, BF10 = 0.327  ±  ∼ 0.014 or the Observer and the Rope conditions, BF10 = 0.268  ±  ∼ 0.002. Thus, our results suggest that the scrub-jays did not adjust their re-caching behaviour in response to the video playback presented.The Bayes factor was In this study, we used California scrub-jays’ cache protection strategies to assess the use of video playback when presenting social stimuli to corvids. Scrub-jays are known to protect their caches against potential pilferage by conspecific observers by caching out of the conspecific’s view  or by reMore specifically, in Experiment 1, the scrub-jays did not prefer to cache in an “out-of-view” tray over an “in-view” tray when presented with a video playback of a conspecific compared to the two non-social conditions. Similarly, in Experiment 2, the birds did not re-cache more when presented with a video of conspecific during an initial caching event compared to the non-social conditions. In a suite of previous studies using live observers with a very similar setup, the scrub-jays cached more in sites that were “out-of-view” of an observer and re-cached their caches once an observer has left the scene, while they did not show the same pattern of behaviours in a non-social condition, i.e., when they were caching in private . Althougnot adjust their behaviour to what was shown on the video. Especially relevant for this present study is the finding that rooks are able to differentiate between individual conspecifics presented on a video screen (Consequently, contrary to the reports of successful use of video playback to display test stimuli to birds e.g., , the biro screen . In addio screen . Hence, o screen . There ao screen . Compareo screen  and are o screen . Additioo screen . In otheo screen . In futuIn our case, it seems likely that the absence of cache-protection strategies reported is associated with factors that might be especially important for the socio-cognitive nature of the caching context. One potentially important feature of a conspecific that was not available to the birds in this study specifically might be a real time response in the behaviour of the video conspecific. The relevance of a conspecific’s behaviour has previously been reported in the successful recognition of and discrimination between conspecifics . Similarwhich features of an observer trigger cache-protection strategies in corvids. One possibility might be the ‘interaction’ with the conspecific. In contrast to the dynamic relationship between a cacher and a live conspecific observer, the conspecific in the video did not respond to the cacher’s behaviour in any way. This lack of interaction may have been an unusual situation for the birds being tested and might have led them to believe that their caches were safe, or at least that circumstances were ‘unusual’. Relatedly, scrub-jays have been shown to treat a mirror-image of themselves during caching as if they were in private, rather than as if the observed image in the mirror was a live observer. In other words, the ‘conspecific’ reflected by the mirror image was not sufficient to elicit cache protection strategies (behaviour of an observer are necessary conditions for cache-protection strategies to occur (While not the main focus of the present study, our results raise the additional question of rategies . In addirategies . Consequto occur .In conclusion, our results suggest limitations in using video playback to present social stimuli to corvids. Depending on the context, different stimuli of a conspecific might be necessary in order to recognise conspecifics, and to behave accordingly. Specifically, there might be some limitations in regards to the use of video playback to mimic real-life situations involving social agents, such as being observed by a conspecific, to a sufficient extent to influence the birds’ decision making such as their cache-protection behaviour.


Recent data show that some patients will have insufficient weight loss or experience weight regain after sleeve gastrectomy. Dilation of the sleeve over time or use of an inadequate technique may contribute to relapse of morbid obesity. Repeat sleeve gastrectomy is the most obvious option but requires stapling of scarred tissue, has a higher risk of leakage, and is prone to re-enlargement with time. We herein describe a simple and innovative technique with which to revise vertical sleeve gastrectomy (VSG) into functional single-anastomosis gastric bypass (f-SAGB).Twelve VSGs were converted to f-SAGB by placing a GaBP Ring  at the base of the “sleeve” and performing the anastomosis above the ring. The length of the biliopancreatic loop was chosen according to the volume of the pouch and the patient’s residual eating capability.2 at the 12-month follow-up. No ring-related complications were reported.All procedures were completed by laparoscopy and were uneventful. The average decrease in the body mass index was from 41.0 to 29.5 kg/mf-SAGB is a low-risk and effective option with which to revise VSG in patients with inadequate weight loss. Avoiding detachment of the pouch from the antrum assures full reversibility of the procedure and preserves the chance to explore the remnant stomach and biliary tree. Bariatric surgery is an evolving science, and the perfect surgical intervention does not exist. Although the effectiveness of bariatric surgery is no longer questionable, recurrence of obesity remains a major concern for bariatric patients. We have thus assisted in the decline in performance of adjustable gastric banding and the progressive disappearance of vertical-banded gastroplasty. The rising number of patients undergoing a second bariatric procedure has captured the attention of the bariatric surgery community, making revision surgery one of the main research topics in the recent years , 2.Poor results after vertical sleeve gastrectomy (VSG) may be related to various factors, such as incorrect indications, patient behavior, and biology, or use of a poor operative technique . Repeat sleeve gastrectomy is the most obvious option, but stapling of scarred tissue leads to an increased risk of leakage; additionally, the re-sleeved pouch is prone to re-enlargement with time , 3.Another strategy involves revision of VSG into Roux-en-Y gastric bypass or a malabsorptive procedure , 4, 5. TSingle-anastomosis gastric bypass (SAGB) has gained popularity, and excellent results have been reported in terms of high rates of weight loss and weight maintenance and a very low rate of complications –13. SAGBWe herein describe the original functional SAGB (f-SAGB) technique as our revision strategy after VSG.2. After VSG, the average BMI decreased to 35.0 ± 14.3 kg/m2 and later increased to 41.0 ± 11.7 kg/m2 at the time of revision surgery. The mean delay from VSG to f-SAGB was 37 months . Five patients had previously undergone gastric band removal; in five cases, the primary surgery was performed by laparotomy.Twelve patients  who previously underwent VSG were referred to our unit for insufficient weight loss or weight regain. The average body mass index (BMI) at the time of VSG was 48.4 ± 12.4 kg/mn = 5) and those with a dilated gastric fundus   Fig. . Eating Each patient received a detailed description of the potential collateral effects and complications related to this surgery, and each patient provided written informed consent.Cefazolin (2 g) was administered as prophylaxis. An antithrombotic agent  was administered 4 h after the end of the procedure and discontinued after 15 days.The procedure was a three-trocar laparoscopy; the trocars were placed according to the patient’s anatomy, surgeon’s comfort, and previous scars. After establishment of pneumoperitoneum with a Veress needle in the left subcostal margin, access to the peritoneal cavity was gained with a 5-mm optic trocar in the epigastric region, about 15 to 20 cm from the xiphoid and 2 to 3 cm to the right of the midline. A 12-mm trocar was placed four to five fingers from the left subcostal margin at the level of the anterior axillary line; the last 5-mm trocar was placed near the right subcostal margin 3 to 4 cm from the midline, trespassing the falciform ligament. A 30-degree, 5-mm camera was positioned in the epigastric trocar and fixed to an autostatic holder .The surgeon stood on a 15-cm-high footboard on the right side of the patient. The patient was placed in the head-up position and rotated to the right side to approximate the superior pole of the spleen and the left 12-mm trocar to the surgeon. The gastric sleeve is usually involved in the formation of firm adhesions among the left lobe of the liver, the omentum, and the staple line; moreover, the esophagogastric junction is often involved in the formation of dense scar tissue in patients who have previously undergone gastric banding. A 40-Fr orogastric tube was moved forward, pushing the pouch and exposing the angular incisure. Dissection was confined to this portion of the gastric wall and extended about 3 to 5 cm on both sides of the lesser and greater curvatures to avoid damage at the esophagogastric junction and bleeding. A GaBP Ring  was then introduced into the peritoneal cavity and placed over the sleeve as distally as possible. The gastrotomy was opened just above the ring, 12 to 15 cm from the esophagogastric junction. No stitches were released to maintain the position of the ring . After marking a point 300 cm proximal to the ICV, an absorbable clip was released as a marker on both sides of the mesentery.The surgeon again moved to the right side of the patient and began measuring from the ligament of Treitz. Measurement of the entire bowel was completed when the mark previously placed 300 cm from the ICV was reached.Medtronic).The bowel was progressively moved upward during measurement, keeping the inferior pole of the spleen as a target. An enterotomy was made, and a 30-mm Tri-Staple Endo GIA  was introduced within the bowel and directed proximally. The bowel was rolled 270° with rotation on the major axis of the stapler to spread the tension on the intestinal wall. With a counterclockwise translation, the limb was approximated to the gastric pouch. While the patient was placed in the reverse Trendelenburg position, the pouch was pulled down; the ring was handled with the surgeon’s left hand. Laterolateral anastomosis was then performed. The service hole was closed with a two-layer barbed suture  , 16, 23.The f-SAGB technique we have herein described involves placement of a non-adjustable ring (Fobi ring) at the base of the sleeve instead of stapling and dividing the pouch from the antrum. The ring is responsible for light compression of the gastric wall; the increased pressure above this neo-pylorus diverts food and liquids through the anastomosis into the bowel loop. The size of the ring is chosen according to the size of the pouch.Prosthetic devices have been used in bariatric operations to control the outlet of the gastric pouch and thus maintain weight loss. A complication of these prostheses is erosion or migration into the gastric lumen. Fobi et al.  reportedNotably, when performing f-SAGB, the Billroth II gastroenterostomy creates a low-pressure system above the ring and facilitates transit through the anastomosis and not through the ring. Thus, we can imagine that the stress on the gastric wall at this level is minimal. Additionally, no new staple lines are in contact with the foreign body, and the integrity of the serosa is preserved because no sutures are released, reducing the risk of slippage and migration.Furbetta  describeThe preserved duodenal continuity in this f-SAGB technique allows for exploration of the remnant stomach and biliary tree in patients with malignancy, bleeding, or lithiasis. Reversibility is also assured and only requires cutting of the ring and division of the anastomosis.We intentionally avoid re-sleeving the pouch to reduce the risk of leakage, and dissection involves only a limited portion of the gastric wall . The lenRevision surgery is technically demanding and may be associated with a high complication rate. The f-SAGB is our revision strategy after VSG. Re-sleeving of the pouch is avoided, and the anastomosis is placed above a non-adjustable ring without division of the pouch from the gastric antrum. The length of the bypass is tailored according to the size of the pouch to modulate the degree of malabsorption of the procedure. The access to the biliary tree and remnant stomach is preserved, and full reversibility is easy to obtain. Device-related complications must be considered but have not yet been reported. Although our preliminary results are encouraging, larger series and longer follow-ups are necessary.


Cofactor engineering plays an important role in metabolic engineering but is rarely reported in the sterols biotransformation process owing to its use of multi-catabolic enzymes, which promote multiple consecutive reactions. Androst-4-ene-3, 17-dione (AD) and androst-1, 4-diene-3, 17-dione (ADD) are important steroid medicine intermediates that are obtained via the nucleus oxidation and the side chain degradation of phytosterols by Mycobacterium. Given that the biotransformation from phytosterols to AD (D) is supposed to be a NAD+-dependent process, this work utilized cofactor engineering in Mycobacterium neoaurum and investigated the effect on cofactor and phytosterols metabolism.Cofactor engineering is involved in the modification of enzymes related to nicotinamide adenine dinucleotides (NADH and NAD+/NADH ratio and the AD (D) production of M. neoaurum TCCC 11978 (MNR M3) were higher than in the control. Moreover, the NADH: flavin oxidoreductase was identified and was supposed to exert a positive effect on cofactor regulation and phytosterols metabolism pathways via comparative proteomic profiling of MNR cultured with and without phytosterols. In addition, the NADH: flavin oxidoreductase and a water-forming NADH oxidase from Lactobacillus brevis, were successfully overexpressed and heterologously expressed in MNR M3 to improve the intracellular ratio of NAD+/NADH. After 96 h of cultivation, the expression of these two enzymes in MNR M3 resulted in the decrease in intracellular NADH level  and the increase in NAD+/NADH ratio . Phytosterols bioconversion revealed that the conversion ratio of engineered stains was ultimately improved by 58 and 147%, respectively. The highest AD (D) conversion ratio by MNR M3N2 was 94% in the conversion system with soybean oil as reaction media to promote the solubility of phytosterols.Through the addition of the coenzyme precursor of nicotinic acid in the phytosterols fermentation system, the intracellular NAD+/NADH is an important factor for the transformation of phytosterols. Expression of NADH: flavin oxidoreductase and water-forming NADH oxidase in MNR improved AD (D) production. Besides the manipulation of key enzyme activities, which included in phytosterols degradation pathways, maintenance the balance of redox also played an important role in promoting steroid biotransformation. The recombinant MNR strain may be useful in industrial production.The ratio of NAD Escherichia coli (E. coli) [Lactococcus lactis [Saccharomyces cerevisiae [Klebsiella pneumoniae [Serratia marcescens [Torulopsis glabrata [Colletotrichum lini [+) is an important cofactor pair that acts in plenty of oxidation–reduction (redox) reactions and regulates various enzyme activities and genetic processes [+ and NADH has a critical effect on the maintenance of the intracellular redox balance, which is a basic condition for the growth and metabolism of microorganisms [+ manipulation system has been successfully used to improve the production of primary metabolites such as ethanol, 1, 2-propanediol [+/NADH ratio is by introducing an NADH or NAD+ regeneration system. Intracellular NADH or NAD+ is easily regenerated in situ by expressing an NAD+-dependent formate dehydrogenase (increase of intracellular NADH availability) or an NADH oxidase (high NAD+/NADH ratio), respectively [pncB, which encodes NAPRTase, results in increased total NAD+ level and ratio of NAD+/NADH [Metabolic engineering studies generally focused on genetic manipulation (overexpression or disruption) of the genes that encode enzymes involved in a particular pathway. However, the flux of a cofactor-dependent pathway is controlled through the availability of enzyme, as well as the cofactor and ratio of the reduced to the oxidized form of the cofactor. Therefore, cofactor manipulation is a potentially powerful tool for metabolic engineering . CofactoE. coli) , 3, Lacts lactis , Saccharrevisiae , Klebsieeumoniae , Serratircescens , Torulopglabrata , and Colhum lini . Nicotinrocesses –7, 9. Thrganisms . The NADpanediol , acetoinpanediol , 6, 11, ectively . Further2O + 4 ATP + 7 GDP + 7 Pi + 10 FAD + 21 NAD+ = AD (D) + 21/2 CO2 + 4 AMP + 4 PPi + 7 GTP + 10 FADH2 + 21 NADH + 21 H+ [+/NADH ratio to the AD (D) production are few.Steroid medications are used widely in clinical applications and form an important and large category in the pharmaceutical industry. Androst-4-ene-3, 17-dione (AD) and androst-1, 4-diene-3, 17-dione (ADD) are two versatile C19 steroid precursors that are obtained via the nucleus oxidation and the side chain degradation of phytosterols by microorganisms , 14. The + 21 H+ , 21. Mai+/NADH ratio in Mycobacterium neoaurum TCCC 11978 (MNR M3) was analyzed by adding nicotinic acid (NA) in the broth. Then, the key enzymes that related to NAD (H) regulation and phytosterols degradation were identified according to the analysis of comparative proteomic of M. neoaurum cultured with and without phytosterols. Afterward, the cofactor engineering was conducted by introduction of NAD+ regeneration system into MNR M3 to increase the availability of NAD+/NADH ratio for AD (D) production based on the proteomic analysis.In this work, cofactor manipulation was used in phytosterols biotransformation. First, the connection of phytosterols biotransformation with the intracellular NADThe strains, plasmids, and primers used in this study are listed in Table Mycobacterium neoaurum TCCC 11978 (MNR M3) obtained from Tianjin University of Science and Technology Culture Collection Center (TCCC), Tianjin, China, was a spontaneous mutant of M. neoaurum TCCC 11028 (MNR) strain. AD accumulated in the broth as a major product by MNR M3. The amount of ADD was too low, less than 5% of the total product in the medium. The AD (D) concentration is the total sum of the two products. MNR M3C2 is the ksdD gene replacement strain MNR M3ΔksdD::ksdD-MNR which was constructed by homologous recombination [nox-1 gene was amplified through the polymerase chain reaction from the total DNA of MNR M3, using the primers nox-1-f and nox-1-r that generate BamHΙ and HindIIΙ sites. The nox-2 gene was amplified from the total DNA, which was isolated from Lactococcus lactis subsp. cremoris NZ9000 (L. lactis subsp. cremoris NZ9000), using the primers nox-2-f and nox-2-r that generate BamHΙ and SalΙ sites. Mycobacterial replicating vector pMV261 which harbored the Kanamycin (kan) resistance was the common plasmids used in Mycobacterium for gene expression [BamHI and HindIII or BamHI and SalI, respectively. Then the treated fragments were inserted into the vector pMV261, which was treated with the same restriction enzymes to generate the vector pMV261-nox-1 and pMV261-nox-2. The pMV261-nox-1 and pMV261-nox-2 vectors were imported into MNR M3 via electroporation. The transformants were screened in a Luria–Bertani medium agar plate containing kan. The selected recombinants were then designated as MNR M3N1 and MNR M3N2 for further characterization.bination . The noxpression . The amp4 0.5, K2HPO4 0.5, (NH4)2HPO4 3.5, citric acid 2, and ammonium iron citrate 0.05 with pH of 7.2. Experiments were conducted under different culture conditions with phytosterols (5 g/L) or soybean oil (16%) and Tween 80 (0.5%) in minimal medium. Different concentrations of NA were added to the medium at the start of fermentation. In the phytosterols-free medium, the growth of cell was measured through optical density. However, the cell growth in the phytosterols-contained culture broth is difficult to measure by using this method. In this study, the cell growth measurement in the medium with phytosterols was conducted following the method by Meyers et al. [The phytosterols substrate used is a sterol mixture contained (by weight percentage) 51.7% β-sitosterol, 27.2% stigmasterol, 17.1% campesterol, and 4.0% brassicasterol . Standards AD and ADD were purchased from Sigma-Aldrich Co. (USA). All chemical solvents and salts were of analytical grade or higher. The cultivation and bioconversion of microorganisms and the preparation and analysis of transformation products, were performed following the procedures described by Shen et al. . The mins et al. . The amo+ were determined as described by Zhang et al. [The intracellular concentrations of NADH and NADg et al. .+ per minute. Protein concentrations were measured by the method described by Bradford.0.2 g wet cells of the strains were washed with Tris–HCl buffer  twice, and resuspended in the same buffer. Cells were then disrupted by sonication at 4 °C for 10 min by a cell sonicator. The homogenate was centrifuged at 12,000 r/min for 30 min at 4 °C. The supernatant was stored in − 80 °C for further research. NOX activity was determined through a photometer assay at 340 nm using 0.1 mM NADH and 0.1 M potassium phosphate buffer at pH 7.0. The reaction was initiated by adding 0.1 mL of cell extracts to the 0.9 mL reaction mixture, and the decrease in absorbance at 340 nm was determined to calculate the NADH concentration. A unit of NOX activity is defined as the amount that catalyzes the oxidation of 1 μmol NADH to NADMNR M3C2 was grown in minimal medium. Experiments were conducted under different culture conditions with phytosterols and without phytosterols in minimal medium. The cells were harvested after 60 h cultivation. The collected sediment was washed with phosphate buffer saline at pH 7.2 thrice, sonicated on ice using a high intensity ultrasonic processor (Scientz) in lysis buffer , and centrifuged at 20,000g for 10 min at 4 °C. The protein contents in supernatural fluids were estimated using 2D Quant kit. Proteins were prepared and analyzed using tandem mass tag (TMT)—based LC–MS/MS. More specifically, the protein solution was reduced with 5 mM dithiothreitol for 1 h at 37 °C and alkylated with 20 mM iodoacetamide for 45 min at room temperature in darkness. For trypsin digestion, the protein sample was diluted by adding 100 mM triethylamine borane. Finally, trypsin was added at 1:50 trypsin-to-protein mass ratio for the first digestion overnight and 1:100 trypsin-to-protein mass ratio for a second 4 h-digestion. Approximately 300 μg protein for each sample was digested with trypsin for the following experiments. After trypsin digestion, peptide was desalted by Strata X C18 SPE column (Phenomenex) and vacuum-dried. Peptide was reconstituted in 0.5 M triethylamine borane and processed according to the manufacturer’s protocol for 6-plex TMT kit (Pierce). TMT-labeled samples were diluted to 10 mM HCOONH4 buffer  before HPLC on Waters XBridge Shield C18 RP column, 3.5 μm, 4.6 × 250 mm. The flow rate used for reversed-phase column separation is 1 mL/min with mobile phase A (10 mM HCOONH4) and mobile phase B . A solvent gradient system was used: 0–5 min, 2–10% B; 5–55 min, 10–35% B; 55–65 min, 35–90% B; 65–70 min, 90% B; 70–75 min, 90–2% B; 75–80 min, 2% B. In total, 20 fractions were pooled for each sample and dried by vacuum centrifuge. The peptides were separated by a linear gradient formed from 2% ACN, 0.1% FA (mobile phase A), and 80% ACN, 0.1% FA (mobile phase B). A solvent gradient system was used: 0–5 min, 2% B; 5–8 min, 2–11% B; 8–53 min, 11–20% B; 53–55 min, 20–80% B; 55–58 min, 80% B; 58–60 min, 80–2% B; 60–65 min, 2% B. at a flow rate of 300 μl/min. MS analysis was performed on a Thermo Scientific Q Exactive plus. The electrospray voltage applied was 2.0 kV. MS spectra were acquired across the mass range of 350–1500 m/z in high resolution mode using 250 ms accumulation time per spectrum. For data analysis, raw data (.raw) was converted into peak lists (.mgf) by Proteome Discoverer. The database used in searching was uniprot reference proteome M. neoaurum VKM Ac-1815D (Uniprot: UP000018763) [p values of < 0.01 were selected to ensure the authenticity of up- and down-regulations. Gene ontology (GO) (http://www.geneontology.org/) was used to investigate the potential functions of peptide precursors. The ontology covers three domains, as follows: cellular component, molecular function, and biological process. Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.genome.jp/kegg/) was used to annotate the protein pathway.MNR M3 was grown under different culture conditions with phytosterols and without phytosterols in minimal medium. The cells were harvested after 60 and 72 h cultivation (late logarithmic phase) and preserved at − 80 °C. Total RNA was isolated using Eastep® Super Total RNA Extraction Kit  according to instructions of supplier. The resulting mixture was treated with DNAse I. For reverse transcription, cDNA synthesis was performed using the PrimeScript™ RT reagent Kit with gDNA Eraser  (Takara) with 0.3 μg of total RNA following the manufacturer’s instructions. Quantitative RT-PCR analyses of cDNA samples were performed on the StepOneTM RealTime PCR System (Applied Biosystems). The above cDNA sample was completely mixed with 0.2 μM specific oligonucleotides, 10 μL of AceQ® qPCR SYBR® Green Master Mix, and 0.4 μL of ROX Reference Dye 1 in 20 μL of reaction mixture. The nucleotide sequences of primers used in this study for target and reference genes are listed in Table −ΔΔCt algorithm [lgorithm . Each geMNR M3, the intracellular NAD+/NADH ratio and AD (D) production during biotransformation were examined at regular intervals; on the other hand, NA (vitamin precursor of NAD+) was added to the fermentation system to change the intracellular redox status.To better understand the connection of the intracellular NAD (H) with AD (D) production in +/NADH constantly changed during the fermentation. This outcome is consistent with the studies of Ji et al. [+/NADH ratio in MNR M3 increased. Following, the NAD+/NADH ratio decreased quickly after 96 h and the highest NAD+/NADH ratio was obtained at 96 h. The dynamics of the accumulation of AD (D) was also studied. From 48 to 72 h, the productivity of AD (D) is 7.5 mg/(L h), which increased to 10.4 mg/(L h) at the next stage (72–96 h). After 96 h, the rate remarkably decreased to 2.9 mg/(L h). The productivity of AD (D) showed a similar trend with the change of NAD+/NADH ratio. Thus, we preliminarily inferred a correlation between the NAD+/NADH ratio and the AD (D) productivity during the late biotransformation period (after 96 h) in MNR M3.As shown in Fig. i et al.  and Wu ei et al. . During + level through the salvage pathway in M. tuberculosis [MNR M3. From Fig. +/NADH ratio during the late biotransformation period as well. When 12 g/L NA was added into the fermentation system, the level of NAD+/NADH ratio was 25% higher than that of the control (without NA). However, the weak growth was observed with increasing NA concentration in the fermentation medium, which is in accordance with that in Colletotrichum lini [+/NADH ratio was obtained and the production of AD (D) was enhanced with the addition of NA. We could get that the regeneration activity of NAD+ had a positive effect on the production of AD (D).NA is used to elevate the intracellular NADrculosis . A suitarculosis , 28. Thu+ cofactor by genetic modification in MNR M3. In order to investigate whether the proteins regulate the redox balance of cofactor and bring a positive influence on phytosterols metabolic pathway, we detected the changes in proteome of MNR M3C2 cultured with and without phytosterols in minimal medium.It’s necessary to establish an efficient way to generate NAD+ from NADH was mainly through electron transfer chain (ETC), oxygen was used as the final electron acceptor and ATP was produced [2 to produce NAD+ and H2O, was also detected in MNR M3C2. It was noteworthy that the expression of this protein was significantly up-regulated by 3.1-fold with phytosterols in minimal medium. By analyzing the proteome of MNR M3 with and without phytosterols, we speculated that the up-regulated protein NOX-1 performed important functions in phytosterols metabolism and cofactor regulation pathways.A total of 19025 peptides and 3727 proteins were identified, among which 299 proteins showed significant changes, as follows: 174 proteins were up-regulated and 125 proteins were down-regulated. The changed proteins included in cofactor metabolism were analyzed. The accession number, protein name, and fold change (with phytosterols/without phytosterols) for proteins were available in Table produced . Apart fMNR M3 with and without phytosterols was assayed by quantitative RT-PCR. As shown in Fig. MNR M3 cultured without phytosterols, the relative transcription of NOX-1 increased by 4.56 and 2.89 times after cultivation for 60 and 72 h, respectively. This finding indicated the credibility of the up-regulated ratio of NOX-1 with phytosterols showed in proteomic analysis.To validate the results from proteomic analysis, the transcription level of NOX-1 in + by using molecular oxygen as the electron acceptor [+/NADH ratio for phytosterols biotransformation, NOX-1 was overexpressed in MNR M3. Moreover, the NOX-2 from L. lactis subsp. cremoris NZ9000 was also chosen to express in MNR M3 as it can lead to a dramatically increased NAD+/NADH ratio [MNR M3N1 and MNR M3N2 were obtained.Both NOX-1 and NADH oxidase (NOX-2) catalyze the oxidation of NADH to NADacceptor , 32. In DH ratio , 34. As + and NADH were measured when the cells entered the end stage of exponential phase. As shown in Table MNR M3N2 was higher than that of MNR M3N1 (0.32 U/mg protein), whereas the NADH oxidase activity in the parent strain was not detected. This finding indicated that NOX was actively produced in the recombinant strains, and the expression of NOX-2 in MNR M3N2 had a higher catalytic activity.The specific activity of NOX and the content of NADMNR M3, MNR M3N1, and MNR M3N2. As shown in Fig. MNR M3N1 was better than that of MNR M3N2 with higher NOX expression. The NOX expression had a negative effect on cell growth.The effect of NOX expression on cell growth was studied during aerobic cultivations of strains MNR M3 was expected to increase the overall intracellular NAD+ pool and NAD+/NADH ratio, thereby improving the flux of NAD+-dependent pathways. As shown in Fig. + continuously changed during various stages of the cell growth process among the parent and the engineered strains. In the three strains, levels of NAD+ were increased during growth phases and when cells entered non-growth phase, the NAD+ levels in these three strains were decreased. No remarkable difference on the NAD+ level among these three strains was observed. However, levels of NADH in the three strains were different. The NADH levels remain unchanged at the first 72 h, and when cells entered the end stage of exponential and the non-growth phase, the NADH levels in these three strains were increased. The increased rate of NADH level in the engineered strains was lower than that in the parent strain, and MNR M3N2 with the higher NOX expression has the lowest increase of NADH level among these three strains. NOX expression led to a decrease of NADH pools comparing with the parent strain. Meanwhile, the ratio of NAD+/NADH ratio among these three strains was studied  in MNR M3, MNR M3N1, MNR M3N2 were determined. The bioavailability of steroid hormone in the bioconversion process is low because of the remarkably high hydrophobic nature of steroids. Furthermore, most of the enzymes involved in phytosterols degradation are promoted by oxygen. To improve the production of steroid products in aqueous systems, natural oils have been widely introduced to microbial transformations of steroids [MNR M3, MNR M3N1, and MNR M3N2 were studied. The highly expressed NOX-2 in MNR M3N2 increased the conversion ratio (94%) when compared with that in the parent strain (38%), and the strain with moderate expression of NOX-1 (MNR M3N1) increased the conversion ratio (60%) as well. When compared with MNR M3, the AD (D) conversion ratio in MNR M3N1 and MNR M3N2 increased by 58 and 147%, respectively. Moreover, the productivity of AD (D) were analyzed in the phytosterols conversion to AD (D). Among these three strains, MNR M3N2 with the highest NOX production showed the highest productivity at every fermentation stage. Moreover, all the substrate of phytosterols (5 g/L) was run out in MNR M3N2 fermentation at 144 h. This indicated that the fluxes towards oxidative and reductive metabolism of phytosterols increased upon introduction of NAD+ regeneration system in MNR M3. Hence, the increase of the NAD+/NADH ratio induced by the expression of NOX is important for AD (D) production.The recombinant strains both could produce NOX and disturb the redox balance of cofactor Table , Fig. 3,steroids , as it csteroids , 36. So + as a cofactor and oxidizes 3β-hydroxysterols to 3-ketosteroids [+ to NADH ratio could be a promoting factor for the AD (D) conversion ratio from phytosterols through the side chain cleavage. As shown in Fig. +/NADH ratio and the AD (D) production were increased with the increasing of NA concentration (0–9 g/L). The result was similar to that obtained in the Torulopsis glabrata [+ level and NAD+/NADH ratio increased in the presence of NA.AD and ADD are important steroid medicine intermediates. They are generally obtained via the side chain degradation of phytosterols by microorganisms. The analysis of metabolic pathways associated with AD production from phytosterols showed that either cholesterol oxidase or 3β-hydroxysteroid dehydrogenase is responsible for the first step of the cholesterol degradative pathway, which uses NADsteroids . Furthersteroids , and thesteroids , 21. Theglabrata , in whicMNR M3 was identified and supposed to the key enzyme to regulate the NAD+/NADH balance for AD (D) production through the analysis of comparative proteomic of MNR cultured with and without phytosterols  production were got in MNR M3N2.Cofactor engineering has been widely used to promote the production of important primary metabolites, such as 1, 2-propanediol , acetoin+/NADH ratio due to the introduction of the NAD+ regeneration system triggered a dramatic variation of phytosterols metabolism. As shown in Fig. +, with NADH, propionyl-CoA, and acetyl-CoA, is generated [+ was essential to the phytosterols degradation pathway [+ from NADH in MNR (Table + regeneration for AD (D) production. Finally, the enhancement of intracellular NAD+ regeneration system resulted in the strengthening of phytosterols degradation and improvement of AD (D) production and productivity in aerobic fermentation.The large increase of the NADenerated , 38. Mor pathway , 21. By +/NADH ratio could be beneficial to the production of AD (D) and that the introduction of NAD+ regeneration system into MNR is a powerful engineering tool to enhance the metabolic flux for the desired metabolites. The successful expression of NOXs in MNR M3 resulted in a large increase in NAD+/NADH ratio, thereby markedly enhancing the phytosterols metabolism pathways. The obtained data proved that, besides the manipulation of key enzyme activities, which are included in phytosterols degradation pathways, the maintenance of redox balance and the regeneration activity of cofactors also played an important role in promotion the steroid biotransformation. This study provides a useful strategy for enhancing the accumulation of NAD+-dependent microbial metabolites in the microbial biotransformation of steroids.Increasing the intracellular NAD


Saturation of carrier occupation in optically excited materials is a well-established phenomenon. However, so far, the observed saturation effects have always occurred in the strong-excitation regime and have been explained by Pauli blocking of the optically filled quantum states. On the basis of microscopic theory combined with ultrafast pump-probe experiments, we reveal a new low-intensity saturation regime in graphene that is purely based on many-particle scattering and not Pauli blocking. This results in an unconventional double-bended saturation behaviour: both bendings separately follow the standard saturation model exhibiting two saturation fluences; however, the corresponding fluences differ by three orders of magnitude and have different physical origin. Our results demonstrate that this new and unexpected behaviour can be ascribed to an interplay between time-dependent many-particle scattering and phase-space filling effects. et al. reveal a new saturation regime at low excitations, resulting in a double-bended saturation behaviour.Saturable absorption, a technologically relevant property of graphene, is usually explained with Pauli blocking of optically driven carriers in the strong-excitation regime. Here, Winzer  Saturation of light absorption is a long-known central phenomenon in nonlinear optics. It results from the fermionic character of optically driven electrons exhibiting Pauli blocking in the excited states. As a result, with increasing light fluence, the probability to increase the carrier occupation in the excited state due to light absorption is reduced. The saturation behaviour can be exploited in optical devices for the modulation of ultrashort laser pulses. In particular, mono- or multilayer graphene can be used as passive mode-locking medium1234515781The widely used approach for modelling the saturation behaviour is the two-level saturable absorber model1011In this work, we present a joint theory-experiment study on the saturation behaviour of the maximal optically induced carrier occupation in graphene. We apply a sophisticated theoretical approach based on graphene Bloch equations813The saturation behaviour has been mostly studied by exploiting the two-level saturable absorber model101112ρc after continuous wave excitation with the intensity I. The saturation intensity Is is determined by Is/I=γΓ/4Ω2, with the Rabi frequency Ω , carrier recombination (Γ) and dephasing (γ). Here Pauli blocking governs the high-intensity regime ρc is proportional to the intensity I/γΓ, where the slope is given by the intrinsic time scales γ−1 and Γ−1 of the two-level system  plays the crucial role rather than radiative recombination. In particular, many-particle-induced scattering channels sensitively depend on the filling of the surrounding phase space that is driven by the intensity of the excitation pulse8The standard saturation model from Tmax/T0 measured in pump-probe experimentsk0 describes the optically excited momentum state in the conduction band. We demonstrate a new and surprising saturation regime of the carrier occupation in the excited state. The observed scenario goes beyond the standard Pauli-blocking-based saturable absorber model. We theoretically predict and experimentally confirm double-bended saturation of the maximal optically induced carrier occupation by measuring the differential transmission of a probe pulse. A first illustration of our results is shown in −2, suggesting a linear relation between optically induced carrier occupation and pump fluence. In contrast, our calculations reveal that the unconventional saturation-like behaviour can be ascribed to Coulomb-induced out-scattering processes that efficiently balance the pump-pulse-driven occupation in the state k0 (k0) that balance out the impact of out-scattering , the microscopic two-band polarization pk and the phonon occupation q for different optical and acoustic phonon modes j  is the free electron charge (mass), Mk the carrier–light coupling strength and A(t) is the vector potential of the applied laser pulse. The in- and out-scattering rates γk, which for symmetric conduction and valence bands reads with the transition Tmax/T0 of the probe pulse is proportional to the maximal occupation σ. In this regime, the competition between in-scattering, out-scattering and the optical pump is most important.In the following, we consider pulsed excitation and probe at 1.5 eV where the initial thermal carrier occupation can be neglected, so that the saturation of differential transmission Δrd model  over eigDegenerate pump-probe experiments were performed at room temperature on multilayer epitaxial graphene (50 layers) grown by thermal decomposition on the C-terminated face of SiCρk(t) and pk(t) by solving the graphene Bloch equations quasi-stationary (Now we turn to the microscopic explanation of the double-bended saturation observed in I(t) and also the formal counterpart of the saturation intensity, denoted with Ik(t). Their ratio reads t), t) and γk(t)=t)+t) is considered, the quasi-stationary solution from with the time-dependent pulse intensity k0 are possible. For graphene, there is a linear relation between energy and momentum with ɛ0=ħνF|k0|. The first term in γk(t) appears instead of a pure out-scattering contribution Γγ in the two-level case. Most importantly, an additional purely many-particle term occurs. This new contribution scaling with the ratio of the in-scattering rate t) and the total scattering rate γk(t) turns out to be of crucial importance to reproduce the experimentally observed differential transmission saturation behaviour  including the qualitative double bending as well as the quantitative saturation intensities. The neglect of this many-particle term leads to only a single saturation in the range of 3 μJ cm−2 at very low occupations  and a dominating contribution proportional to accumulated carriers with t ∝I(t)) itself, finally balancing the optical excitation. To understand this in more detail, we consider c is a numerical factor depending on the material properties. That is, I) saturates due to the denominator. The interplay between the optically driven increase of the carrier occupation I) starts to become important and causes the deviation from the linear dependence and thus leads to a bending. Note that this bending has nothing to do with the regular Pauli-induced saturation, since at the considered very low fluences, the carrier occupation of the excited state is below 0.01 , the Pauli-blocking regime, is reached. Here in- and out-scattering are in balance and the conventional Pauli blocking of the excited state results in a standard saturation behaviour.As shown in −2), where the maximal optically induced carrier occupation scales linearly with the intensity I)∝I/Γ0 with nearly intensity-independent scattering rate Γ0  that efficiently depopulates the optically excited state causing a deviation from the linear scaling (I)≈4(Ω(I)/I))2 and the bending results from the strong increase of the intensity-dependent out-scattering rate. (C) Intermediate regime (few 100 μJ cm−2), where the Coulomb-induced in-scattering balances out the out-scattering resulting again in an approximately linear increase of the carrier occupation due to the optical pump  (with respect to the centre of the excitation pulse). The discrete steps of tmax arise from our calculation beyond the rotating wave approximation and corresponds to a single-cycle duration of the exiting intensity, tmax shifts to earlier times due to the faster increasing out-scattering rate compared to the optical drive. Consequently, the time where out-scattering overbalances the excitation shifts towards tmax≈0 fs. As soon as in-scattering becomes significant the delay between pulse centre and tmax rises again. In the Pauli-blocking regime, tmax is constant.Now, we can summarize our results from a microscopic point of view by distinguishing four distinct intensity regimes : (A) lin scaling . Here eqcal pump . (D) Secpump −2; . The dif−2 range, which corresponds to the regime of the predicted unconventional saturation behaviour.In summary, based on a joint theory-experiment study, we have found a yet undiscovered double-bended saturation of the optically excited carrier occupation in graphene for optical pulse excitations having a duration in the range of the predominant scattering channels. This unconventional behaviour beyond the standard saturable absorber model is explained by the universal structure of Coulomb-induced many-particle scattering processes, clarified via in- and out-scattering and their fundamental scaling with the pump fluence. In particular, we have identified both in theory and experiment a surprising low-intensity saturation-like behaviour of the optically excited carrier occupation that is not due to Pauli blocking, but can be ascribed to the out-scattering-induced depopulation of electronic levels. Even if our accompanying analytical approach is an approximation, it covers important aspects of the dynamics. Most importantly, we find that in solids, a sub-linear relation between the optically excited carrier occupation and the pump fluence is not a sufficient criterion for an absorption saturation by means of fermionic Pauli blocking. We note that due to the universal structure of Bloch equations for solids, the observed effects are not restricted to graphene. The gained insights will be also valuable for the technological application of graphene as saturable absorber in lasers generating ultrashort pulsesThe data that support the findings of this study are available from the corresponding author upon request.How to cite this article: Winzer, T. et al. Unconventional double-bended saturation of carrier occupation in optically excited graphene due to many-particle interactions. Nat. Commun.8, 15042 doi: 10.1038/ncomms15042 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Concurrent spinal dural arteriovenous fistula (AVF) and cecal arteriovenous malformation (AVM) are very rare. A 6-year old boy presented with lower limb paresis after trauma. On imaging work-up spinal dural AVF was found. It was managed with endovascular glue embolization. After two years, the boy presented with severe anemia and occult gastrointestinal tract (GIT) bleed. Cecal AVM was diagnosed and managed with embolization. Spinal dural AVF are vascular malformations located in the dura mater of the spinal cord that result in arteriovenous shunting. Although rare, they account for approximately 80% of all spinal vascular abnormalities.[1] Vascular malformations of the gastrointestinal tract are also rare in children.[2] Concurrent spinal dural AVF and cecal AVM have not been reported till now.A 6-year-old boy presented with weakness of both lower limbs after trauma after fall from 8 feet height. On examination, he had power of 1/5 in both lower limbs with hyporeflexia and hypotonia. Bowel and bladder functions were also affected. MRI of spine was suggestive of abnormal flow voids in cervico-dorsal spine (C6-D4) with intramedullary bleed . Digital subtraction angiography suggested dural arteriovenous fistula with arterial feeder from radiculopial branches of left D6 intercostal artery with venous drainage into multiple perimedullary veins . Endovascular glue embolization of dural AVF was done with 0.4 ml of N-butyl cyanoacrylate-lipiodol. Angiography post-embolization showed glue cast at fistula site and no opacification of arterial feeder . Child had gradual improvement in lower limb weakness on follow up. Two years later, the child again presented with progressive pallor for one month duration. On examination, he had severe pallor and residual lower limb weakness with power of 2/5. Other systemic examination was normal. On investigation child had haemoglobin of 2.9 gm/dl. Peripheral smear showed microcytic hypochromic anemia. He was transfused with packed red cells. There was decline in hemoglobin even after blood transfusion. Further work up for anemia suggested stool positive for occult blood. As child already had AV malformation of one site, AV malformation of gut was also suspected. CT angiography of mesenteric vessels showed AVM of Iliocolic artery . Glue embolization of AVM was done under digital subtraction angiography . Check angiogram of intracranial vessels showed no abnormality. Child has normal hemoglobin after one year follow up.Spinal cord vascular malformation is a rare entity, and it accounts for 2–4% of spinal diseases.[3] Vascular malformations of the gastrointestinal tract are also rare in children.[2] Concurrent spinal and gut AVM is a rarity. Multiple AVMs are usually associated with hereditary disorders such as Rendu–Osler–Weber syndrome or hereditary hemorrhagic telangiectasia. It is extremely uncommon to find concurrent multiple AVM not associated with such syndromes. Multiple AVMs may be result of yet unrevealed pathogenesis or strong embryogenetic anomaly, which may be different from that involved in single AVM.[4]AVMs arise because of failure of embryogenesis during the differentiation of vascular channels into mature arteries, capillaries, and veins, resulting in direct arteriovenous shunts without intervening capillary beds.[5] Spinal cord AVM are shunts, which are fed by arteries normally supplying the neural tissue, whereas spinal dural AVFs are fed by radiculo-meningeal arteries.[6] Screening for multiple AVMs is warranted in cases when a single lesion does not explain the presenting features.[7]Common presentation of colonic vascular malformations in children are lower intestinal bleeding, intussusception, intestinal obstruction or intestinal perforation. The diagnosis is often delayed until severe anemia or chronic hemorrhage ensues. A negative initial workup should be followed by angiography to prevent delay in diagnosis and its complications.[8]Main aim of treatment is complete angiographic obliteration of the AVM. Modalities like microsurgery, endovascular embolization, and stereotactic radiosurgery have an established role in the treatment of patients with AVM, and a staged approach has been proposed for patients with multiple AVMs.[9] The case deserves attention for variety of reasons including a rare association of spinal dural AVF and cecal arteriovenous malformation, its non-syndromic association and potential for cure.Source of Support: NilConflict of Interest: None declared


Scratch card games are incredibly popular in the Canadian marketplace. However, only recently have researchers started to systematically analyze their structural characteristics and how these in turn affect the gambler. We present two studies designed to further understand the underlying physiological and psychological effects that scratch cards have on gamblers. We had gamblers  play custom made scratch cards involving a small win, a regular loss and a near-miss—where they uncovered two out of the three symbols needed to win the top prize. Our predictions were that despite near-misses and losses being objectively equivalent (the gambler wins nothing) gamblers’ reactions to these outcomes would differ dramatically. During game play, skin conductance levels and heart rate were recorded, as well as how long gamblers paused between each game. Gamblers’ subjective reactions to the different outcomes were then assessed. In both studies, near-misses triggered higher levels of physiological arousal (skin conductance levels and heart rates) than losses. Gamblers paused significantly longer following small wins than other outcomes, and reported high arousal, positive affect and urge to gamble—a constellation of results consistent with their rewarding properties. Importantly near-miss outcomes were rated as highly arousing, negative in emotional tone, and the most frustrating of all three outcome types examined. In Experiment 2, when we measured subjective urge to gamble immediately after each outcome, urge to gamble was significantly higher following near-misses than regular losses. Thus, despite not rewarding the gambler with any monetary gain, these outcomes nevertheless triggered higher arousal and larger urges to gamble than regular losses, a finding that may explain in part, the allure of scratch cards as a gambling activity. Lottery products are an exceptionally popular form of gambling. For example, in our home jurisdiction of Ontario, Canada, in a single fiscal year (2013/2014) the Ontario Lottery and Gaming Corporation (OLG) reported that lottery sales accounted for approximately 3.3 billion dollars in revenue OLG . A numbeLottery products are available at almost 10,000 retailers across the province OLG . These rOf the existing gambling literature examining scratch cards, the majority has looked at the use of these products by youth populations . Indeed, this gambling activity  appear to be popular among this demographic  is especially important. Clark et al.  periodicIn a scratch card game, a near-miss outcome would consist of uncovering two of the three jackpot symbols needed to win a large prize. Although researchers have identified near-miss outcomes in scratch cards as a potentially problematic characteristic of this gambling medium . We also sought to provide new evidence that both small wins and near-misses promote increases in the urge to continue gambling on scratch card games—a finding that could partially account for their overwhelming popularity.Based on our previous experiment, the general aim of Experiment 1 was to test the following hypotheses: we predicted that gamblers would show larger PRPs following wins  than following losses or near-misses. For near-misses we predicted that SCLs would increase as gamblers uncovered two of the three jackpot symbols and that such SCL changes would be greater than comparable changes for regular losses (or perhaps even wins). We predicted that we would show similar effects on gambler’s HR, such that HR would be elevated as gamblers successively uncovered the two jackpot symbols. We predicted that near-misses would be more subjectively arousing than regular losses and as arousing as wins, but for different reasons . Consequently we predicted frustration ratings should be highest for near-miss outcomes, followed by regular losses, with wins being the least frustrating. Lastly, we predicted that small wins should trigger the urge to continue gambling but so too would near misses (more so than regular losses).The aim of Experiment 2 was also to confirm the above hypotheses, offering a built-in replication of our findings. As in our previous experiment . Participants were all prescreened to ensure that they were: (1) at least 18 years of age , (2) not currently in or seeking treatment for problem gambling, (3) had experience playing scratch cards, (4) not currently in treatment for an anxiety disorder or taking medication for an anxiety disorder (since some anxiolytic medications can interfere with skin conductance recordings), and (5) not allergic or sensitive to gels, adhesives, or sanitizing agents (as such compounds are used in attaching electrodes). All prescreening criteria were confirmed at the time of consent, before the experiment began. One participant who by chance selected the top-prize winning card was excluded from all analyses, and another was excluded from the SCL analyses due to a technical error involving the SCL data.Following the informed consent procedure, all participants completed the Problem Gambling Severity Index (PGSI), part of the Canadian Problem Gambling Index , subjective arousal and valence were measured using Self-Assessment Manikins .Participants’ game play was recorded to allow accurate time locking of physiological responses with outcome delivery. Videos were recorded using the built-in FaceTime camera on a MacBook computer used to record the physiological data. The computer was arranged in a laptop stand such that only participants’ hands and the cards they were scratching were included in the video.SCL was recorded using non-gelled passive electrodes attached to participants’ index and ring fingers of their non-dominant hand. These electrodes were connected to an ADinstruments PowerLab (model 8/30) with a Galvanic Skin Response amplifier. A 1000 Hz sampling rate was used. LabChart 7.0 analysis software was used to analyze SCLs based on the precise timing of outcome delivery. Specifically, SCL was measured from the time the first symbol of the scratch card game was scratched, to the end of the last symbol in the game being uncovered. This time period was measured for all outcomes that participants experienced.HR was recorded using three electrodes placed on participants skin in a modified Mason-Likar arrangement  was counterbalanced.To closely approximate real scratch card gambling, the rules of our game were similar to those found in existing scratch cards. To win a prize, the participant needed to uncover three matching symbols within one game. Therefore, a win would consist of three matching symbols and three non-matching symbols within the six-symbol matrix. A near-miss outcome would consist of two matching (top-prize) symbols and four non-matching symbols, and a loss would contain six non-matching symbols within the matrix. The symbols in our game, like those in Cash for Life© were monetary amounts, with the exception of the top prize amount, which was denoted by the word “MONTH”. This was chosen to emulate the “LIFE” symbol in Cash for Life©. Thus, the near-miss outcome contained only two of the three “MONTH” symbols needed to win the top prize, and the small win consisted of three matching $5.00 symbols, interspersed among other non-matching symbols.At the beginning of the Experiment, all participants read and signed an informed consent letter. The University of Waterloo’s Office of Research Ethics approved all procedures in this Experiment. Following consent, participants completed the PGSI on a laptop computer using Qualtrics survey software. Next the participant selected the scratch cards that they would be playing in the game play portion of the Experiment. Participants were informed that they would be choosing two cards, and that one of the cards within the display case contained the top prize of Cash for a Month, equivalent to $100.00 CAD. The researcher removed both pullout card trays, presented them to the participant, and allowed them to freely choose one card from each tray.Once they had selected their cards, the researcher explained the rules of the game. For clarification purposes, the researcher used an oversized version of the scratch card game as an example. The researcher explained that each scratch card contained three separate games, and that each game contained six symbols. Participants were told that to win a prize, they had to uncover three matching symbols within one game. The matching symbols denoted the prize that was won. The researcher also explained that to win the top prize of Cash for a Month, three “MONTH” symbols had to be uncovered within a given game. Participants were also asked to scratch each game in three rows, starting from the top, and moving from the leftmost symbol to the rightmost symbol (to ensure consistent outcome delivery between participants). Additionally, they were asked to only move onto the next game once they were finished scratching the preceding game in its entirety.After choosing their cards and going over the rules of the game, participants were escorted to another lab room to wash their hands with Ivory soap to ensure clear SCL recordings. Participants were shown how to attach the three HR electrodes and given a mirror, a reference diagram, and a private space behind a curtain to apply them. A same-sex experimenter was available if participants needed assistance with electrode application. Next, the SCL electrodes were attached to the upper phalanges of the first and third fingers of the participants’ non-dominant hand. Participants were instructed to keep their non-dominant hand as still as possible while they were scratching the cards in an attempt to limit the amount of movement artifacts in the HR and SCL data.Following this set-up phase, the researcher placed one of the cards that the participant had chosen into a secure scratching platform  and answered the subjective arousal, valence, frustration and urge questions to gauge their subjective reactions to these displayed outcomes.Recall that the game orders were as follows: loss, small win, loss, and loss, near-miss, loss, with the order of card presentations counterbalanced. For all analyses, the comparisons of interest were reactions to the win, reactions to the near-miss, and reactions to a loss. Although there were four losing outcomes, only one of these was selected for analysis, namely the first loss on the second card played by the participant. By choosing this epoch we ensured that for each analyzed outcome the proceeding epoch was a loss. As such any effects of outcome type shown in the analysis could not be differentially affected by the previous outcome . Thus for all analyses with all participants there were three data points .For the SCL data, the recording epoch started from the time they began scratching the first symbol in the game matrix to the time they uncovered the last symbol revealing the outcome. To assess whether there were changes in SCL levels as gamblers revealed the symbols , we used LabChart software to record the slope of the SCLs over the entire recording epoch. For each participant the slope of the winning outcome, the near-miss outcome, and the first loss on card 2 was calculated.For HR, we used the same epochs as above, and calculated the beats per minute in the winning, near-miss and loss epochs. For PRPs, the time between outcome delivery (uncovering the last symbol in a particular game) and the initiation of the next game (scratching the first symbol in the next game) was recorded.t tests (Fischer’s least significant difference test [LSD]). For all repeated measures analyses, in the event of sphericity assumptions being violated (assessed using Mauchly’s test), a Greenhouse-Geisser correction was applied prior to calculating the F-ratios. In these instances, corrected degrees of freedom are reported.The data was subjected to an outlier trimming procedure using a cutoff of three standard deviations from the mean of that outcome condition. The remaining data were then submitted to a repeated-measures analyses of variance (ANOVA), with outcome type as the repeated measures factor. Follow up analyses were conducted using paired-samples Participants’ scores on the PGSI classified 41 participants as non-problem gamblers, 20 participants as low-risk, 1 participant as a moderate/at-risk gambler, and no problem gamblers. Note that the PGSI was used to characterize our sample—no specific predictions were made concerning gambling status.F = 18.45, p < .001, η2 = .238. Post hoc Fisher’s LSD comparisons indicated that winning slopes were steeper than losing slopes t(59) = 4.31, p < .001, near-miss slopes were steeper than losing slopes, t(59) = 5.59, p < .001, but that there was no difference between winning and near-miss slopes t(59) = −1.57, p = .12.Data from one participant could not be analyzed due to a recording error. Table F = 20.36, p < .001, η2 = .355. Fischer’s LSD t-tests demonstrated that HR was significantly higher across winning epochs than losing epochs t(37) = 4.75, p < .001. Near-miss epochs had higher HR than losing epochs t(37) = 6.59, p < .001, but there were no significant differences in HR for winning versus near-miss epochs. t(37) = −1.57, p = .13.Data from 24 participants could not be analyzed due to excessive movement artifacts associated with the scratching movements during play. For the remaining 38 participants, the average BPM was calculated for winning, near-miss and losing epochs. Table F = 15.58, p < .001, η2 = .209. The PRP was significantly longer following wins versus losses, t(59) = 5.41, p < .001, and wins versus near-misses, t(59) = 3.13, p = .003. These t-tests also showed that PRP’s following near-misses were marginally longer than for losses, t(59) = 2.43, p = .018.Table F = 59.10, p < .001, η2 = .492. Paired samples t-tests revealed significantly higher subjective arousal ratings for winning outcomes compared to losing outcomes, t(61) = 10.07, p < .001, and near-miss outcomes, t(61) = 3.70, p < .001. Additionally, near-misses were rated as higher in subjective arousal than losses, t(61) = 7.87, p < .001.Subjective arousal ratings in Table F = 94.44, p < .001, η2 = .608 (since the assumption of sphericity was violated Mauchly’s test: X2(2) = 6.30, p = .043, a Greenhouse-Geisser correction was applied). Winning outcomes were rated as significantly more positive in valence than both losing outcomes, t(61) = 11.33, p < .001, and near-miss outcomes t(61) = 10.95, p < .001. There were no significant differences between losing and near-miss outcomes, t(61) = .948, p = .35.Valence ratings in Table F = 36.96, p < .001, η2 = .385. Paired samples t-tests revealed that near-miss outcomes were rated as significantly more frustrating than wins, t(59) = 7.75, p < .001, and (most importantly) significantly more frustrating than losing outcomes, t(59) = 3.94, p < .001. Finally, losing outcomes were rated as more frustrating than winning outcomes, t(59) = 5.27, p < .001.Table F = 3.82, p = .025, η2 = .06. Paired samples t-tests revealed that winning outcomes were associated with a higher rating of urge to continue gambling compared to losing outcomes, t(60) = 2.56, p = .013, and compared to near-miss outcomes, t(60) = 2.10, p = .04. No significant difference was found between the urge ratings for losing and near-miss outcomes, t(60) = −.444, p = .66.The final subjective measure, urge to continue gambling, revealed what appear in Table Both SCL and HR measures showed that revealing the symbols that lead to a regular loss was an experience that was the least physiologically arousing. By contrast, revealing the symbols in both winning and near-miss outcomes triggered significantly greater arousal. Here the contrast between losses and near-misses is paramount, as these outcomes are objectively equivalent in that the player gains nothing, yet they generate very different physiological responses. Consistent with research in slot machine play  we added subjective measures assessing positive and negative valence, as well as disappointment, and used Likert scales for all subjective questions , and (2) immediately after gamblers completed an outcome, they pressed a button on a response box indicating they were ready to answer questions pertaining to that outcome. Subjective assessments of that outcome were then administered. Thus subjective assessments were gathered immediately after each experienced outcome.Sixty-eight University of Waterloo undergraduate students were recruited for this experiment and received one course credit in appreciation of their time. The average age of the sample was 19.87 years, with age ranging from 18 to 35 . Participants were prescreened to ensure that they met inclusion criteria (defined in Experiment 1). Of the 68 students recruited, four participants were excluded from all analyses due to idiosyncratic, non-left-to-right scratching patterns, and another three were excluded from SCL and HR analyses due to technical errors. One additional participant was excluded from HR, SCL, and PRP analyses due to a video recording error compromising the analysis of time-locked responses.As in Experiment 1, all participants completed the PGSI as part of the Canadian Problem Gambling Index  for the participant and then recording the participant’s verbal response (i.e. a number between 1 and 5). Four unique question orders were created and randomly assigned to each participant to account for order effects.The scratch cards used in this experiment were identical to the cards used in Stange et al.  and ExpeThis experiment utilized the same apparatus as in Experiment 1.Procedures were identical to Experiment 1 except that immediately after completing each game, participants were instructed to press a button placed next to the scratching area. Once the button press had been completed, subjective questions were answered verbally. Participants responded to all 6 subjective scales  immediately following each outcome. This interleaved pattern of game play, followed by subjective questions continued for all six games.Participants’ scores on the PGSI revealed that 41 participants were non-problem gamblers, 21 participants as low-risk, and 2 participants as moderate/at-risk gamblers. No problem gamblers were identified within this sample.F = 4.58, p = .015, η2 = .072. Paired samples t-tests revealed that SCL’s leading up to losing outcomes were not significantly different from those leading up to wins, t(59) = −1.10, p = .278. SCL’s leading up to near-miss outcomes were significantly greater than those leading to winning outcomes, t(59) = 2.09, p = .041, and those leading to losing outcomes, t(59) = 2.59, p = .012.Average slopes of SCLs over the time leading up to outcome delivery are shown in Table F = 5.17, p = .01, η2 = .112. Subsequent paired samples t-tests showed that the average HR for near-miss outcomes and winning outcomes were not significantly different, t(41) = .28, p = .78. However, average HR leading up to wins was significantly higher than HR leading up to losses, t(41) = 3.38, p = .002. Likewise, HR leading up to near-misses was significantly greater than for losses t(41) = 2.87, p = .006.Data from 18 participants could not be analyzed due to excessive movement artifacts. As shown in Table F = 10.19, p < .001, η2 = .149. Wins led to longer PRPs than losses t(58) = 4.50, p < .001. PRPs were longer following near-misses compared to losses, t(58) = 3.44, p = .001. Interestingly, there were no significant differences between PRPs following wins and near-misses, t(58) = 1.46, p = .15.The PRPs in Experiment 2 were the pause durations between the outcome reveal to pressing the button to initiate the answering of the subjective questions. Table F = 49.50, p < .001, η2 = .440. Wins were significantly higher in subjective arousal than near-miss outcomes, t(63) = 5.27, p < .001, and losing outcomes, t(63) = 9.07, p < .001. Subjective arousal ratings of near-miss outcomes were significantly higher than losing outcomes, t(63) = 5.28, p < .001.Subjective arousal ratings  = 64.98, p < .001, η2 = .508. Wins were higher in positive valence than both losses, t(63) = 9.60, p < .001, and near-misses, t(63) = 8.67, p < .001. Near-misses and losses did not differ significantly, t(63) = −.80, p = .428.Table F = 19.40, p < .001, η2 = .247. Near-misses were rated as significantly more negative than losses t(59) = 2.19, p = .033, and significantly more negative than wins t(59) = 5.18, p < .001. Additionally, losing outcomes were rated as significantly more negative than winning outcomes, t(59) = 4.64, p < .001.Subjective negative valence ratings were highest for near-misses and lowest for wins. There was a main effect of outcome type, F = 25.32, p < .001, η2 = .293. Near-miss outcomes were rated as significantly more frustrating than both winning, t(61) = 5.98, p < .001, and losing outcomes, t(61) = 2.34, p = .022. Losses were also rated as more frustrating than winning outcomes, t(61) = 5.32, p < .001.Table F = 58.82, p < .001, η2 = .491. As shown in Table t(61) = 8.78, p < .001, and more disappointing than losing outcomes, t(61) = 4.41, p < .001. Losses were rated as more disappointing than winning outcomes, t(61) = 7.56, p < .001.Subjective ratings of disappointment showed a similar pattern. There was a main effect of outcome type, F = 7.87, p = .001, η2 = .111. Wins were rated as provoking a stronger urge to continue gambling than losses, t(63) = 3.72, p < .001, but not near-misses, t(63) = .683, p = .497. Crucially near-misses led to higher urges to gamble than losses, t(63) = 3.02, p = .004.For urge, Table The majority of our findings from Experiment 1 were replicated in Experiment 2. Specifically, as in Experiment 1, HR was elevated leading up to winning and near-miss outcomes, and lowest leading up to regular losses. SCLs in Experiment 2 showed an overall downward trend throughout outcome delivery; however, the least negative SCLs were found leading up to near-miss outcomes, and these SCLs were significantly less negative than those for regular losses, thus replicating the pattern found in both our previous investigation . One interpretation of the near-miss PRP data is that it was during the PRP period following a near-miss that gamblers were ruminating on how frustratingly close they were to the grand prize and how disappointed they felt at not having won. Such ruminations may have elevated near-miss PRP lengths over and above those of regular losses.Arguably the most important finding in this series of studies involves the urge to gamble. In both studies small wins triggered the urge to gamble—a finding that may account for the popularity of scratch cards. In our studies, any time a gambler won a small reward, they experienced the urge to play again. It is not unreasonable to assume that a substantial number of gamblers may act on this urge, and purchase more scratch cards with their winnings in a real gambling environment.In slot machine research near-misses have been shown to trigger the urge to continue gambling. Clark et al.  periodicimmediately after game play may have elevated their physiological arousal—with a reduction in SCLs occurring as they scratched the symbols in the next game. It also remains unclear if near-miss outcomes affect urge to continue gambling above and beyond the other outcome types. While our hypotheses based on slot machine literature predicted that this would be the case, our data suggest otherwise and draw attention to the fact that small wins found in this form of gambling may be more influential than we imagined.While the present studies help shed light on what currently is an under-researched area of gambling behaviour, there are still many questions unanswered. For example why were all SCL slopes negative in Experiment 2 ? Perhaps the procedural differences between the studies are partially responsible: asking participants to gauge their affective responses on a number of dimensions urge to continue gambling—it remains unclear whether or not this urge would translate into the repurchasing of more scratch cards, possibly illustrating the potentially detrimental effects that these types of outcomes may have on real-world gambling behaviour. Additionally, our understanding of these gambling activities would be deepened by analyzing the influence of gambling frequency , problem gambling severity, or participant gender on the physiological and subjective effects of scratch card play, and future research studies designed to target a population with a wider range of gambling problems than a sample of typical University students could answer these questions.Other limitations include the fact that participants were not investing their own money in the experiment, as would be the case in the real-world. Additionally, the jackpot prize of our game was significantly smaller than those found in real scratch card games. Yet despite this, our results still indicated significant effects of near-miss outcomes on arousal and the urge to continue gambling. Perhaps these effects would be more pronounced if gamblers were using their own money or had the chance of winning an even larger prize. In line with this notion, it is also important to consider that we only measured