CB glomus cells have also been found to detect hypoglycemia in both non-primate mammals and humans. O2 and low-glucose responses share a common final pathway involving membrane depolarization, extracellular calcium influx, increase in cytosolic calcium concentration, and neurotransmitter secretion, which stimulates afferent sensory fibers to evoke sympathoadrenal activation. On the other hand, hypoxia and low glucose induce separate signal transduction pathways. Unlike O2 sensing, the response of the CB to low glucose is not altered by rotenone, with the low glucose-activated background cationic current unaffected by hypoxia. Responses of the CB to hypoglycemia and hypoxia can be potentiated by each other. The counter-regulatory response to hypoglycemia by the CB is essential for the brain, an organ that is particularly sensitive to low glucose. CB glucose sensing could be altered in diabetic patients, particularly those under insulin treatment, as well as in other medical conditions such as sleep apnea or obstructive pulmonary diseases, where chronic hypoxemia presents with plastic modifications in CB structure and function. The current review will focus on the following main aspects: (1) the CB as a low glucose sensor in both in vitro and in vivo models; (2) molecular and ionic mechanisms of low glucose sensing by glomus cells, (3) the interplay between low glucose and O2 sensing in CB, and (4) the role of CB low glucose sensing in the pathophysiology of cardiorespiratory and metabolic diseases, and how this may serve as a potential therapeutic target.The carotid body (CB) is a key chemoreceptor organ in which glomus cells sense changes in blood O The first study linking the CB to glucose metabolism was reported more than 25 years ago , CO2 tension, pH, and other stimuli  of electrically excitable neuron-like glomus (type I) cells surrounded by glia-like sustentacular (type II) cells. Type II cells, or a subpopulation of them, have recently been identified as neural stem cells that contribute to the growth of the organ in conditions of chronic hypoxemia  present in glomus cells , as described in lower mammals  potentiates low glucose-induced catecholamine secretion, whereas low glucose further induces Ca2+ influx during hypoxia  is a common clinical syndrome characterized by intermittent hypoxia and sleep fragmentation. OSA is a well-established significant risk factor for cardiovascular disease and mortality. As indicated above Intermittent Hypoxia and Glucose Sensing, chronic intermittent hypoxia results in CB chemoreceptor over-stimulation and augmentation of CB sensory responses in rats (Peng et al., Type 2 diabetes is a major chronic disease associated with high morbidity, mortality, and economic burden. Glucose sensing is essential for insulin-treated diabetic patients to counter-regulate insulin-induced hypoglycemia. It has been proposed that the CB dysfunction, increasing sympathetic tone and catecholamines in the blood, could possibly contribute to the pathogenesis of type 2 diabetes and essential hypertension (Nimbkar and Lateef, OSA syndrome and type 2 diabetes are also strongly linked to each other. Patients with OSA have an increased incidence of impaired glucose metabolism and are at an increased risk of developing type 2 diabetes (Tasali et al., 2 levels. Indeed, defects in CB function have been associated with several respiratory disturbances, particularly in the newborn (reviewed by Lopez-Barneo et al., 2 sensitivity of the respiratory center and, in some cases, long term resting hypoventilation and hypercapnia (reviewed by Timmers et al., 2-sensing mechanisms or CB growth in chronic hypoxia (Platero-Luengo et al., Carotid chemoreceptors work in coordination with other glucose sensing organs to counter-regulate hypoxia and hypoglycemia. The responses to hypoxia and hypoglycemia could be potentiated by each other. Failure to respond to these stresses could lead to malfunction of organs, such as the brain, which is highly sensitive to glucose and OThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Pulmonary arterial hypertension (PAH) is a vascular disease characterized by persistent precapillary pulmonary hypertension (PH), leading to progressive right heart failure and premature death. The pathological mechanisms underlying this condition remain elusive. Analysis of global metabolomics from lung tissue of patients with PAH (n = 8) and control lung tissue (n = 8) leads to a better understanding of disease progression. Using a combination of high-throughput liquid-and-gas-chromatography-based mass spectrometry, we showed unbiased metabolomic profiles of disrupted arginine pathways with increased Nitric oxide (NO) and decreased arginine. Our results also showed specific metabolic pathways and genetic profiles with increased Sphingosine-1-phosphate (S1P) metabolites as well as increased Heme metabolites with altered oxidative pathways in the advanced stage of the human PAH lung. The results suggest that PAH has specific metabolic pathways contributing to the vascular remodeling in severe pulmonary hypertension. Profiling metabolomic alterations of the PAH lung has provided a new understanding of the pathogenic mechanisms of PAH, which benefits therapeutic targeting to specific metabolic pathways involved in the progression of PAH. All. All4]. Metabolomic analysis was carried out according to the procedure previously described . In shorIdiopathic PAH lung tissue and human control lung tissue were used for global profiles. The analyses for total RNA in lung tissue were performed as formerly outlined . ShortlyPAH lung samples displayed broad changes in arginine and sphingolipid metabolism. Significant changes were also observed in the heme metabolism compared to control lungs. We additionally performed transcriptomic analyses for genes encoding enzymes that regulate the affected metabolic pathways.P-value <0.05; FDR 1.1%), suggesting that PAH tissues have a unique pattern of global metabolomic heterogeneity.PAH tissue exhibited a distinct metabolic signature in comparison to the control lung (CL), as shown in the overview of metabolite analysis , Table 1We compared levels of polyamines and other substances involved in arginine metabolism between the PAH and normal lung tissue. Arginine is a key intermediate for extracellular matrix remodeling and for producing polyamines and nitric oxide (NO) in addition to protein synthesis. Polyamines are important in the stabilization of DNA, cell growth and differentiation. The metabolomic analysis shows that arginine was significantly decreased in PAH lung tissue compared to normal lung tissue , while oIn conjunction with our metabolomics study, we also performed molecular analysis. Microarray analysis showed that the gene encoding the enzyme arginase1 (ARG1) was significantly increased while nitric oxide synthase 1 (NOS1) was decreased in PAH. Both enzymes are involved in the homeostatic regulation of arginine and NO levels. We did not find significant changes of genes encoding iNOS (NOS2) and eNOS (NOS3) in PAH compared with controls. Increased expression of ARG1 combined with decreased NOS1 may be the result of a feedback mechanism due to disruption of the arginine metabolic pathway and excessive intracellular and extracellular NO levels. We also found that the PAH lung had significantly increased gene expression for genes involved in the biosynthesis of collagen, such as procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2), collagen type XIV alpha 1 (COL14A1) and collagen type III alpha 1 (COL3A1) In sphingolipid metabolism, sphingolipid metabolites in PAH lung have variation with a scattered distribution pattern with a wider data range in PAH disease . GeneralHeme is synthesized from succinyl-CoA and glycine, and then degraded into bilirubin through biliverdin. Biliverdin is oxidized from heme by heme oxygenase. The byproduct of heme degradation is a vasodilator, CO. Bilirubin (ZZ) and bilirubin (EE), are two of the products from biliverdin that contribute to the yellow color of bruises and urine, the brown color of feces, and the yellow discoloration in jaundice. We found a significant difference in heme metabolism in PAH samples compared to the normal control. Biliverdin, bilirubin (ZZ), and bilirubin (EE) were found significantly increased in PAH , which iPAH is characterized by increased resistance in the pulmonary vasculature, eventually leading to right heart failure. We aimed to investigate alterations in several metabolic pathways that may contribute to the pathogenesis of PAH. Genetic profiling of enzymes involved in several metabolic pathways showed disrupted arginine metabolism, increased heme degradation and enhanced sphingolipid metabolic pathways in the human PAH lung, indicating that metabolic disruptions play an important role in the progression of PAH in its severe stages.Polyamines are essential for cell proliferation and cell growth; increased levels of polyamines are primarily driven by an increase in polyamine enzymes, such as ornithine decarboxylase (ODC), polyamine transport or AdoMetDC . In our Nitric oxide (NO) is the predominant signaling molecule for vasodilation ,14. NO dStudies have found that excess unbound NO will react with reactive oxygen species to produce peroxynitrite , a very Our metabolic results show there is a low ratio of arginine-to-ornithine levels in the severity of pulmonary hypertension. The increased arginase activity decreases the arginine-to-ornithine ratio, and high arginase activity is linked with progression of pulmonary hypertension . IncreasStudies have shown that collagen synthesis is connected with hypoxia-induced pulmonary hypertension (HPH), and reducing collagen synthesis can limit the progression of HPH towards more severe stages, as well as limiting right ventricular (RV) hypertrophy –23. PAH-Sphingolipids are a class of lipids that have sphingoid bases, and play a key role in maintaining the cell membrane, signal transmission and cell recognition. They are synthesized from serine and palmitoyl-CoA through several steps. It has been shown that alterations in expression of genes involved in sphingolipid metabolism could result in the progression of severe diseases, such as Alzheimer’s disease , which pHeme metabolism consists of two parts: heme synthesis and heme degradation. Heme is synthesized from glycine and succinyl-CoA, and is a part of hemoglobin, myoglobin and cytochromes. Heme degradation converts heme into bilirubin with the help of heme oxygenase. Our results indicate that there was no significant change in heme synthesis, but heme degradation was significantly increased. Our results are in agreement with previous studies showing that serum bilirubin levels were significantly elevated in PAH and could serve as a predictor of PAH mortality ,38. The Heme degradation is correlated with intravascular hemolysis, as lysis of red blood cells releases free hemoglobin, which further disassociates into free heme. Subsequently, heme serves as the source for the heme degradation pathway. Hence increased heme degradation may be the result of increased hemolysis, and may explain why heme biosynthesis was not increased in our study. Studies from Fox et al indicate that high erythrocyte creatine levels are an indicator of hemolysis . In addiIn heme metabolism, (HO)-1 helps protect the lung from oxidative stress and inflammation . SpecifiLimitation: we have analyzed the metabolic changes in PAH patients who are in the severe stage of PAH and require an organ transplantation. By analyzing end-stage PAH lung tissue, it is difficult to make conclusions about mechanisms that are pathogenic  versus response to disease . In addition, because these are patients who were scheduled to undergo transplantation, it is impossible to distinguish whether the metabolic changes were a consequence of disease development or a drug effect- as the patients surely were on multiple PAH-specific therapies, including prostanoids.In summary, our metabolomic profiles showed a disrupted arginine pathway, with increased NO, decreased arginine, increased S1P metabolites, and heme metabolites in severe human PAH lungs. The results suggest that PAH has specific metabolic pathways contributing to the vascular remodeling process in the advanced stage of pulmonary hypertension. Profiling metabolomic changes may provide a new understanding of pathogenic mechanisms involved in PAH and will aid in therapeutic targeting to specific metabolic pathways in the progression of PAH.


Background. Iatrogenic biliary injuries are considered as the most serious complications during cholecystectomy. Better outcomes of such injuries have been shown in cases managed in a specialized center. Objective. To evaluate biliary injuries management in major referral hepatobiliary center. Patients & Methods. Four hundred seventy-two consecutive patients with postcholecystectomy biliary injuries were managed with multidisciplinary team  at major Hepatobiliary Center in Egypt over 10-year period using endoscopy in 232 patients, percutaneous techniques in 42 patients, and surgery in 198 patients. Results. Endoscopy was very successful initial treatment of 232 patients (49%) with mild/moderate biliary leakage (68%) and biliary stricture (47%) with increased success by addition of percutaneous (Rendezvous technique) in 18 patients (3.8%). However, surgery was needed in 198 patients (42%) for major duct transection, ligation, major leakage, and massive stricture. Surgery was urgent in 62 patients and elective in 136 patients. Hepaticojejunostomy was done in most of cases with transanastomotic stents. There was one mortality after surgery due to biliary sepsis and postoperative stricture in 3 cases (1.5%) treated with percutaneous dilation and stenting. Conclusion. Management of biliary injuries was much better with multidisciplinary care team with initial minimal invasive technique to major surgery in major complex injury encouraging early referral to highly specialized hepatobiliary center. Iatrogenic biliary injuries during cholecystectomy are a serious surgical complication that can have devastating consequences, including a significant risk of early death , 2.Iatrogenic biliary injuries are feared complications reported to occur in approximately 0.2-0.3% in open cholecystectomy Era, but with incidence figures increasing following the introduction of laparoscopic cholecystectomy, with a mean figure of bile duct injuries when including both minor and major injuries up to 0.9% ; this isApproximately 17–20% of biliary injuries were recognized intraoperatively .The long-term implications for the patient, surgeon, and healthcare system along with the rising cost of litigation continue to mitigate this otherwise excellent procedure .Traditionally, surgery has been the gold standard for the management of biliary injuries. Recently, various endoscopic and radiological intervention methods have been used as the preferred modalities of these patients , as theyThe management outcome of iatrogenic biliary injuries when it occurs has been shown to be better when such injuries are managed at specialized hepatobiliary center equipped with multidisciplinary service , 11.The availability of surgical expertise to repair small caliber bile ducts high within the porta-hepatis and the availability of specialized radiological and endoscopic support are the main factors that contribute to the better outcome .The choice of surgical reconstruction and timing of surgical repair are decisive for long-term course. Numerous surgical and interventional treatment modalities that are available require close interdisciplinary cooperation of gastroenterologists, radiologists, and surgeons , 14.In this setting, we analysed the multidisciplinary management approach of iatrogenic bile duct injuries following cholecystectomy with emphasis on the improvement of long-term outcome in a major hepatobiliary referral center.This retrospective study included 472 patients with iatrogenic bile duct injuries following cholecystectomy (open and Laparoscopic) referred to the Department of Hepatobiliary Surgery at National Liver Institute, Menophyia University, Egypt  from January 2002 to January 2012 and treated by multidisciplinary approach team including hepatobiliary surgeons, gastroenterologists, and interventional radiologists. The multidisciplinary team was established after ethical and scientific approval from Hepatobiliary Department and National Liver Institute committees. All cases of iatrogenic bile duct injuries should undergo this multidisciplinary team approach to set up a road map management of such cases.All patients complained of postcholecystectomy biliary tract injuries encountered with variable presentation and timing from the surgical insult until they were referred to our center for further evaluation and management. thorough detailed history taking; meticulous clinical examination.Cases were subjected to the following:Operative details of the previous cholecystectomy should be revised with surgical team of referring hospital.Investigation needed to diagnose the problems such as liver function tests and abdominal ultrasound were done for all cases as routine preliminary workup.Computed tomography or magnetic resonance imaging was done in some cases.Cholangiogram was done for all cases  as a trans-tube cholangiogram (with a T-tube in place), an endoscopic cholangiography endoscopic retrograde cholangiopancreatography (ERCP) in most cases, or percutaneous transhepatic cholangiogram in some selected cases in which endoscopic approaches failed.After receiving patients data by multidisciplinary team, patient condition was categorized through discussion of detailed results of treatment for each category to reach consensus on which type of modality to start with, either endoscopy or intervention radiology as minimal techniques for definitive treatment or bridging technique for definitive surgery (as complementary tool) prior to surgery or whether surgery still is needed for definitive treatment or surgery is mandatory from the start as definitive treatment.Also the multidisciplinary team approach gave an outreach service for on-table repair of iatrogenic bile duct injuries to nearby hospitals around the tertiary center in 19 cases after receiving emergency call from the surgical team in those hospitals.Patients were categorized according to the presentation into the biliary leakage group and the biliary stricture group as diagnosed by previous tools. Each group was managed according to the road map made by multidisciplinary team, starting with the minimally invasive tools  to more invasive surgical treatment.Biliary leakage group classified according to the classification of Strasberg et al.  was manaBiliary stricture group categorized according to the classification of Strasberg et al.  was treaPercutaneous manipulation was attempted in cases of proximal biliary injuries as in major CBD injuries, transaction, or ligation through percutaneous transhepatic cholangiogram as diagnostic tool prior to surgery, percutaneous manipulations, and guide wire deployment through the CBD prior to combined procedures (Rendezvous) techniques or percutaneous dilatation and stenting for stricture or injuries.emergency surgery for peritoneal lavage and drainage of biliary peritonitis;on-table repair of iatrogenic bile duct injuries in cases diagnosed intraoperatively in our center or as an outreach service in nearby hospitals;primary repair on T-tube splint in a minor laceration injury of the CBD;choldocholithotomy procedure in associated CBD stones;undoing CBD ligation;bilioenteric anastomosis operations were done as a Roux-En-Y loop depending upon the site of injury, in proximal injuries in porta hepatis (Hepp-Couinaud technique), was capitalized on the extrahepatic course of the left main hepatic duct. Hepaticojejunostomy was done (for the injuries above the biliary confluence) in which the repair was done in the common hepatic duct or at the bile duct confluence with widening the stoma by opening the right and left bile ducts together at site of confluence (stomaplasty), or cholodochojejunostomy was done . The bilioenteric anastomosis may be side to side or end to side maneuvers depending upon the site and extent of the biliary injuries, and the anastomosis was tension free, mucosa to mucosa, and good wide stoma, with T-tube or biliary splint  in majority of the cases to decompress the biliary tree in the immediate post-operative period and to obtain postoperative, contrast studies.Surgical approaches: surgical intervention was attempted for the cases not fixed by endoscopy or interventional radiology or cases which deserved surgical intervention from the start ), with the following surgical maneuvers:This study was conducted on 472 cases of postcholecystectomy biliary injuries. The mean age was (46.8 years), with a range of 19–71 years. Out of 472 cases there were 302 cases (64%) females and 170 cases (36%) were males. Biliary injuries cases were 265 (56%) after laparoscope and 207 (44%) after open approach, with most of the cases of the open approach occurring at the late 5 years of study as the learning curve for laparoscopic approach reaches its saturation state and many surgeons, especially the young ones, become master of the laparoscopic technique without gaining good training in the open approach. only 24 cases (5%) were originally operated on in our center and 19 cases (4%) were operated on in the nearby hospitals as part of outreach service program for biliary injuries after urgent consultation from surgical team of those hospitals. Cases presented to our center within a month after operation were considered as early referrals and they were 274 cases (58%) including outstretch service, but the cases presented postoperatively after one month were considered late referrals and they were 208 cases (42%).Cholangiogram was the main line of the diagnosis in cases of biliary injuries and was done in most of our cases. Also cholangiogram was the method of the diagnosing intraoperatively 5 cases in our center and 19 cases of outreach service program as intraoperative cholangiogram was done for those patients during or after the completion of the repair.Cholangiography methods were done by endoscopy (endoscopic retrograde cholangiopancreatography (ERCP)) for 346 patients (73.4%), percutaneous transhepatic cholangiogram (PTC) was done for 24 patients (5%), magnetic resonance cholangiopancreatography (MRCP) was done for 61 patients (13%), intraoperative cholangiogram was done in 24 cases (5%), and complementary tests, combination of all these tests, were done for 17 patients (3.6%).CT scan and MRI of the abdomen were done in most of the cases to detect any abdominal collection. Biliary leakage group includes 288 (61%).According to the results of cholangiogram, the injuries can be classified into biliary leakage and stricture group . Biliary minor leakage in 93 patients (19.7%); major leakage in 52 patients (11%); possible transaction of CBD in 17 patients (3.6%); leakage with CBD stone shadow in 69 patients (14.6%); leakage with CBD stricture in 20 patients (4.2%); undetected leakage by cholangiography in 37 patients (7.8%) that may be due to minor leakage from bile ductules or gall bladder bed; biliary stricture group includes 184 patients (39%).Cholangiogram demonstrated the following injuries: possible CBD ligation in 31 patients (6.5%); stricture in CBDhigh stricture in 26 patients (5.5%),middle stricture in 68 patients (14.4%),low stricture in 24 patients (5%), stricture and stone in 18 cases (3.8%); postoperative bilioenteric stoma stricture in 17 patients (3.7%).Treatment was done by either endoscopic approach (ERCP) alone or in conjunction with percutaneous approach or percutaneous approach alone or surgical approach after failing of the endoscopic or percutaneous approach or surgery from the start according to patient condition assessed by multidisciplinary team.Cholangiogram demonstrated the following injuries:Endoscopy was attempted in 232 patients (49%) using a side viewing videoscope, with regular instruments that were used in sphincterotomy and balloon dilatation and sphincteroplasty. Endoscopic treatments include sphincterotomy in mild cases and/or stenting in moderate to major biliary leakage, with concomitant stone extraction if present within the CBD , and also dilatation and stenting in repeated endoscopic sessions with upgrading of stents until a cure was obtained (after full dilatation of the stricture segment as evident by loss of the waist in the repeated follow-up cholangiogram)  in 18 patients or with other percutaneous techniques in the rest of the cases were attempted, where therapeutic dilatation and stenting for stricture or injuries were used in 14 cases and diagnostic PTC prior to surgery was used in the other 10 cases , and 6.Surgery was attempted in 198 cases (42%) either as an urgent surgery in 62 patients (including in-table repair in 19 patients in outreach service and 5 patients in our center) or as an elective surgery in 136 patients. In urgent surgery (62 patients) slipped cystic duct was ligated in 12 cases while peritoneal drainage and external biliary stents were inserted in 30 cases prior to further definitive treatment; however, it was a definitive treatment in 20 patients (17 patients in outreach service and 3 patients in our center).peritoneal lavage and drainage for biliary peritonitis;drainage and ligation of slipped cystic duct ligature or clip;CBD repair on a T-tube splint in a minor lacerations injury in the CBD;choledocholithotomy procedure in associated CBD stones;undoing ligation and strictureplasty with a T-tube splint if CBD ligation is discovered early;bilioenteric anastomosis by Roux-en-Y hepaticojejunostomy.The surgical maneuvers involved the following , and 10:Routine postoperative management was carried out as follow. Endoscopically and percutaneously treated cases were regaining oral feeding 6 hours after the procedure and were discharged at the next day after the patient's condition became stable. Surgical cases were followed up in surgical ICU overnight and transferred to the surgical ward for a variable period prior to discharge (7–13 days). All cases were followed up for a period of 1.5–5 years after procedure.There was one (0.5%) mortality postsurgical maneuver due to biliary sepsis with secondary biliary cirrhosis due to long standing biliary stricture and obstruction. Complications were reported in each group of treatment optionally, postendoscopic maneuver complications were cholangitis, pancreatitis, and stent obstruction, and postpercutaneous manipulation complications were bleeding from PTC/PTD, biliary leakage around PTD, or slipped PTD catheter commonly reported; however, postsurgical complications were mainly wound infection, postoperative bile leakage in early postoperative period and postoperative intrahepatic stones, postoperative biliary stricture, and incisional hernia in the long-term follow-up .Iatrogenic bile duct injuries pose a complex challenge to the treating physicians . Simon wInadequate management of bile duct injuries led to severe complications, such as biliary peritonitis leading to sepsis and multiple organ failure in early phase, and biliary cirrhosis during long-term follow-ups, and eventually the need for liver transplantation .Not all forms of diagnostic workup and specialized treatments are available in all hospitals and there should be a low barrier for referral. Unfortunately, lesions will occur, but suboptimal treatment of biliary injuries is not accepted nowadays.Our institute is a major referral center for hepatobiliary surgery with an increase in the flow of referral cases of postcholecystectomy biliary injuries. We adapted the multidisciplinary management approach program to deal with all cases of postcholecystectomy biliary injuries.All cases of biliary injuries were reviewed by the multidisciplinary team following the steps of diagnosis and treatment.In this series, all cases were subjected to a variety of diagnostic workups for diagnosis and delineation of biliary tract before any therapeutic intervention. In 7.8% of the cases diagnostic workup did not reveal any abnormalities which were considered as minor injuries and were treated conservatively without any intervention.Management of biliary injuries detected during cholecystectomy is mainly dependent on the local expertise. If a competent hepatobiliary surgeon is not available, biliary drainage should be performed without exploration and patients should be referred to a highly specialized center as further exploration could lead to proximal extension of the lesion, sacrificing the normal healthy duct tissues, with having a negative impact on its reconstruction in the near future.Multidisciplinary team has outreach service to nearby hospitals around our institute for immediate on-table repair of biliary injuries. In this series, 19 cases (4%) were treated as outreach service. In 17 cases, definitive treatment by repair of the bile duct over T-tube splint and hepaticojejunostomy anastomosis were performed with good long-term follow-up, while in other 2 cases biliary drainage was done for later further definitive treatment.The advantages of immediate on-table repair of biliary injuries include single anesthesia, surgical procedure for the patient, and shorter hospital stay. When a hepatobiliary surgeon provides the service of on-table repair as an outreach service, in addition to the added advantage of better surgical outcome, the need to transfer the patient to a tertiary center is also abolished.As opposed to a delayed repair, an immediate on-table repair nullifies the need for prolonged external biliary drainage and associated increases risk of sepsis. The disadvantages of such an outreach on-table repair of bile duct injures are that these injuries are often complex, requiring high hepaticojejunostomy reconstruction for nondilated, normal diameter  ducts with thin wall.With our experience in living liver transplant at our center since 2003, our surgical team used to operate on normal bile ducts and becoming familiar with access to the site of injury was achieved satisfactorily since our outreach team brought a long suitable abdominal wall retractor and other instruments that are used for hepatobiliary surgery.The extent of the ischaemic injury suffered by the bile duct is less apparent in the immediate repair setting , 21. To The higher rate of injuries with laparoscopic method was initially attributed to the learning curve. This has, however, remained the same, a decade after the wide spread acceptance of the procedure , 23.In this series, biliary injuries after laparoscopic approach were 56% of the total cases and 44% for open approach, with most of the cases of open approach occurring at the late 5 years of study as the learning curve for laparoscopic approach reaches its saturation state and many surgeons, especially the young ones, become masters of the laparoscopic technique without gaining good training in the open approach.Bile leakage was a common presentation among our patients (61%), usually the leakage that originated from the liver bed or biliary injuries as documented by various studies , and canBile leakage was demonstrated by cholangiogram in most of the cases (251 of 288 patients); however, the spillage was very mild and not evident by contrast injection in 37 cases (12.8%), such minimal bile leakage was resolved spontaneously which is concomitant to the stated facts in other literatures .Endoscopic treatment in this series was applied in 232 (49%) cases of biliary leakage and stricture.In this series, endoscopic treatment was achieved in mild and moderate cases up to 96–100%, as explained in the literatures that endoscopic treatment accelerates the healing period by decompressing the biliary system; in addition, it closes the defect physically and acts as a bridge at the site of extravasation. Stenting also acts as a mold and prevents stricture formations during recovery period and should be the preferred treatment .In major leakage (type D&E) Strasberg classification endoscopic treatment with sphincterotomy and stenting was successful in 65% (34 out 52 cases) only. This result was compatible with other reports in –30.Out of 34 cases 10 cases developed later stricture which was treated with upgrading the size of the stent; our results are also comparable with other literature reports .Common bile duct stones were found to be exacerbating the bile leakage in 69 cases and were successfully treated by sphincterotomy and stone extraction in conjunction with stenting in 61 cases (88%) out of 69 cases. This result was in agreement with other reports , 32. AlsIn biliary strictures after biliary injuries the endoscopic treatment was successful in 67 patients with sphincterotomy, boogies or balloon dilatation, and convenient stenting. It was performed in conjunction with common bile duct stone extraction in 7 cases out of 18 cases and in repeated ERCP sessions to replace or subsequently upgrade the stent in 49 cases, in agreement with other previous reports stating that ERCP and stenting have good results with lower rates of morbidity and mortality , 33, 34.Endoscopy is the preferable initial therapy in biliary leakage and stricture , 36, butStents should be replaced every 3 months before possible clogging could cause cholangitis, and the patient should be informed about the risk of stenting and duration of the treatment –40.Otherwise, surgery is indicated as the treatment of choice, especially in surgically suitable patient . HoweverUnfortunately, the role of endoscopy is weak in common bile duct transection injuries with leakage as only 4 cases out of 17 patients were endoscopically treated, in agreement with other studies demonstrating this low incidence of endoscopic treatment of such problems , 37.Diagnostic percutaneous transhepatic cholangiography (PTC) was done in 10 cases prior to surgery in high proximal injuries not delineated by endoscopy and percutaneous transhepatic drainage (PTD) was inserted for 5 patients in bad condition for preoperative preparation for surgery in high ligation of common bile duct.Stenting of stricture with leakage in high proximal injuries was done in 3 patients out of 20 patients. However, Rendezvous techniques plus endoscopy were performed in cases which failed endoscopy in 18 cases, in agreement with other reports in , 42.Percutaneous dilatation and stenting for stenosis and stricture in post-bilioenteric repair was successfully performed in 6 cases out 17 cases with good long-term results, in agreement with several reports of the treatment of such postoperative biliary stricture at stoma side in bilioenteric anastomosis , 42.Surgery was done in 198 cases (42%) of this series as an urgent surgery for the 62 cases, ligated slipped cystic duct (open or laparoscopic)was done in 12 cases, and peritoneal lavage with external biliary drainage was carried out in 30 cases; however, the surgery was definitive (on table-repair) in 20 cases and 13 cases were common bile duct repair over T-tube and in 7 cases bilioenteric anastomosis was done on-table repair as practiced by other authors , 44.On other hand, surgery was needed as elective in 136 patients, especially after failure of other minimal invasive techniques , and surgery was effective in common bile duct repair over a T-tube splint, choledocholithotomy, and common bile duct repair over T-tube splint, choledocholithotomy, strictureplasty and T-tube splint, and bilioenteric anastomosis, which was done in 96 cases as the operation of choice in most documented studies –47.In this series, we used transanastomotic stents, the rationale that leaks of small bilioenteric anastomosis promote stricture and the rationale that both lowering of the intraductal pressure and adequate flow through the anastomosis were warranted by stents, as practiced by other authors , 48, 49.Redo surgery in the post-bilioenteric anastomotic stricture or stenosis was done in 11 cases out of 17 cases with good long-term outcome.The operation of choice in this series is Roux-en-Y hepaticojejunostomy as good long-term surgical results are obtained in this type of technique as documented in most literatures –47.No mortality occurred in this series after endoscopic treatment, which is consistent with most reports in the literature . But somAll complication were treated conservatively except incisional hernia which was treated with hernia repair with mesh and postoperative anastomotic stricture, managed by percutaneous dilatation and stenting as it is very beneficial in such cases documented by other authors , 42.The management of patients with biliary injuries should be ideally performed/discussed in a multidisciplinary team approach that consists of a gastroenterologist, radiologist, and surgeon. Better outcomes of such cases are mainly the result of multidisciplinary care and changes in technical aspects which have changed considerably through the time of learning curve with growing experience of the team. Early referral to high volume tertiary care center with experienced hepatobiliary surgeon, skilled gastroenterologist, and interventional radiologist would appear to be necessary to assure optimal results and should be encouraged.


Major discussion is included on the current spectrophotometric and imaging based techniques used for quantifying, and studying the clearance and biodistribution of IONPs. Several invasive and non-invasive quantification techniques along with the pitfalls are discussed in detail. Finally, critical guidelines are provided to optimize the design of IONPs to minimize the toxicity.Increasing biomedical applications of iron oxide nanoparticles (IONPs) in academic and commercial settings have alarmed the scientific community about the safety and assessment of toxicity profiles of IONPs. The great amount of diversity found in the cytotoxic measurements of IONPs points toward the necessity of careful characterization and quantification of IONPs. The present document discusses the major developments related to  The commercial use of engineered nanoparticles (NPs) has increased extensively in recent years to include applications in areas such as advanced nanoelectronics, optics, enhanced automation and robotics, nanostructured chemical catalysts, textile, oil and gas industries ,2. Moreo3O4 and maghemite, γ-Fe2O3) have been used in numerous applications in biomedicine, such as magnetic resonance imaging (MRI) [Superparamagnetic iron oxide MNPs  , drug deng (MRI) , and theng (MRI) . IONPs cng (MRI) . By conjng (MRI)  or diagnng (MRI) . Superpang (MRI) . Moreoveng (MRI) ,14 and gng (MRI) .in vivo. Yet, the off-target impact of IONPs exposure is largely unknown because the mechanisms of IONPs interactions with otherwise healthy cells, tissues, and organisms are not well understood [As the development of IONPs in biomedicine and manufacturing industries continue to rise, so do the risks of environmental damage and biological toxicity as these particles accumulate in the environment, the food chain, and the work force . Indeed,derstood . Becausein vitro and in vivo characterization of NPs. Specifically, NCL carry out pre-clinical characterization of nanomaterials intended to diagnose or cure cancer [in vitro tools. The need to evaluate the biotransformation of IONPs in the body and their pharmacokinetic profiles was strongly emphasized in the NanoTEST project [Considering the potential and growing applications of MNPs, many government and non-government agencies have introduced regulatory policies facilitating the scientific advancement and promoting safe use of nanotechnology based medicine. A plethora of information is being generated to explain the impact of physiochemical properties of IONPs on toxicity and bio-distribution pattern of IONPs. The Nanotechnology Characterization Laboratory (NCL), which is a combined effort by FDA, National Cancer Institute (NCI), and National Institute of Standards and Technology (NIST) has developed a standardized analytical cascade that include physical e cancer  In anoth project . Many unin vitro/in vivo toxicity. Considering the significant impact of physicochemical parameters on the absorption, distribution, metabolism and excretion (ADME) pattern of IONPs, it is required to quantify each unique IONP containing nanomedicine. Thus, this document also discusses a variety of invasive and noninvasive quantification techniques along with their detection limits and will assist readers and scientists to choose an appropriate technique for in vitro/in vivo quantification of bare and coated IONPs.The current review aims to explain the correlation between the mechanism of toxicity of IONPs and major physicochemical factors responsible for Findings from the majority of nanotoxicity studies conducted with IONPs have found the production of reactive oxygen species (ROS) as a major reason behind cell death. A variety of stress factors such as temperature, interaction with pathogens, and foreign materials can be responsible for the generation of ROS. ROS such as anions, hydroxyl radicals, and hydrogen peroxide are byproducts of oxidative metabolism, which occurs in mitochondria. Cells respond to increased levels of ROS by a “detoxification” mechanism, which involves enzymes such as superoxide dismutase and glutathione peroxidase. Glutathione has been considered to play a major role in defense against ROS . It oxidSystematic administration of IONPs in the blood stream faces the initial uptake by the liver and spleen. IONPs uptake is mediated by the mononuclear phagocytic system via endocytosis in kupffer cells of the liver and macrophages of spleen. IONPs are degraded in the lysosomes of kupffer cells and macrophages, thereby releasing the free iron from IONPs, which affect the iron homeostasis. IONPs can be enzymatically degraded in the lysosomes (pH~4.5) and released iron ions can participate in the Fenton reaction to produce hydroxyl radicals ,33. Free3O4  and Fe3O4  have toxicity in A549 cells in terms of cell death, mitochondrial damage, and DNA damage. But, no significant difference was found between the toxicity response by Fe3O4 (20–30 nm) and Fe3O4 (5 μm) [2O3) showed a higher degree of necrosis in mouse macrophage cells than spherical IONPs did. Rod-shaped IONPs were mostly accumulated in the cytoplasm, while spherical IONPs aggregated in vacuoles. Higher surface area/volume, nonspecific endocytosis, and membrane damage due to their rod shape can explain the higher toxicity compared to spherical shaped IONP [Physicochemical parameters of IONPs such as size, shape, and surface chemistry also contribute towards ROS induction in cells. The higher surface area of smaller IONPs has been linked to increased toxicity of IONPs ,36. Howe4 (5 μm) . The shaped IONP . The surped IONP . The dirDue to this diversity, it has been extremely difficult to assign a definite toxicity profile that can be followed while choosing the proper IONPs. Several reports discussed the higher toxicity of bare IONPs than coated IONPs; however, some research studies have found the toxicity of bare IONPs to be less than that of the (oleate) coated IONPs. Oleate itself did not show any cytotoxicity, which shows that the toxicity might have been associated with the interaction and cellular uptake of oleate coated IONPs . Incorpo2O2 production by the higher ratios of iron  at neutral pH levels [2+ and Fe3+ [3+ occupies all the tetrahedral sites, and Fe3+ or Fe2+ occupies the octahedral sites. However, in maghemite all the octahedral sites have cationic vacancies and mostly contain Fe3+. Thus, dissolution of magnetite IONPs releases Fe3+ or Fe2+ while maghemite releases Fe3+ only [2O3 and α-Fe2O3 have shown a varying degree of hydrogen peroxide degradation, later being more catalytic [The chemical composition and crystalline nature of IONPs have also mediated ROS linked redox reactions. The Fenton-like reaction was significantly affected in terms of increased HH levels . Moreoveand Fe3+  and oxidand Fe3+ ,44. Recee3+ only . Differeatalytic .et al. suggested that the adsorption of IONPs on cell surfaces can be responsible for disturbing the structure and function of the cell membrane. Size and time dependent disruption of junctional complexes in the epithelium of caco-2 cell lines was verified by transepithelial electrical resistance measurements (TEER). The disruption was found to be maximum with high doses (300 mg/L) and 26 nm IONPs, and the γ-catenin, which is a key protein in adherence junctions, was also affected [et al. described the impaired cell integrity of HMEC-1 and HUVEC cells upon incubation with IONPs. Decreased impendence and increased intracellular gap junction was observed, which is an indication of altered cytoskeleton and plasma membrane, and impaired endothelial barrier [Exposure of IONPs can cause alterations in cell morphology, cytoskeleton of cells, and cell motility ,48. Endoaffected . The samaffected . Astanin barrier .Genotoxic effects of IONPs are mainly influenced by direct interaction with leached iron ions from IONPs or various indirect factors, such as excessive ROS  and IONPin vivo toxicity of IONPs. Once IONPs come in contact with blood, various proteins are adsorbed on the surface of IONPs, forming a protein corona. Adsorption of proteins can be affected by size, charge [Even though the physicochemical properties of IONPs have a major influence on the toxicity of IONPs, the role of cell-nanoparticle interactions and protein corona has also affected the , charge , source , charge , incubat, charge  and type, charge . Studies, charge . HoweverRecently, a group of researchers studied the effect of slight temperature changes on the protein corona formation of IONPs. Protein coronas formed at different temperature showed different responses towards ROS generation in HeLa cells. Positively charged IONPs generated more ROS compared to neutral and negatively charged IONPs, highest being at 39 °C. These results were further supported by analysis of neutral, negatively and positively charged IONPs in HeLa cells using LysoTracker assay and electron microscopic analysis . The resUndoubtedly, the catalytic properties of iron play a major role in the induction of ROS formations resulting into oxidative stress. Passivating the surface of IONPs can certainly alter the ROS production but, is not the only factor to control it. In addition to physiochemical properties of IONPs, various experimental conditions also influence the production of ROS and require careful monitoring to narrow down the responsible factor.in vivo research is currently underway to explore the feasibility of using IONPs in a delivery and ablative treatment capacity. Magnetic localized delivery of drug-loaded NPs for cancer therapy is advantageous due to the nanoparticle’s design in bypassing cellular uptake barriers. Efficient treatment utilizing magnetic ablation has been shown in xenograft models [Toxicity studies of IONPs started to appear during late 1980s when IONPs were introduced as MRI contrast agents ,71. Initt models .in vitro, in vivo, and clinical nanotoxicological studies. Even though in vitro studies do not substitute for in vivo or clinical studies, it is considered a good starting point for toxicity studies. Currently, toxicity studies are considered a mandatory factor while demonstrating the use of IONPs intended to diagnose or treat illnesses in the human body. Primary in vitro studies conducted with IONPs shed light on changes in membrane integrity, metabolic activity, and genetic material of cells upon reacting with IONPs. In vitro nanotoxicity assessments can produce reliable and reproducible results without the use of animals, and are of simple, rapid, and inexpensive nature. The -2,5-diphenyltetrazolium bromide) (MTT) or -5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) assay, which measures the mitochondrial function, and lactate dehydrogenase (LDH) assay, which measures the cell membrane integrity, are widely used cell-based assays in studying the toxicity of IONPs. Reproducibility is highly affected by certain parameters such as types of NPs, cells, cell culture conditions, and assay protocols. Results of in vitro assays can also be affected by the large surface area and chemical nature of IONPs, which can interact with MTS [Researchers aim to solve the puzzle of nanotoxicity by collecting the data obtained from with MTS , affect with MTS , and intwith MTS ,81.in vitro toxicity studies. The size, shape, charge, surface area, and aggregation of IONPs have a significant impact on in vitro toxicity studies. Increased toxicity was observed with polyethylimine coated IONPs (50 μg/mL), whereas inclusion of PEGylation and acetylation eliminated cytotoxicity in KB cells (MTT assay). Authors claimed that the increase in toxicity can be attributed to the strong electrostatic interaction between the negatively charged cell surface and positively charged IONPs at higher doses [in vitro tests. Present toxicological information reflects the importance of meticulous physicochemical characterization of IONPs and its effect on er doses . Numerouer doses ,84,85. Din vitro test results. Due to the widespread use of positively charged, amine modified IONPs and negatively charged, carboxyl group modified IONPs in biomedical applications, majority of toxicity studies were performed with these types of IONPs. Amine-modified IONPs have been found to be more lethal in in vitro tests due to their strong interaction with negatively charged cell surface [Furthermore, the type of functional groups adds another complication to the  surface ,87. Howe surface . This be surface . Attempt surface .in vitro toxicity results. Selection of cell lines is influenced by the major exposure routes of IONPs. For example, the lung is one of the main exposure routes, so it would be more logical to use the murine alveolar macrophage cells to obtain reliable in vitro cytotoxic results. IONPs have been tested with a variety of cell lines such as human epidermal keratinocytes, human lung epithelial cell lines, BRL3A rat liver cells, and Cos-7 monkey fibroblasts [in vitro studies. Clearly, the difference between the in vitro and clinical dose of IONPs is significant and can be explained by the motive of studying the tolerance and biocompatibility of IONPs at higher doses. Knowing the toxicity of IONPs at higher doses could provide guidelines for further biodistribution studies and also shed light on the possible mechanism of toxicity generation.Different types of cytotoxicity assays, cells/tissues, and culture media can also provide variations in roblasts . Howeverroblasts  whereas roblasts . Dose deroblasts ,92,93. Troblasts  found throblasts  which isIn vitro assays such as lactate dehydrogenase (LDH) measures leakage of LDH from the cell membrane upon incubation with IONPs and indicative of damage to cell membrane Some of the other assays that can provide information about cell membrane integrity are annexin V which labels phosphatidylserine, propidium iodide staining, and neutral red staining [Physical damage by IONPs can also cause toxicity by inducing oxidative stress in cells. Incubation with IONPs affects the cell surface roughness which could also change the shape and alter the response by cellular cytoskeleton . Morpholstaining .in vivo [et al. found the significantly lower toxicity of bismuth NPs, quantum dots, and IONPs in microtissues grown in 3D media compared to the tissues grown in 2D media. The 3D microtissue culture forms a protective layer that may inhibit the uptake of NPs [Several studies appeared in the literature discussing the effect of the type of culture medium on the cytotoxic results of IONPs. The 3-dimensional culture medium has been increasingly preferred by scientists due its ability to mimic a natural cell environment, which provide a larger surface area for interaction with drugs or NPs. Also, it has been found that cell-cell interaction in 3D culture medium is similar to behavior of cells in vivo . In contin vivo . Toxicitin vivo . On the e of NPs , and thee of NPs .in vitro toxicity investigations of IONPs [in vitro toxicity studies of IONPs focusing on physicochemical parameters in in vitro report; selections were primarily made on the basis of type of coating agents used for IONPs. Apart from the conflicting results generated by in vitro testing, it still provide a primary direction in the long run of toxicity studies and hints at the complexity involved in IONPs mediate toxicity. Impact of several physicochemical properties of IONPs on in vitro toxicity assays increases the probability in variations of results. This process has become more efficient with the help of HCS, which have provided relief by generating larger set of data in a small amount of time. But, a proper characterization of IONPs must be performed to explain the variations in in vitro studies.Recently, multiparametric toxicity evaluations by high content screening (HCS) are being implemented to perform of IONPs . Multipaof IONPs . We havein vitro studies, little correlation has been made between in vitro and in vivo studies [in vitro studies to mimic the complex environment and homeostasis mechanism maintained by clearance organs such as the kidney and liver. However, to proceed with registration of a drug with FDA for human clinical trials, in vivo studies explaining biocompatibility, biodistribution, and bioclearance in animal models must be completed. In vivo studies are considered a critical step to study the pharmacokinetic parameters such as absorption, distribution, metabolism, and excretion of IONPs. Irrespective of the expenses and ethical issues involved with in vivo studies, they remain to be an integral part of research studies aiming towards gaining a better understanding of IONPs in the body.Despite the rapid, inexpensive and reproducible nature of  studies . This isin vitro studies, various physicochemical factors such as surface chemistry, size, shape, and charge of IONPs play an important role in animal studies [meso-2,3-dimercaptosuccinic acid (DMSA) to PEG-IONPs doubled the residence time upon intravenous administration [In vivo studies conducted with positively charged IONPs have shown improved penetration of placenta in CD-1 mice than negatively charged COOH modified IONPs [Similar to  studies . To redu studies ,124. Constration . In vivoed IONPs . Size-deed IONPs .Different exposure routes can affect primary interactions and intracellular entry of IONPs in cells. Organs, which are enriched with reticuloendothelial systems , take up the majority of IONPs introduced by most of the administration routes. IONPs administered by inhalation route and intravenous route accumulated in the liver, spleen, brain, testis, and lung, whereas IONPs administered by an intravenous route in mice were accumulated in the kidney, spleen, and brain ,127. Intin vivo study with pluronic coated IONPs showed normal levels of AST and ALT after 3 days of intravenous injection in rats [Different types of animal models have also displayed variable toxicity profiles of IONPs. Changes in the levels of aspartate transaminase (AST) and alanine transaminase (ALT), which is indicative of liver function, were reported in different types of animals upon intravenous administration of IONPs. Increased levels of AST and ALT were found after 1 month of intravenous injection in mice. Moreover, hematology studies conducted on mice showed slight increase in the neutrophils content after one day which returned to normal one month post injection . Another in rats . The typin vitro/in vivo toxicity studies of IONPs can be considered as an important factor for successful transition to clinical setting. However, comprehensive understanding of the quantitative distribution of IONPs in the various organs of the body to determine the iron clearance holds a key to successfully extrapolate nanomedicines to clinical setting. Thus, it is required to equally emphasize the in vitro/in vivo quantitative analysis of IONPs in different organs to assign pharmacokinetic profile to IONPs nanomedicines.Data obtained from physicochemical characterization and in vivo/in vitro quantification of IONPs with better sensitivity and reproducibility [The significant amount of work poured into developing nanodelivery systems have raised the need to study the pharmacokinetic parameters of nanomaterials, which can be used to improve the efficacy of the system with minimal side effects. Accurate quantification of nanodelivery systems containing IONPs is a critical step that provides information about biodegradation and bioclearance patterns of IONPs. Quantitative analysis of intracellular IONPs in various cellular compartments such as mitochondria, lysosomes, and nuclei may provide directions towards their biological impact, and assist in designing potential nanocarriers. Iron quantification techniques can be broadly classified on the basis of spectrophotometric and imaging based measurements . Inducticibility ,115.2+ or Fe3+, digestion of IONPs is required to release Fe2+ and Fe3+ prior to analysis, thereby making these technique strictly useful for in vitro or ex vivo purpose. Most importantly, these techniques fail to differentiate between the iron from IONPs and endogenous iron, which is in the form of ferrin, ferritin, and hemoglobin. Additionally, the expensive nature and unavailability of ICP-OES/AES or ICP-MS in biomaterials laboratories limits the access to this quantification approach. Precaution should also be taken in preparation of proper calibration standards, which should mimic the environment in tissues and body fluids.Inductively coupled plasma-optical emission spectroscopy (ICP-OES) or inductively coupled plasma-mass spectrometry (ICP-MS) are considered as a “gold standard” technique to quantify IONPs due to their high detection limit . ICP-OESin vivo biodistribution pattern of 14C NPs [14C labeled IONPs administered by the inhalation route. The AMS successfully detected 14C labeled IONPs in the lungs, liver, and spleen with a detection limit of 1 ng/mg [Biodistribution of IONPs can also be tracked using sensitive isotope-based labeling accelerator mass spectrometry (AMS). AMS is commonly used for radiocarbon dating, which accelerates the ions to extremely high kinetic energies followed by separation of isobars according to their respective mass to charge ratios . The sam 14C NPs . AMS was 1 ng/mg .in vitro quantification of IONPs [2+ ions to yield a magenta colored solution which absorbs light at 562 nm. The complex is stable at a pH range of 4–10, and has a detection limit in the range of 2 to 10 ppm. Ferrozine also forms colored complexes with Cu, Co, Ca, Mg, Pb, Ag, Mo, Al, Ni, Zn, As, Mn, Cr, oxalate(>500 mg/L), cyanide, and nitrite [et al. noted the interference of Fe3+ in ferrozine measurements in the absence of light, which resulted in an increase in the absorbance upon increased incubation [2+, precaution should be taken while performing the measurements [in vitro iron uptake in cells. Upon reduction of Fe3+ to Fe2+, the chromegen reacts with Fe2+ and forms a blue colored complex, which can be measured at 590 nm [2+ that can be measured at 525 nm. Ascorbate treatment is used for the reduction of Fe3+ to Fe2+ prior to its reaction with bathophenathroline [In vitro quantification of IONPs can also be performed by using Prussian blue staining, which involves reaction between potassium ferrocyanide and Fe3+ to form a blue product called Prussian blue. Prussian blue labeled Fe3+ ions can be measured at 680 nm using a microscope as well as a spectrophotometer [et al. [et al. Authors attributed this disparity to differences in protocol and use of thicker slices [In order to perform rapid and affordable quantification of IONPs, many laboratories rely on colorimetric techniques that involve the use of easily available UV-Vis spectrophotometer or fluorescence spectrophotometer. The majority of colorimetric methods modify the iron ions with the reagents that can absorb light at certain wavelengths. Colorimetric ferrozine assay has been acknowledged as a reliable method for measuring intracellular iron uptake. The simplicity and affordable nature of this technique has gained popularity for of IONPs ,128,129. nitrite ,136. Inicubation . Additiourements . Anothert 590 nm ,140. Anothroline ,138. In otometer . In ordeotometer . In anototometer . Conflic [et al. . High ler slices .Optical imaging techniques such as fluorescence have been preferred by scientists due to its sensitivity and ease of use over radiolabeling techniques. To quantify the IONPs by fluorometric methods, the IONPS are encapsulated with coating agents containing fluorophores  conjugatNear infrared (NIR) fluorescent dye labeled IONPs can be quantified by fluorescence reflectance imaging (FRI). However, due to weak tissue penetrating ability of 2D FRI, a 3D fluorescence molecular tomography (FMT) was introduced and reported to have spatial resolution of ~500 μm with ~10–20 mm tissue depth . FMT canT2 and T2*) and show negative contrast have made them a favorable choice to use as an MRI contrast agent [T2 and 1/T2*) and the quantity of iron at low concentrations [R2*-based quantification has shown some pitfalls such as overestimation of relaxation rates due to air tissue interface [Ability of superparamagnetic IONPS to shorten transverse relaxation  have been preferred for in vivo quantification of IONPs [M(H) curves of endogenous iron and IONPs. The saturation magnetization (Ms) can be easily calculated by using the Langevin function. The Ms calculations were used for in vivo quantification of dextran-coated IONPs in the liver and lungs of Balb-c mice, and the values were found to be 0.25 and 2.4 μg, respectively [in vivo and ex vivo quantification of IONPs [To avoid the interference of endogenous iron in of IONPs ,160. Upoof IONPs . Anotherectively . Electroof IONPs . In a coof IONPs .R2* based measurements of MRI signal at higher concentrations have directed researchers towards T1 quantitative mapping sequences, such as ultra-short echo time (UTE) and sweep imaging with Fourier transformation (SWIFT). The UTE and SWIFT can reduce the time interval between excitation and acquisition to microseconds, thereby minimizing the T2* signal loss. The sweep imaging with Fourier transformation using variable flip angles (VFA-SWIFT) allows the ability to perform T1 measurements by varying the flip angle close to Ernst angle, the peak signal intensity for a given T1. This ability of VFA-SWIFT can allow the T1 quantification of IONPs with higher concentration (1–7 mM) [MRI quantification has proven to be effective at low concentration since higher IONPs concentration can cause MRI signal loss and image distortions. Pitfalls of (1–7 mM) ,166.In another technique called magnetic particle spectroscopy (MPS) or magnetization response spectroscopy, frequency and large amplitude is applied to IONPs and the non-linear magnetization response of IONPs in the presence of an oscillating magnetic field is monitored . The MPS59Fe monodisperse IONPs were synthesized and tested in both in vitro and in vivo systems. The 59Fe labeled IONPs were found to be stable in various organic solvents and biological media , and no morphological changes were observed. The 59Fe IONPs were injected in mice via tail vein injection with minimum dose (to avoid radio toxicity) to study size and time dependent half-life of polymer-coated IONPs. The 59Fe IONPS were found to be stable in biological media , and no morphological alterations were observed. The same work also observed an entirely different organ uptake pattern by 14C labeled IONPs raised a possibility of separation of the 14C label in biological media [et al. while studying the in vivo integrity of IONPs labeled with 111In. Some degree of dissociation of 111In from IONPs was also reported [59Fe and 111In labeled IONPs showed a similar biodistribution pattern; however, 51Cr labeled IONPs showed excretion in feces rather than in urine which can be attributed to a different path of metabolism of 51Cr [“Hot spot” techniques such as positron emission tomography (PET) and single photon emission computed tomography (SPECT) can be combined with MRI or computed tomography (CT) to provide qualitative and quantitative information about anatomical and morphological changes. PET utilizes positron emitting radionuclides for visualization and quantification with detection limit in picomolar range. Radionuclides can be introduced in the IONPs during synthesis steps either in the coating layer ,173 or ial media . The simreported . The 59F of 51Cr . Widesprin vitro and ex vivo settings [4 NPs per spot. In ex vivo approach can also preserve spatial distribution, which renders it effective for in vivo applications [Quantitative photo acoustic imaging was demonstrated to accurately quantify IONPs in both settings . When ansettings . Using aications .Non-invasive and sensitive nature of imaging based techniques certainly has an edge over spectrophotometric based quantitative techniques. No single imaging technique can single handedly provide good depth, resolution and sensitivity, which explains the emergence of multimodality imaging agents.in vitro and in vivo cytotoxicity results can also be rooted in the physicochemical properties of IONPs, and more importantly affected by the behavior of IONPs in biological media. The interaction of IONPs with cells and tissues can alter the physicochemical properties, mainly the aggregation of IONPs. Thus, knowing the altered surface chemistry of IONPs in the in vivo setting can help to develop a link between the surface chemistry of IONPs and its relevant induced toxicity.An enormous amount of cytotoxic studies appeared in the literature to improve the current understanding of toxicity of IONPs. However, inconsistencies keep growing with an increasing number of research studies. A striking disparity found between the shape, size, and coating dependent toxicity of IONPs directs the focus towards a meticulous physicochemical characterization of IONPs. Discrepancies between in vitro or in vivo use of metal oxide NPs. IONPs should be tested for microbial contaminations  to assure the safety of IONPs, and to eliminate a parameter, which could lead to ambiguous cytotoxic results. The majority of IONPs can be considered sterile due to harsh synthesis procedures; however, contamination can be introduced in various downstream processes such as surface functionalization. Sterilization can be performed by filtration, irradiation, and autoclaving or treating with various organic solvents containing disinfectant properties [Sterilization of IONPs is one of the ignored factors that contribute towards the toxicity of IONPs. The importance of sterilization of pharmaceuticals is highlighted and prioritized in various pharmacopoeias. However, the majority of studies lack or fail to report the sterilization data prior to operties .in vivo experiments, and the toxicity studies should be conducted in an environment, which mimics the cellular environment.Passivating the hydrophobic IONPs with a hydrophilic layer is a common practice during synthesis of biomaterial agents, which improves the solubility and stability in aqueous and biological solvents. However, the integrity of a hydrophilic layer needs to be maintained at various pHs in the cellular environment. Various biological environments can remove this hydrophilic coating, and the bare IONPs can be directly exposed to cells. Considering the altered and higher toxicity of bare IONPs, it is important to monitor the stability of biocompatible IONPs in The majority of IONPs induce toxicity in cells via ROS production, which varies according to physicochemical properties of IONPs. Considering the role of physicochemical properties of IONPs in ROS production, stricter rules to determine the oxidative properties of IONPs should be followed. The high reactivity and short half-life of ROS are major obstacles faced while studying the mechanism of IONPs.in vivo quantification of IONPs, and have helped to gain a better understanding of the in vivo distribution and clearance patterns of IONPs. However, many biomaterials laboratories do not have access to expensive MRI instruments and prefer spectrophotometric techniques for quantification. Choosing an appropriate quantification technique from a large pool of options depends on a variety of circumstances. Considering the pros and cons of every quantification technique, a combination of multiple techniques may be necessary for the absolute quantification of IONPs in in vivo setting. In addition to quantification of intracellular IONPs, surface ligand quantification [Advanced, sensitive MRI and PET/CT scanners have changed the face of fication  should aSome encouraging research studies have been published about the pulmonary and dermal toxicity of IONPs. When inhaled, engineered airborne NPs can cause severe harm such as asthma, chronic obstructive pulmonary disease, pulmonary inflammation, and lung cancer . From thin vivo bio-distribution and clearance pattern. Unfortunately, the toxicity and biodistribution studies have failed to keep up with the rapid pace of synthesis of novel nanomaterials and require similar attention. As presented in this review, we hope to provide a clear understanding of the physicochemical factors that need to be monitored during in vitro and in vivo toxicity studies of IONPs. Additionally, this document will also assist researchers in selection of the combination of proper quantification techniques to monitor the fate of IONPS in the body.Current progress in the applications of IONPs promises upcoming excitements, with a hope of successful transformation of under trial and pre-clinical nanomedicines to a successful pharmaceutical agent. Significant barrier between the research based nanomedicine and commercialization is the vast structural complexity of nanomaterials that differentiates them from marketed drugs in terms of lacking a clear “definition” and is of utmost importance in studying their


Pancreatic ductal adenocarcinoma (PDAC), the most common form of pancreatic cancer, is one of the most lethal human diseases. PDAC is now the fourth leading cause of cancer mortality in both men and women and deaths due to PDAC are projected to increase dramatically. Novel targets and agents for chemoprevention are urgently needed and will most likely arise from a more detailed understanding of the signaling mechanisms that stimulate the promotion and progression of sub-malignant cells into pancreatic cancer cells and from the identification of modifiable risk factors for PDAC. Many epidemiological studies have linked obesity and long-standing type 2 diabetes mellitus (T2DM) with increased risk and worse clinical outcomes for developing PDAC. These diet-related metabolic disorders are multifaceted but characterized by peripheral insulin resistance, compensatory overproduction of insulin and increased bioavailability of insulin-like growth factor-1 (IGF-1). Mounting evidence indicates that the insulin/IGF-1 receptor system plays a critical role in PDAC development and multiple studies support the notion that crosstalk between the insulin receptor and heptahelical G protein-coupled receptor (GPCR) signaling systems is an important element in the biological responses elicited by these signaling systems, including cell proliferation. This article highlights the central role of the mechanistic target of rapamycin (mTOR) in mediating crosstalk between insulin/IGF-1 and GPCR signaling in pancreatic cancer cells and proposes strategies, including the use of metformin, to target this signaling system in PDAC cells. KRAS oncogene which appears in ~90% of PDACs as well as inactivating mutations in tumor suppressors genes, including p53, p16, and SMAD4 , the most common form of pancreatic cancer, is one of the most lethal human diseases. Indeed, the overall 5-year survival rate is a dismal 6% and the median survival period of 4–6 months. The incidence of this disease in the US is estimated to increase to more than 44,000 new cases in 2014 and is now the fourth leading cause of cancer mortality in both men and women , a membrane lipid second messenger that coordinates the localization and activation of downstream effectors, including the isoforms of the Akt family  which are critical for Akt activation. Specifically, Akt translocated to the plasma membrane in response to products of PI3K, is activated by phosphorylation at Thr308 in the kinase activation loop and at Ser473 in the hydrophobic motif. The PI3K/Akt/mTOR pathway plays a pivotal role in promoting the proliferation and survival of PDAC cells  and Shc which propagate downstream signals  and TSC1 (hamartin) represses mTORC1 signaling by acting as the GTPase-activator protein for the small G protein Rheb (Ras homolog enriched in brain), a potent activator of mTORC1 in its GTP-bound state. Phosphorylation of TSC2 by Akt and/or ERK/p90RSK (at different sites) uncouples TSC1/TSC2 from Rheb, leading to Rheb-GTP accumulation and mTORC1 activation Figure . Phospha636/639 by mTORC1 and Ser307/636/1001 by S6K  abrogated Akt phosphorylation at this site in PDAC cells , PKD/ERK  and mTORC1  in PDAC cells potently stimulates DNA synthesis and proliferation of these cancer cells, and thus provide potential targets for chemotherapeutic intervention Figure . Since bMetformin  is the most widely prescribed drug for treatment of T2DM worldwide. Although it has been in clinical use for decades, its precise molecular mechanism of action remains incompletely understood. The primary systemic effect of metformin is the lowering of blood glucose levels through reduced hepatic gluconeogenesis and improved insulin sensitivity by increasing glucose uptake in peripheral tissues, including skeletal muscles and adipose tissue  activation , which disrupts its association with mTOR  AMPK stimulates TSC2 function via phosphorylation on Serin vitro with multiple cell types have used high concentrations of this agent to elicit effects , a condition that can lead to off-target effects. In addition to inhibit mTORC1, our results demonstrated that metformin prevented ERK activation in PDAC cells  by which metformin targets cancer cells is key for advancing the field as can lead to novel therapeutic strategies, including the identification of specific patient populations that ultimately will benefit from metformin administration, the generation of preliminary biomarker evidence of target inhibition, will stimulate the development of second generation drugs and the design of combinatorial interventions.The Associate Editor Guido Eibl declares that, despite having collaborated with the author Enrique Rozengurt, the review process was handled objectively and no conflict of interest exists. The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Photoreceptors are highly specialized neurons that have evolved to optimally capture photons. Understanding their demise, in order to develop therapeutic strategies, has the potential to improve the quality of life of millions of patients.Retinitis pigmentosa (RP) is the most frequent form of inherited photoreceptor degeneration. In many cases, the genetic defects are specific to the night active rod photoreceptors; however, cones, which mediate daytime and high acuity vision, die as well . The depWe recently proposed a new model where cone loss is caused by a nutrient shortage (particularly glucose) induced by the collapse of the retinal architecture as rods die . In the Pten) and the tuberous sclerosis complex 1 (Tsc1), both significantly prolonged cone survival while deletion of Raptor, the accessory protein of the mTOR complex 1 (mTORC1), accelerated cone death during disease [Pten and Raptor, as well as Tsc1 and Raptor, and Tsc1 and Rictor, the mTORC2 accessory protein, confirmed that the insulin-mediated effect on cone survival was indeed driven by mTORC1. Because the effect on cone survival upon loss of Tsc1 was the most robust and long lasting described thus far, the data supports the idea that nutrient shortage may be the overarching problem cones face as rods die. This can be explained by the high-energy demand of photoreceptors. In a healthy retina in addition to re-equilibrating membrane potentials photoreceptors need to synthesize membranes and proteins at a rate equivalent to one cell division per day, as these are lost in the daily shedding of their photosensitive structure [To circumvent the feedback-loop, test if insulin acted directly on cones, and test the long-term therapeutic potential of the pathway on cone survival we took a genetic approach using various conditional alleles of genes downstream of the insulin receptor, that when deleted either increase or ablate mTOR activity. Deletion in cones of the two negative regulators of mTOR, the phosphatase and tensin homologue ( disease , 4. Conctructure . Additiotructure .Tsc1 increased the expression of these genes in cones in addition to other metabolic genes [Tsc1 may have promoted cone survival by improving glucose uptake, retention, and utilization. Interestingly, all of these genes appeared already upregulated in cones during disease, albeit to a lesser extend. Loss of Tsc1 further increased their expression making cones more resistant to the nutrient shortage. Consistent with that, loss of mTORC1 activity during disease accelerated cone death, as cones fail to balance demand with supply, while combined loss of Tsc1 and mTORC1 not only failed to promote cone survival but resulted in the same acceleration of cone death as seen upon loss of mTORC1 alone. In contrast, loss of mTORC1 in wild-type cones had no effect suggesting that mTORC1 is mainly required when the metabolic equilibrium is disturbed [To sustain their metabolic needs photoreceptors have a high glycolytic rate that is accompanied by secretion of lactate in the presence of ample oxygen, a phenomenon known as the Warburg effect and typically found in cancer cells. Like proliferating cells, photoreceptors thus express high levels of hexokinase II and pyruvate kinase-2. In dividing cells these genes are regulated by mTORC1, which controls a wide range of metabolic pathways including glycolysis, fatty acid synthesis and the pentose phosphate pathway. It is thus not surprising that loss of ic genes . Therefoisturbed .Tsc1 can promote cone survival in other models of RP, we repeated the experiments in the slow progressing rhodopsin knockout mouse. Cone death was significantly delayed in this model too, indicating that increasing mTORC1 activity has broad therapeutic implications for RP [To test if loss of s for RP . While m


Arabidopsis thaliana (Arabidopsis) with a total of 49 members. In the last years several members of such transport proteins have been analyzed allowing to present a more detailed picture of nucleoside and nucleobase transport and the physiological function of these processes. Besides functioning in nucleotide metabolism it turned out that individual members of the before named transporters exhibit the capacity to transport a wide range of different substrates including vitamins and phytohormones. The aim of this review is to summarize the current knowledge on nucleobase and nucleoside transport processes in plants and integrate this into nucleotide metabolism in general. Thereby, we will focus on those proteins which have been characterized at the biochemical level.Nucleotide metabolism is an essential process in all living organisms. Besides newly synthesized nucleotides, the recycling  of partially degraded nucleotides, i.e., nucleosides and nucleobases serves to keep the homeostasis of the nucleotide pool. Both types of metabolites are substrates of at least six families of transport proteins in  During de novo nucleotide synthesis nucleoside monophosphates are synthesized from amino acids, bicarbonate, organic acids, and phosphoribosyl pyrophosphate (PRPP). In addition, the reducing equivalents adenosine-5′triphosphate (ATP) and tetrahydrofolate (THF) are required  pathway . In addition nucleosides can be phosphorylated by nucleoside kinases (NKs). Both pathways are less energy consuming than de novo synthesis from PRPP and glutamine (Gln) to inosine monophosphate (IMP) involves nine enzymatic steps . In Arabidopsis three ATase genes are present. Knockout of ATase2 is characterized by pale green mosaic leaves, also known as reticulated leaf mutant and a 50% reduction in leaf cell size indicating the role of purine de novo synthesis in cell division  They can be recycled to nucleoside monophosphates by PRTs or NKs. (2) They are subjected to complete degradation by which nitrogen is liberated in form of ammonia and may then be reassimilated to synthesize amino acids. According to the results of the analysis of mutants in catabolic enzymes as well as flux measurements using radiolabeled nucleosides and nucleobases it can be concluded that nucleosides are more effectively salvaged compared to nucleobases. In other words, the hydrolysis of nucleosides to nucleobases by nucleoside hydrolases (NSHs) results in a shift toward catabolism. This allows the assumption that nucleoside import into cells mainly supports salvage whereas nucleobase import supports catabolism.In the cell relative adenylate contents determine the cellular energy charge whereas outside ATP seems to be the primary signal  or phosphatases. Which enzymes are in charge for this process has not been clarified so far. However, a complete degradation of ATP down to the nucleobase adenine was shown to proceed in potato tuber apoplastic extracts  exhibit broad substrate specificity and transport the purine nucleosides adenosine and guanosine, as well as the pyrimidine nucleosides cytidine and uridine. The apparent KM values were in the range from 3 to 94 μ M (Table Arabidopsis ENT proteins.Equilibrative nucleoside transporters (ENTs) represent a family of integral membrane proteins present in a wide range of eukaryotic organisms and mediating transport of hydrophilic nucleoside substrates. Substantial progress has been made in characterization of mammalian ENTs in the last decades due to their medical importance . This lead to the identification of AtENT3 as the major pyrimidine importer in Arabidopsis seedlings  were transported  with an Arabidopsis cDNA expression library —one of the three compounds that can be called vitamin B6  belong to a family of small, highly hydrophobic membrane proteins. They are related to nucleotide-sugar transporters known from bacteria, archae, fungi and diverse eukaryotes within the drug metabolite transporter (DMT) super family Jelesko, . HomologArabidopsis, whereas three of them  have been characterized. The name originated from the ability of AtUPS1 to complement a yeast mutant defective in allantoin uptake  form a protein superfamily of plant membrane transporters with five members in Glycine max) or french bean (Phaseolus vulgaris) ureides play a dominant role in primary nitrogen metabolism. After the initial fixation into glutamine, almost all fixed nitrogen is subsequently converted to the ureides allantoin and allantoic acid via inosine monophosphate  were characterized as high affinity allantoin transporters (Table PvUPS1) encoding an allantoin transporter was identified and detected at the transcript level throughout the plant body. Expression in root tissues increased markedly upon nodulation indicating the importance of UPSs from legumes for delivery of ureides to the vasculature . The uridine transporter FUI1 and the uracil transporter FUR4 from Saccharomyces cerevisiae  family consists of over 1000 proteins from bacteria, archaea, yeast, fungi and plants. Also known as purine-related transporters, they generally transport purines in a proton symport mode and consist of 12 transmembrane domains , rice (Oryza sativa), wine (Vitis vinifera) and poplar (Populus trichocarpa) also possess a PLUTO homolog while Brachypodium distachyon even harbors two PLUTO homologs  family, belonging to the nucleobase:cation symporter 2 (NCS2) family, represents the largest and most conserved class of nucleobase transporters (De Koning and Diallinas, Arabidopsis 12 members of the NAT family, AtNAT1-12, have been identified and analyzed concerning their expression patterns during growth and development (Maurino et al., Arabidopsis (Maurino et al., In Aspergillus nidulans strains lacking functional NATs, heterologous expression studies in yeast, oocytes or measurements of radiolabeled substrates in whole plants (Maurino et al., AtNAT3 and AtNAT12 in E. coli cells lacking the endogenous uracil transporter UraA allowed for a detailed biochemical characterization of these two NAT proteins showing that they mediate high affinity uptake of uracil, adenine and guanine (Niopek-Witz et al., The biochemical characterization of these proteins was not successful by the complementation of Aspergillus nidulans, AzgA has been identified as a proton-symporter specific for hypoxanthine, guanine, and adenine (Cecchetto et al., Aspergillus nidulans, namely AtAzg1 and AtAzg2, have also been identified in Arabidopsis. These proteins share 36.5 and 38.5% identical amino acids with AzgA, respectively (Mansfield et al., AtAzg1-1 and AtAzg1-2 showed increased resistance on 8-azaguanine and 8-azaadenine compared to the wildtype and this effect was even more pronounced in AtAzg1 and AtAzg2 double mutants. Growth tests of S. cerevisiae cells expressing AtAzg1 and AtAzg2 on medium supplemented with the toxic analogs 8-azaadenine or 8-azaguanine as well as direct uptake studies with radiolabeled [3H]-adenine and [3H]-guanine indicated that AtAzg1 and AtAzg2 are capable of transporting adenine and guanine (Mansfield et al., The AzgA proteins define a group of membrane proteins which may be distantly related to the NAT family and homologs of unknown function are found in plants, fungi, bacteria and Archaea. In In the last years it became apparent that nucleoside and nucleobase transporters accept a diverse range of substrates, far more than judged by the given name of these proteins. Consequently, the adjudicated physiological functions also increased. To briefly summarize, ENTs represent the sole family of nucleoside transporters in plants, mediating transport of purine and pyrimidine nucleosides across the plasma membrane and the tonoplast. Within the large number of nucleobase transport families, PUPs exhibit a broad substrate spectrum including purine nucleobases, cytokinins, vitamins and alkaloids. UPS proteins play a major role in ureide long distance transport in tropical legumes whereas in non-legumes pyrimidine nucleobase transport predominates. From the NCS1 protein family only one member is present in higher plants with PLUTO, the plastidic nucleobase transporter. In contrast, NCS2 members reside at the plasma membrane where they catalyze transport of purine and pyrimidine nucleobases. It can be anticipated that research in the field of nucleoside and nucleobase transport will continue to develop fruitfully in future, implementing aspects from crop plants.The Guest Associate Editor Ilka Haferkamp declares that, despite being affiliated to the same institution as the authors, the review process was handled objectively and no conflict of interest exists. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In experiments replicated within two subestuaries of the Chesapeake Bay, USA across multiple seasons and non-consecutive years, we comprehensively censused mesopredators and epifaunal communities using complementary quantitative methods. We found that community properties, including abundance, species richness, Simpson and functional diversity, and composition were generally unaffected by the number of patches and the size of the landscape, or the intensity of sampling. Additionally, an index of competition based on species co-occurrences revealed no trends with increasing patch size, contrary to theoretical predictions. We extend conclusions concerning the invariance of animal communities to habitat fragmentation from small-scale observational surveys and artificial experiments to experiments conducted with actual living plants and at more realistic scales. Our findings are likely a consequence of the rapid life histories and high mobility of the organisms common to eelgrass beds, and have implications for both conservation and restoration, suggesting that even small patches can rapidly promote abundant and diverse faunal communities.Human-driven habitat fragmentation is cited as one of the most pressing threats facing many coastal ecosystems today. Many experiments have explored the consequences of fragmentation on fauna in one foundational habitat, seagrass beds, but have either surveyed along a gradient of existing patchiness, used artificial materials to mimic a natural bed, or sampled over short timescales. Here, we describe faunal responses to constructed fragmented landscapes varying from 4–400 m As in Experiment 1, community properties were strongly influenced by season, with mean abundance significantly decreasing throughout the year , suggesting temporal changes were consistent among landscape sizes.For core-sampled epifauna in Experiment 2, the only community metric on which landscape size had a marginally significant effect was community evenness (P = 0.04), based on analysis of deviance. The remainder of the predictors had no significant associations with the index (0.25 < P < 0.89).Species pairs appeared to be randomly distributed in the experimental treatments, once again indicated by the lack of standardized effect sizes falling outside of the  range , and wasIdotea balthica) and caprellid and gammaridean amphipods, and increases in the abundance of ampithoid amphipods and the lunar dovesnail (Astyris lunata)  .2 unfragmented and 400 m2 fragmented plots . Thus, the high degree of collinearity precludes us from independently testing both, and since the experiments were initially designed to test the effects of total area [If many hypotheses explaining fragmentation effects have been structured around the amount of edge habitat, why did we not test for statistical effects of edge area? Unfortunately, our experimental design confounds total area with edge area: Pearson correlations between total area and edge area range from tal area ,34, we htal area . To thatOne of the enduring debates in conservation biology is “single large or several small,” referring to the amount and configuration of habitat set aside or created to protect and maintain existing diversity. Others have proposed that several small seagrass beds may be equally or more effective for conserving the diversity of macroinvertebrates and fishes , and infNevertheless, we show that the primary mechanism by which seagrass fragmentation may affect faunal communities in this estuary is not likely to be via changes in the inherent structure of the remaining communities, but rather by reducing the total habitat area and thereby the total potential productivity of the system, consistent with emerging consensus about fragmentation effects in seagrass systems in general . InsteadS1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 FigControl refers to an adjacent natural eelgrass bed, sampled to determine whether natural faunal communities resembled ones recruiting to the experimental transplants.(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S1 File(ZIP)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.