Endocytic pathways are broadly classified into clathrin dependent and independent on the basis of the requirement for the coat protein, clathrin. The molecular pathways and mechanisms underlying the formation of clathrin-independent pathways are still being explored, and this review summarizes recent advances and emerging functional roles of these diverse pathways. In particular, this review will discuss the growing consensus on the role of BAR domain proteins and the actin machinery in different clathrin-independent pathways and its significance to the functions fulfilled by these endocytic pathways. The formation of clathrin-coated pits and associated molecular machinery has been relatively well studied along with its functions in many physiological contexts5. This review will deal with recent findings about the clathrin-independent pathways.Endocytosis involves vesicular carriers that bud from the plasma membrane carrying membrane components, including signaling ligands, receptors, nutrients, or growth factors that are delivered to intracellular sites where they are further processed, recycled, or degraded. Secretion and endocytosis are closely integrated with the cellular protein synthesis and degradation machinery such that the flux of material through these processes is a key element in maintaining homeostasis in a cell. Naturally, the diversity in the types of cargo and the kinetics of their trafficking demand a diversity in the types of endocytic machinery employed. The convenient classification based on the requirement for the coat protein, clathrin, divides these endocytic processes into clathrin-dependent and clathrin-independent pathways2. Recently, a role for BAR domain proteins has emerged in multiple clathrin-independent endocytosis (CIE) pathways. The members of the BAR domain protein endophilin—primarily endophilin-A2 and endophilin-A1—directly bind to and facilitate the endocytosis of selected G-protein-coupled receptors like β1-adrenergic receptor (β1-AR) and dopaminergic and acetylcholinergic receptors6. Endophilin and lamellipodin have previously been implicated in the clathrin-mediated endocytosis of epidermal growth factor receptor (EGFR)7. During the CIE of β1-AR, rapidly formed vesicles labeled by endophilin and its binding partners, synaptojanin and dynamin, internalized β1-AR from the leading edge of cells in culture and mediated the downregulation of its signaling. This novel endocytic pathway was named the fast endophilin-mediated endocytic pathway (FEME) and requires the lipid phosphatidylinositol 3,4-bisphosphate—PIP2—as well as the protein lamellipodin, which binds both PIP2 and the SH3 domain of endophilin for its recruitment to the leading edge. The class I PI(3)K pathway and associated phosphatases, including the 5′ phosphatase SHIP1/2, act near the plasma membrane to locally enhance the PIP2 levels. This assists in the formation of FEME vesicles enabled by the recruitment of endophilin6.Endocytic pathways that do not use clathrin can either use the scission GTPase dynamin or be independent of it. They are known to be more sensitive to the dynamics of the cytoskeletal actin machinery for stabilization and scission8. These toxins induce local membrane curvature upon binding to their receptor, following which the BAR domain-containing protein is recruited to the cholesterol-dependent curved membrane surfaces9. Cortical actin dynamics were previously shown to be necessary for scission of STxB endosomes10. With reconstituted model membranes, endophilin-A2 was observed to stabilize the tube-shaped StxB vesicles. Actin, endophilin-A2, and dynamin independently and additively contribute to the scission of these vesicles, and the authors propose a model wherein endophilin-A2-stabilized invaginations are acted upon by the pulling forces of cytoskeletal motors (possibly microtubule-associated motors11) to lead to scission at a controlled rate and length8.Endophilin-A2 has also been recently implicated in the CIE of glycosphingolipid (GSL)-binding toxins Shiga (STxB) and cholera toxin BDrosophila cell lines and tissues16. The CG endocytic intermediates carrying GPI-APs and fluid phase markers were visualized by electron microscopy to reveal uncoated, tubular intermediates17 called clathrin-independent carriers (CLICs), which eventually fuse to form GPI-AP-enriched early endosomal compartments (GEECs)18. Arf1, its GEF (guanine exchange factor), GBF1/Garz (gartenzwerg), and membrane cholesterol are necessary for forming the CG endocytic vesicles in this pathway19. In a screen in S2R+ insect cell lines to identify molecular players regulating this pathway, Arf1-COP1 machinery, BAR domain-containing proteins, vacuolar ATPase, lysosomal genes involved in vacuole biogenesis, and actin remodeling factors, including Slingshot, Coronin, Arcpc1, and Capping protein, were among the major hits19. Actin inhibitors used at concentrations that disrupt the cortical actin network without affecting the long-lived structures like stress fibers selectively perturb CG endocytosis while not affecting clathrin-dependent uptake12. This acute sensitivity to actin dynamics also translates to the regulation of CG endocytosis by Cdc42, a modulator of actin polymerization.The reliance on actin cytoskeletal machinery has also been studied in a clathrin- and dynamin-independent, CLIC/GEEC (CG) pathway. This is the major route of internalization of a large fraction of glycosylphosphatidylinositol-anchored proteins (GPI-APs) and fluid phase in several mammalian and20. While reaffirming the role of ARF1-GBF1, the actin nucleator Arp2/3, and associated GTPase Cdc42 at the nascent sites of endocytosis, this study also identifies the role of a BAR domain protein, IRSp53, and an ARP2/3 inhibitor, PICK1. Imaging the direct recruitment of these molecular players also helps develop a mechanistic picture of how a GEEC is formed: initial recruitment of ARF1/GBF1, IRSp53, and Arp2/3 to the cell surface is followed by the arrival of Cdc42 that activates Arp2/3 and IRSp53 to catalyze the formation of F-actin and membrane buckling, leading to the formation of the CG endosome20. The ARF1/GBF1 (Garz)-dependent CG pathway was also shown to be active inDrosophila wing disc tissue, wherein class I PI3-kinase activity is necessary for the recruitment ofGarz to the cell surface and the initiation of the CG endocytosis16.Recently, the recruitment of these early molecular players at nascent endocytic vesicles was studied at high spatial and temporal resolution to understand the sequence of events leading to the formation of a CG endocytic vesicle2. Perturbations in cholesterol and sphingolipid levels affect the distribution of GPI-APs, normally present as actively maintained nanoclusters on the cell surface21, and subsequently the formation of GEEC endosomes12. The CIE of cholera and Shiga toxins too is initiated upon their binding to specific GSLs, which induces membrane bending and endosome formation22. Endogenous galectins, which can bind glycan chains and GSLs, are postulated to use a similar mechanism for concentration and multimerization leading to membrane bending, potentially facilitating the internalization of a large variety of endogenous glycosylated proteins. This GSL-galectin-3-mediated mechanism (termed GL-Lect22) can mediate the CIE of β1-integrin and CD44, dependent on their glycan chains23. Recently, galectin-3 binding was also implicated in the endocytosis of the GPI-AP CD59 and major histocompatibility complex (MHC) class I protein, suggesting a more global utilization of this axis of interaction to lend specificity to CIE24. Interestingly, the degree of glycan branching affects the internalization rates in contrasting ways. CD59 internalization is obstructed by a highly branched glycan lattice. This raises the possibility of clathrin-independent endocytic fluxes being selectively altered by factors affecting N-glycosylation, like metabolic substrates24.Besides the role of the actin machinery, membrane composition is a key component that determines the enrichment of clathrin-independent cargoes in endocytic pitsCaenorhabditis elegans neurons25 and also in mouse hippocampal neurons26 is necessary for compensating and coordinating membrane retrieval with exocytosis rates27. This ultra-fast endocytosis is triggered by exocytosis, and recently synaptojanin-1 and endophilin-A have been implicated in the rapid maturation of these endocytic pits so as to facilitate membrane turnover in milliseconds—an essential feature of synapses28.Emerging understanding of the molecular machinery behind non-canonical endocytic processes provides a view of the versatility of CIEs depending on the context. Clathrin-independent rapid endocytosis (~100 ms) at the synapses of29.Plasma membrane cholesterol is necessary for the functioning of many CIEs, including CG and Shiga toxin endocytosis. Cholesterol-dependent CLICs were recently found to be enriched at the mid-body during cytokinesis at the intercellular bridge by electron microscopy. This postulates a role for CLICs to stabilize and maintain the membrane reservoir during cell division30. A recent role for promoting Wingless/Wnt signaling in developingDrosophila wing discs highlights the potential role of CIEs during development and cell fate determination in metazoans16. The previously described CG pathway internalizes and brings together the apically localized ligand Wingless with its basolaterally localized receptor Dfrizzled2  to activate full-strength signaling. This novel mechanism of cooperating endocytic pathways that promote signaling could be functional in other contexts where the dosage of signaling needs to be graded rather than act as an on/off switch. The requirement for class I PI3-kinase activity for CG endocytosis and hence Wingless signaling illustrates how this endocytic pathway introduces new nodes of control and modulation from the single modality of ligand–receptor interaction in a tissue16. The well-documented roles of GPI-APs (cargo for CG endocytosis), Dally, and Dlp as potential co-receptors for multiple signaling molecules during development33 also make this pathway an exciting candidate as an integrator of signaling with cues from the actin cytoskeletal machinery and membrane composition in the growing organ.Down-regulation of signaling by CIE has also been documented for EGFR and other ubiquitinated cargoes: at low ligand concentration, clathrin-mediated uptake is employed; at higher ligand concentration, the CIE pathway operated to down-regulate signaling36. This pathway shares some similarities with the CG pathway in its cargo: the fluid phase and its molecular players, actin and PIP3. However, it is also able to accommodate large dextran (>70 kDa), is amiloride sensitive, and appears specifically upregulated by Ras-mediated activation of Rac and Cdc42, triggered in some cases by AMPK (5′-AMP-activated protein kinase)36.An actin-dependent macro-pinocytic pathway is upregulated in cancer cells in a nutrient-sensitive manner. Specifically, in Ras-driven tumors, growth factor signaling appears to activate this mechanism. This helps in nutrient scavenging from surrounding extracellular material and even necrotic cell debris to supplement increased energy demand37. The extreme reliance of CIEs on the actin cytoskeletal machinery makes them a good candidate to transduce information from the mechanical tension experienced by the cell to its homeostatic machinery. A recent study has explored the dependence of the flux in the CG pathway of endocytosis on mechanical tension and finds a reciprocal relationship between the two38. The CG pathway is specifically upregulated transiently to buffer a decrease in mechanical tension during stimulated stretch-relax cycles as well as during normal de-adhering of cells. Conversely, inhibiting CG endocytosis reduced the membrane tension within the cell. This newly uncovered function of the CG endocytic pathway makes it a crucial cog in the homeostatic machinery that maintains cellular mechanical tension and, by extension, its shape, size, and response to the environment. Depleting a molecular player implicated in CIE-Graf1 leads to increased cancer cell blebbing and enhances its invasiveness in culture39, which could be linked to the function of CIEs in maintaining membrane tension and also highlights the many ramifications this process could have in multiple tissue contexts if perturbed. The extent to which the functions of these pathways influence signaling networks and their interdependence remains to be explored, and recent work indicates that there is significant cross-talk28.CLICs are reported to internalize a large fraction of the total plasma membrane surface area; their endocytic volume is capable of internalizing the entire surface area of the plasma membrane in 12 minutes in fibroblastsThus, the common emerging theme among CIEs is their reliance on the cell cytoskeletal machinery, especially the cortical actin network, for the early steps. This dependence also provides these endocytic pathways with the ability to respond to rapid changes in the cellular environment. Perhaps the cortical actin network provides the feedback for the control of these endocytic pathways, integrating the endocytic machinery with the dynamic cortical actin mesh.


To describe the first case of bilateral retinal angiomatous proliferation (RAP) in a patient with a variant of retinitis pigmentosa (RP). An 85-year-old man with RP presented with visual acuity decrease and metamorphopsia in the left eye (LE). Fundus examination revealed typical signs of RP in both eyes, associated with intraretinal macular hemorrhage in the LE. Multimodal imaging, using Colour fundus Photography, Fluorescein (FA), and Indocyanine Green Angiography (ICGA) as well as Spectral-Domain Optical Coherence Tomography (SD-OCT) and Optical Coherence Tomography Angiography (OCTA), revealed a type 3 neovascular lesion in the involved eye. Genetic testing  was performed to search for genetic variants correlated with the disease phenotype displayed by the patient. The patient was treated with intravitreal injections of bevacizumab, according to a fixed protocol of bimonthly injections plus a booster dose at second month. After 9 months, he was referred for visual acuity decrease and metamorphopsia in the fellow eye, where SD-OCT/OCTA showed a type 3 neovascular lesion in the right eye (RE). He was scheduled for intravitreal injections of bevacizumab. In both eyes, treatment with intravitreal bevacizumab was successful. Retinitis pigmentosa (RP) is a complex of inherited retinal dystrophies characterized by the loss of photoreceptors and characterized by retinal pigment deposits in the peripheral retina with a relative sparing of the central retina. The most common form of RP is a rod-cone dystrophy, in which there is a primary degeneration of the photoreceptor rods, with secondary degeneration of cones, thus leading to night blindness, followed by the progressive loss in the peripheral visual field in daylight, and eventually blindness after several decades . RetinalCNV is very rarely associated with RP . To dateAn 85-year-old with RP was referred to our center in April 2016 for decrease in visual acuity and metamorphopsia in the left eye (LE) for a few weeks. The patient had the clinical diagnosis of retinitis pigmentosa from the age of 15 years old and for this reason did not do military service. He reported a history of night blindness and narrowing of the subjective visual field in both eyes with a recent visual impairment in the LE. Best corrected visual acuity was 0.1 LogMAR (80 LN) in the right eye (RE) and 0.7 LogMAR (50 LN) in the LE. The visual field test was not reliable due to the poor collaboration of the patient.Fundus examination revealed arteriolar attenuation, peripapillary atrophy, optic disc pallor, macular edema and typical retinal pigment deposits involving the midperipheral retina (bone spicules) of both eyes, associated with intraretinal hemorrhage in the macular region of the LE.Fluorescein angiography (FA)  revealed a continuous retinal-retinal anastomosis and the presence of well distinct area of intense leakage temporally to the fovea corresponding to the neovascularization with a late pooling of dye due to intraretinal cystoid edema . In the Indocyanine green angiography (ICGA)  revealed a well-distinct “triangular shape” area of intense hypercyanescence (“hot spot”) corresponding to the neovascularization and a feeder and draining vessel, pathognomonic for RAP. In the fellow eye, ICGA did not show any signs of neovascularization.Spectral-Domain Optical Coherence Tomography (SD-OCT)  in correspondence of the hyperfluorescent area of the LE revealed a hyperreflective intraretinal lesion located at the outer retinal layers apparently connected with the sub-RPE space, characterized by intraretinal and subretinal exudation and intraretinal fluid . In the Optical Coherence Tomography Angiography (OCTA)  is a rapid, noninvasive technique that showed clearly the intraretinal neovascularization with capillary proliferation and retinal-retinal anastomoses .In the segmentation of deep capillary plexus, OCTA revealed a high- flow third- order vessel descending into a tuft-shaped high-flow lesion toward the outer retinal layers, characterized by a retinal-retinal anastomosis and bordering the sub-RPE space  Figure .Taking into consideration the clinical picture of patient we decided to perform a genetic testing to search for genetic variants, which may be correlated with the RP-like phenotype observed in the patient. To this purpose, the patient was asked to sign an informed consent and to give a saliva sample for genetic testing . The DNANGS analysis revealed the presence of two missense variants  located in SEMA4A (1q22) and in CNGA1 (4p12), respectively. In particular, SEMA4A encodes a member of semaphorin family of transmembrane proteins and modulate the transport of retinoid-binding proteins in RPE cells (REF4). CNGA1 codes for the homonymous protein that is crucial for phototransduction pathway , meaningSuccessively, the patient was screened for AMD-related variants, in order to estimate the genetic susceptibility to disease, considering the observation of a neovascular lesion and the advanced age of the patient. In particular, three polymorphisms were tested, namely rs1061170 (T/C), rs10490924 (G/T) and rs2227306 (C/T) . They arWe decided to treat the patient with 3 consecutive monthly intravitreal injections of bevacizumab adopting the Treat and Extend (TE) strategy after the loading phase.In January 2017, 9 months later the first visit, patient referred for sudden visual acuity impairment in the fellow eye. Best corrected visual acuity decreased from 0.1 LogMAR 80 LN to 1.0 LogMAR 35 LN.Fundus examination revealed macular hemorrhage, exudates and intraretinal edema in the right eye (RE); these clinical findings supported the diagnosis of type 3 CNV in the RE that was then confirmed by SD-OCT and OCTA imaging. OCTA showed clearly two distinct intraretinal neovascularizations located in the segmentation of choroid capillary, both bordering the sub-RPE space . We admiTo date, we decided to stop the anti-VEGF therapy because SD-OCT did not show any CNV activity in both eyes.CNV rarely occurs in patients affected by RP . Only feThe underlying pathogenesis of RP-associated CNV remains uncertain. Malik et al hypothesized that retinal pigment epithelium changes, constantly present in retinitis pigmentosa, associated with concomitant photoreceptor cell involvement and chorio-capillaris damage could lead to development of CNV . We deci SEMA4A and CNGA1, respectively. Both genes are known to be associated with autosomal recessive RP. However, the clinical significance of these variants is ambiguous, and cannot be utilized to provide a certain genetic diagnosis of RP in this case. Further studies will be probably decisive to clarify the genetic background of peculiar phenotypes and of RP in general.Genetic testing revealed the presence of two variants (c.1265C>T and c.1162T>C) in ARMS2 and IL-8 susceptibility variants). ARMS2 and IL-8 have also been associated with angiogenesis, Extra-Cellular Matrix (ECM) organization, alteration of Bruch's membrane, inflammatory and immune response overactivation observed in AMD [ SEMA4A may play a critical role in immune response activation and angiogenesis [ CNGA1 and other phototransduction related-genes to more severe AMD, especially the neovascular form of disease [ SEMA4A and CNGA1 share some biological pathways with ARMS2 and IL-8, suggesting that these genes may interact together and contribute to increase the susceptibility to develop neovascular lesions, which may be more facilitated to grow in RP cases because of photoreceptor cells degeneration, RPE changes and choriocapillaris damage.Concerning the AMD screening, the patient revealed a moderate risk to develop the disease  may be purely coincidental. Further research is required to examine the mechanism associating these two conditions and to establish the effective treatment for RP-related CNVs.


Background: The risk of developing invasive cancer in the remnant pancreas after resection of multifocal high-grade pancreatic precursor lesions is not well known. We report three patients who were followed up after resection of multifocal high-grade pancreatic intraepithelial neoplasia (PanIN)-3 or intraductal papillary mucinous neoplasia (IPMN), two of whom eventually developed invasive carcinoma.Presentation: 1) 68-year-old woman who had a laparoscopic distal pancreatectomy for multifocal mixed-type IPMN, identified as high-grade on final pathology, with negative surgical margins. During semiannual monitoring, eight years from the first surgery, the patient developed suspicious features prompting surgical resection of the body with final pathology revealing invasive ductal adenocarcinoma in the setting of IPMN. 2) 48-year-old woman who had a distal pancreatectomy for severe acute/chronic symptomatic pancreatitis, with final pathology revealing multifocal high-grade PanIN-3, with negative surgical margins. Despite semiannual monitoring, two years from the first surgery, the patient developed pancreatic adenocarcinoma with liver metastasis. 3) 55-year-old woman who had a Whipple procedure for symptomatic chronic pancreatitis, with multifocal PanIN-3 on final pathology. The patient underwent completion pancreatectomy due to symptomatology and her high-risk profile, with final pathology confirming multifocal PanIN-3.Conclusion: Multifocal high-grade dysplastic lesions of the pancreas might benefit from surgical resection. Pancreatic cancer develops from precursor lesions. It is theorized that such lesions progress in a step-wise manner from low grade to high grade to invasive adenocarcinoma. Pancreatic intraepithelial neoplasia (PanIN) and intraductal papillary mucinous neoplasia (IPMN) are two types of precursor lesions. Although identical at the microscopic level, they differ is size, genetics, and detectability with better imaging.1 Some studies have focused on the risk of high grade or invasive IPMN recurrence after resection of a unifocal high-grade dysplastic lesion,2 but to our knowledge, there have been no published reports investigating the effect of multifocal high-grade dysplasia on recurrence. In this study, we present three such cases to increase physician awareness regarding the possibility of recurrence and the need for surveillance.The risk of recurrence after resection of IPMN is generally considered to be low. Approximately 20% of patients will develop recurrence in the pancreatic remnant with a mean follow-up of 66 months.A 68-year-old woman had a laparoscopic distal pancreatectomy for multifocal IPMN-mixed type with two foci of high-grade dysplasia detected on final pathology analysis . SurgicaA 49-year-old woman presented with moderately severe pancreatitis. She required three hospitalizations. This was traumatic in etiology as she was kicked by a horse. The patient experienced multiple complications including thromboembolic events and a pancreaticopleural fistula. Owing to escalation of symptoms and failure to thrive, she presented to medical attention, and a distal pancreatectomy was pursued. Surgical pathology analysis revealed multifocal high-grade dysplasia PanIN-3, with evidence of chronic pancreatitis in the specimen . Three fA 55-year-old woman was referred to our clinic for further management after having had a Whipple procedure elsewhere for chronic pancreatitis. The specimen demonstrated multifocal PanIN-3 with evidence of chronic pancreatitis in the background. Four foci of high-grade dysplasia were observed; surgical margins were negative. The patient was symptomatic with intermittent epigastric pain radiating to the back, which was thought to be related to pancreatitis. The pain was severe enough to affect her daily activities, resulting in a chronic narcotic-dependent status and hospitalizations for pain control. Recent outside CT imaging confirmed changes compatible with chronic pancreatitis in the remnant pancreas, as well as the presence of a retained pancreatic stent thought to be partially contributing to her pain. Her family history is significant for pancreatic cancer, her sister developing it in her 60s. Owing to her symptomatology, the retained stent, significant family history, the initial surgical pathology report, and our experience with the mentioned two cases, completion pancreatectomy was recommended and pursued. Final pathology report disclosed the presence of multifocal PanIN-3 in the setting of pancreatitis.3 Tan et al. examined the mutation status of adjacent areas within the same patient's invasive IPMN specimen. For 28% of the cases, they detected different mutations of the same gene in adjacent areas of varying dysplastic grade from the same patient; moreover, they reported the absence of mutations in specimens of higher levels of dysplasia that were present in specimens of lesser dysplasia from the same patient. These findings support a multifocal nature of the tumorigenic process within the pancreas. The authors propose that a field defect exists in which the entire pancreas may be prone to the accumulation of distinct genetic mutations and errors that may eventually evolve into cancer.The presence of multifocal high-grade dysplastic lesions raises intriguing questions about pancreatic tumorigenesis. Two possible scenarios may explain the simultaneous occurrence of multiple lesions. First, all the lesions may share a similar genetic or molecular background leading to malignant progression, also known as the field theory; or second, these lesions may arise independently and not share a common tumorigenic pathway or event. In a recently published study aimed at establishing a relationship between histological subtypes of IPMN and frequencies of specific genetic mutations,4 Specifically, 1552 subjects were followed up for a minimum of 2 years, and of these, 29  had evidence of cancer of the pancreas 2 or more years after the diagnosis of pancreatitis during a mean follow-up (±standard deviation) of 7.4 ± 5.6 years. In another case–control study, the relative risk in patients with chronic pancreatitis was found to be as high as 13.3 compared with the control group.5In addition, the presence of chronic pancreatitis in two of our cases highlights the effect that pancreatitis may have on the outcome. In one cohort study, patients with chronic pancreatitis were found to have an increased risk of pancreatic cancer.6 However, risk factors for local recurrence of noninvasive lesions are not well defined. Furthermore, the risk of recurrence of multifocal high-grade dysplasia has not been studied extensively. Prior study evaluating the association between unifocal high-grade dysplasia and recurrence has shown that the degree of dysplasia seen on the specimen is a risk factor.2 The rates of recurrence significantly increased as the grade of dysplasia increases. After a median of 44.4 months of follow-up, 39 out of 366 (10.7%) patients experienced disease recurrence with a median of 18.8 months, and of these 16 (41%) patients did not initially have an invasive IPMN. Analysis of these recurrences shows that six patients with high-grade dysplasia developed recurrence—one of them had an adenocarcinoma, two had recurrent IPMN, and three had distant metastasis. Therefore, thorough postoperative surveillance is needed not only for invasive IPMN but also for benign noninvasive lesions and especially those with high-grade dysplasia.The presence and extent of invasive IPMN have consistently been identified as risk factors for poor outcomes.8 Complications after total pancreatectomy may include brittle diabetes, a very challenging form of diabetes that is resistant to insulin treatment, thought to originate from a lack of glucagon secretion as a result of the surgery. The patient may also develop pancreatic exocrine insufficiency with its associated fat-soluble vitamin deficiency as well as malnutrition, typically addressed by adding supplemental pancreatic enzymes.At the Indiana University Pancreatic Cyst and Cancer Early Detection Center, high-risk patients  undergo surveillance that includes annual EUS-FNA as well as semiannual magnetic resonance imaging (MRI)-MRCP. Unfortunately, the surveillance strategy failed for Case 2, prompting a more aggressive approach in managing Case 3, potentially preventing malignant recurrence. Therefore, based upon our experience, we recommend that total pancreatectomy be strongly considered in fit patients with multifocal high-grade lesions, whether or not pancreatitis is present. However, pancreatic surgery is high risk and has a morbidity rate of 45–55% and a mortality rate of 2–4%.1 Therefore, the guidelines recommend indefinite close surveillance after achieving clear surgical margins. However, the risk of progression from multifocal high-grade PanIN or IPMN into invasive cancer is unclear, and no clear recommendations for the management of these patients exist to date. Based upon our experience, we are recommending strong consideration of completion pancreatectomy in fit patients. In the event, completion pancreatectomy is not pursued, we would recommend quarterly surveillance with serum markers, MRI-MRCP, and semiannual EUS-FNA.Although the surveillance protocol for a resected invasive IPMN is well established, the follow-up for a noninvasive cystic lesion depends on many factors and is still a subject of controversy. The rates of recurrence for unifocal noninvasive IPMN are reported to be ∼20%.


The layered structure of this novel nanoporous structure has been analyzed unambiguously by Rutherford backscattering spectroscopy, scanning electron microscopy, and X-ray diffractometer. The cell using the heterostructure as photoanode manifests an enhancement of about an order in the magnitude of the short circuit current and a seven-fold increase in efficiency, over pure TiO2 photoanodes. Characterizations further reveal that the Zn-TONT is preferentially oriented in [001] direction and there is a Ti metal-depleted interface layer which leads to better band alignment in DSSC.The paper reports the fabrication of Zn-doped TiO The results emphasize the significance of TONT/ZnO photoanodes over pure TONT photoanodes, for improved DSSC performance.The SSC Fig. c using Z2 nanotubes (band gap ~3.2 eV) decreases the recombination rate of the electrons owing to the small energy barrier created by them [The improvement in the efficiency of DSSC could be explained based on the more effective processes of carrier production and transport in the heterostructure-based DSSC whose band alignment is illustrated in Fig.  by them , 23. In  by them . This in2 nanotubes.In conclusion, a clear picture of the layered structure, morphology, and crystallinity of the Zn-TONT/ZnO heterostructure was gained using RBS, SEM, and XRD. The advantage of using heterostructures of the Zn-TONT/ZnO nanoflake heterostructure in a DSSC constructed by a cost-effective and highly reproducible method was analyzed. This layered heterostructure shows enhanced DSSC efficiency over the pure TiO


A semi-structured questionnaire was used to collect information regarding drugs used, knowledge, history and reasons for antibiotic self-medication. Log—binomial regression analysis was done using STATA 13 to examine factors associated with self-medication. A slightly majority of the respondents (58%) admitted to self-medication. Antibiotics most commonly utilized were amoxycillin (43%) and an antiprotozoal drug metronidazole (10%). The most common symptoms that led to self-medication were cough (51.17%), headache/ fever/ malaria (25.57%) and diarrhoea (21.59%). The most common reasons for self-medication were emergency illness (24.00%), health facility charges (20.33%), proximity of pharmacy to home (17.00%) and no reason (16.66%). Almost all reported that self-medication is not better than seeking medical consultation, 98% can result into harmful effects and 96% can result to drug resistance. The level of self-medication in this study is comparable with findings from other studies in developing countries. Pharmacies were commonly used as the first point of medical care. There is therefore a need for educative antibiotic legislative intervention to mitigate the adverse effects of antibiotic self-medication in Siha district in Tanzania.Self-medication is very common especially in developing countries and is documented to be associated with many health risks including antibiotic resistance. This study investigated the prevalence, determinants and knowledge of self-medication among residents of Siha District in Tanzania. A cross-sectional study was conducted among 300 residents in a rural District of Kilimanjaro region, North-eastern Tanzania from 1 Self-medication is defined as the use of drugs to treat self-diagnosed disorders or symptoms or the intermittent or continued use of drugs for chronic or recurrent disease or symptoms without a prescription or guidance from a physician. AntibioAmong the measures taken by a person who feels unwell in a typical developing country is the use of traditional medication alongside modern medicine. The latThe adverse effects of self-medication include incorrect self-diagnosis, delays in seeking medical advice when needed, infrequent but severe adverse reactions, dangerous drug interactions, incorrect manner of administration, incorrect dosage, incorrect choice of therapy, masking of a severe disease and a risk of dependence and abuse. Many ofThis study was therefore designed to determine the prevalence and predictors of self-medication with antibiotics in North-eastern Tanzania and propose appropriate interventions in mitigating the globally rising crisis of antibiotic resistance.This was a community based cross sectional study which was conducted in SanyaJuu ward, Siha district in Kilimanjaro region, North-eastern Tanzania with a population of 116,313. Siha isZ2P (1-P)/d2.On the basis of the prevalence of self-medication of 71.5% in Ilala district, Dar es Salaam, Tanzania and a reOut of seven districts of Kilimanjaro region Siha district was randomly selected. Sanya Juu ward was randomly selected. Systematic sampling was used to select households in all villages of Sanya Juu ward. Head of the household was selected to participate in the study or, in his absence, any adult member above 18 years present was selected. In the presence of more than one adult, always the oldest was chosen as the respondent. Individuals not willing to participate were excluded. A total of 300 respondents were interviewed. Data were collected using a semi-structured questionnaire developed by a research team.The validity and reliability of the questionnaire were determined by using computer software IBM SPSS Version 20. To maximize its validity, the questionnaire was pre-tested on relevant respondents before data collection. Ten community members were selected. These respondents were excluded during data collection for this study. In addition two experts in the field of survey design approved the quality of the questionnaire. After the pre-test, adjustments in phrasing were made.st to 28th April 2017. The questionnaire captured socio–demographics, self–medication practices with antibiotics and other associated conditions like sources of drugs used, reasons for antibiotic self- medication and type of antibiotics employed for self medication.The principal investigator together with two trained research assistants administered the questionnaire from 1Data were entered into an excel spreadsheet and STATA version 13  was used for statistical analyses. Data were visually examined for distribution, missing values and outliers using univariable analysis. We described baseline characteristics of the study population by using descriptive statistics. Median (inter-quartile range) was used to summarize continuous variables and frequencies and proportions for categorical variables. Log-binomial regression was performed to determine the association between socio-demographic characteristics and self-medication. Crude and adjusted Odds Ratio (ORs) or prevalence ratios together with their 95% confidence intervals were estimated as measures of association. We added all variables from the bivariate log-binomial regression into a multivariate log-binomial regression model regardless of their significance. In the final model, the p-value of <0.05 was considered to be significant.Ethical approval to conduct this study was obtained from Kilimanjaro Christian Medical University College Ethics Committee Certificate number 892. Permission to conduct the study was obtained from the District Executive Director of Siha District and Sanya Juu Ward Executive Officer (WEO). Written informed consent to participate was obtained from all individuals who agreed to participate in the study prior to participation. All measures to protect the privacy and confidentiality were considered in that neither names nor house registration numbers were mentioned and recorded during data collection.A total of 300 individuals participated in this study. The median (IQR) age of participants was 23 (20.5–36.5) years. The majority of the participants, 222 (74.00%) were youth and the proportion of males and females was almost similar among the study participants and comparable to the study population. More than half of the participants had attained primary education or higher and a higher proportion (94.67%) were unemployed. The prevalence of antibiotic self-medication was 58% .Above ninety percent of the participants reported to go to the pharmacy for care when they fall sick. The most common symptoms reported by self-medicating participants were cough (51%), fever (23%) and diarrhoea (22%). The most common self- medicating antibiotics employed were amoxicillin (43%) and an antiprotozoal drug metronidazole (10%). The most commonly reported reasons for self-medication were emergency illness (24.0%), health facility charges (20.33%), proximity of pharmacy to home place (17.00%) and no reported reason (16.66%) .On a bivariate log-binomial regression, being an adult , female , unmarried , having a primary education  and having an income of more than TShs180,000 , were associated with the increased risk of self-medication. Other factors were not statistically significant . On a multivariate log-binomial regression after adjusting for variables , being an adult , female , unmarried , and having a primary education , remained to be associated with the increased risk of self—medication although the association was not statistically significant  .All study participants had heard about antibiotic self-medication and 98% said self-medication couldn’t be practiced with all drugs. When sick, 92% went to pharmacy for care. 98% responded that self-medication could result to harmful effects. Ninety nine percent knew that self—medication was not better than medical consultation and above 95% of the participants reported to know that self-medication could result to delays for one to seek medical care as well as development of drug resistance .The study aimed at determining the prevalence and factors for antibiotic self-medication in a rural area of Kilimanjaro Region. It is important to establish and quantify existence of antibiotic self-medication and associated factors due to its consequent antimicrobial resistance for control and mitigation of the vice. The prevalence of antibiotic self–medication in this population was 58%.Our data indicate that more than half of the study population self-medicated with antibiotics. This may be due to lack of health care facilities that adequately cater for the health care needs of the community. One may resort to self-medication in a rural developing country due to non-sustainable medicine supply in health facilities, difficulties in seeing a clinician due to congestion, lack of money to pay for health services, long distance to the health facility, delaying of hospital services, ignorance of the consequences and lack of antibiotic regulatory control–32. DissThe data show that more than nine out of ten individuals went to pharmacy for care when they fall sick. It is a common practice in developing countries to directly resort to pharmacy visits instead of medical consultation for care. One of Diarrhoea was the second common symptom for self-medication. Most of the management of diarrhoea is supportive by oral rehydration and intravenous infusions. AntibioEscherichia coli resistant against penicillins has been published to be between 30%-81.6%[The antibiotics mostly used for self-medication were penicillins 43%). We observe that penicillins are the most commonly known of many antibiotics and freely available, hence potential for self-medication in many countries since they are believed to treat many ailments–43. It i%. We obs30%-81.6%–49. Amox30%-81.6%, Ghana . An observation on the most frequent self-medication with metronidazole occurs in treatment of diarrhoea. Since dIn Tanzania antibiotics can be available and accessed in rural and peri-urban areas through Accredited Drug Dispensing Outlets (ADDO) Programme–58. DispThe accessibility of antibiotics through ADDO initiative has occasionally negatively resulted to abuse with evidence of non-prescription purchase of antibiotics–61. A moWe found a high level of awareness of the negative consequences of antibiotic self-medication despite a reported high practice of the same. All the study population were aware of harmful effects of antibiotic self-medication including antibiotic resistance. They also reported to have heard of self-medication, but, surprisingly 98% had practiced self-medication. This is cognitive dissonance theory at work. Sixteen percent of the self-medicating respondents had no reason why they practiced it. Clearly, a deliberate and a serious government initiative to enforce the restriction of availability and inappropriate accessibility to antibiotics may not be over emphasized.The most important reasons for antibiotic self–medication given by participants were emergency illness (24%) and health facility charges (20%). Emergency illness are taken care of at home instead of health care facility thereby encouraging self-medication mostly from “left over” antibiotics. Health This study commands strength by involving the general population, which is an attempt to describe in practical terms a reality on the ground in self-medication practice, unlike other studies that involved institutionalized populations like university students, health-care workers or outpatients in a hospital ,33,64–69Self-medication with antibiotics is practiced by more than a half of Siha district residents predominantly by adults than youth and elderly. Majority (92%) of respondents go to pharmacy for care on falling sick. Antibiotic self-medication is mostly due to cough, fever and diarrhoea. Factors associated with increased risk of antibiotic self-medication are being an unmarried adult female with a primary school education. Despite a high awareness of the negative effects of self-medication, self-medication practice remains high in Siha district. The findings of this study warrant government intervention through TFDA by mass education, dispensers’ education and lawfully enforcing restriction on access to antibiotics on prescription only policy. An avenue for further study should envisage identifying, through a qualitative methodology, whether the same respondents in this study understand the consequences of antibiotic resistance caused by antibiotic self-medication.S1 Questionnaire(DOC)Click here for additional data file.


Non-AIDS-related mortality rates among HIV-infected patients still exceed those of their uninfected peers. A major driver of this excess mortality is a higher risk of non-infectious comorbidities, including cardiovascular disease, chronic kidney disease, type 2 diabetes mellitus, osteoporosis and cancer. The prevalence of mental illness and other chronic non-infectious comorbidities is identified as a primary concern of antiretroviral prescribers in Australia.We conducted a cross-sectional, observational study using data from MedicineInsight, a large-scale Australian primary care database comprising longitudinal data from electronic clinical information systems. The HIV-infected cohort included all men with a recorded diagnosis of HIV. The non-HIV-infected cohort comprised all other men from the same practices. The prevalence and risk of cardiovascular disease, chronic kidney disease, type 2 diabetes mellitus, osteoporosis, cancer, anxiety and depression were compared between the groups.We included 2,406 HIV-infected males and 648,205 males with no record of HIV diagnosis attending primary care in this study. HIV-infected men were less socioeconomically disadvantaged and more urban-dwelling than men in the primary care cohort. We found that HIV-infected men attending primary care in Australia are at increased risk of chronic kidney disease, cancer, osteoporosis, anxiety and depression. There appears to be a risk of premature onset of cardiovascular disease, osteoporosis and cancer among younger HIV-infected patients. There is a high prevalence of anxiety and depression among HIV-infected men.Increased prevalence of non-infectious comorbidities among HIV-infected men has broad implications for the effective management of those with these chronic conditions. Education to raise awareness among both HIV-infected men and their care providers, together with a greater focus on risk reduction, monitoring and preventive care, may be effective strategies in primary healthcare settings to further narrow the gap in health outcomes between people living with HIV and their uninfected counterparts. The widespread use of highly active antiretroviral therapy and success in achieving durable viral suppression have led to dramatic demographic and epidemiological shifts in the population of people living with HIV. Non-AIDS-related causes of death now exceed AIDS-related illnesses as the major drivers of mortality among Australians living with HIV . HIV-infHIV-infected patients are at an increased risk of a number non-infectious comorbidities (NICMs) including cardiovascular disease (CVD) , chronicThe specific mechanisms underlying the difference in prevalence and risk of NICMs between HIV-infected and uninfected cohorts are not clear; HIV infection itself, antiretroviral toxicity, chronic immune activation and patient factors such as lifestyle and socio-economic status, among others, are likely to play a role. It remains unresolved whether NICMs occur at a younger age in HIV-infected people or whether there is a higher burden of NICM in HIV-infected people overall without a premature onset , 14.There are no published population-based estimates of the prevalence of NICMs among HIV-infected people in Australia. We undertook this study to assess the prevalence of common NICMs in HIV-infected people in Australia; an area identified as clinically important but about which little has been published. Improving our understanding of NICM prevalence in HIV-infected people represents an opportunity to improve clinical outcomes with effective screening of at-risk patients and early modification of risk factors .MedicineInsight is a national primary care data program developed and managed by NPS MedicineWise and is the first large-scale national primary care data collection program in Australia. MedicineInsight extracts longitudinal de-identified clinical data from primary care clinical information systems, and contains data on patient demographics, encounters, diagnoses, prescriptions, pathology tests and referrals. MedicineInsight includes prospectively gathered records for more than 3.5 million patients, with over 3,500 GPs from over 640 participating primary care practices. The patient population registered on the database is largely representative of the age, sex and geographical distribution of the Australian population .We have used data from MedicineInsight to investigate the prevalence and risk of seven common NICMs  among HIV-infected men compared to men without HIV who attend Australian general practices enrolled in MedicineInsight. We restricted the results and discussion presented here to male patients only, to eliminate sex as a confounder between the two groups.A cross-sectional observational study was conducted using the June 2017 MedicineInsight download. Data were extracted from all relevant available data fields including patient demographics, diagnoses and symptoms, observations and referrals. Progress notes containing free text are not available, as they can incorporate patient-identifying information. Patient location was assigned using the Australian Bureau of Statistics (ABS) mapping of the patient’s postcode of residence to Australian Statistical Geography Standard (ASGS) Remoteness Areas 2011 .Patients were selected for inclusion in the study if they were aged 18 years or over, alive, marked as active by the practice, had 3 or more visits to the practice in the past 2 years, and were treated in a primary care practice that met the following MedicineInsight standard data quality requirements:established as a practice for at least 2 years, to ensure adequate longitudinal data on patientsno gaps of more than 2 months in the previous 2 years in data entry into key data tables , anddata available for at least 50 patients in the 2 years prior to the database build, to exclude practices that did not record clinical data in their CISs.The cohort of HIV-infected patients was defined as those with a recorded diagnosis of HIV at any time in their medical history, in any designated text or code field in the diagnosis tables, reason for prescription, or reason for visit. Patients were excluded if their HIV had been diagnosed within the preceding 6 months, as we were specifically looking at comorbidities associated with longer term HIV infection.The general primary care population cohort was drawn from all other eligible active patients attending the same general practices from which the HIV-infected patient cohort was selected, and for whom there was no indication of a diagnosis of HIV or AIDS.We have investigated the recording and relative risk of specific NICMs in HIV-infected people and the general practice population. We developed coding algorithms to identify conditions and symptoms of interest within the MedicineInsight database, using commonly accepted clinical definitions, terms and synonyms from SNOMED CT-AU . ClinicaFor NICMs, the data were coded based on the dichotomous outcomes of “ever been diagnosed within lifetime” and “no record of diagnosis”. While this is not intended to be representative of the overall lifetime prevalence of the condition, the recorded rates for CVD and T2DM in the MedicineInsight dataset are similar to reported national prevalence rates .Descriptive statistics, including percentages, relative risks and associated 95% confidence intervals (CI), have been used to describe the cohort of HIV-infected patients and the general population comparator according to socio-demographic characteristics and risk factor profile. The significance of the effect of age, rurality or socioeconomic status (as indicated by SEIFA quintiles) was assessed using multivariable analysis and determination of odds ratios (ORs) with associated 95% CIs, based on the final logistic model fitting. Analyses were conducted using SPSS Statistics v23 (IBM).General practice data collection serves the primary purpose of managing patient care. The quality and completeness of MedicineInsight data are dependent on the accuracy and completeness of data recorded in, and available from, the general practice clinical information systems.A limitation of MedicineInsight is that patients are not linked across practices, and patients who regularly visit more than one practice that contributes to MedicineInsight may be recorded more than once. It has been estimated that approximately 4% of general population patients may be duplicates .This study was approved by the Bellberry Human Research Ethics Committee (2016-10-781).We identified 2,630 HIV-infected patients and 1,522,550 general population patients who met the study population selection criteria. The HIV-infected cohort was 91.5% male, with 2,406 males and 222 females. In comparison, the general practice population was 57.3% female and 42.6% male . We therefore restricted the results presented here to male patients only, in both HIV-infected and uninfected cohorts, to eliminate sex as a confounder, ensure adequate statistical power (particularly upon stratification by age) and to reduce the potential risk of patient re-identification due to small cell sizes .The demographic distribution of the HIV-infected cohort, as predominantly urban-dwelling men aged 45–64 years, differs significantly from that of the general practice population, consistent with previously reported HIV demographic and prevalence characteristics in Australia . Almost Male HIV-infected patients had statistically significantly higher (p < 0.001) overall prevalence rates of depression, anxiety, cancer, osteoporosis and CKD compared to the male general practice population . There wWe investigated the age-stratified odds ratio (OR) of these NICMs, adjusting for rurality and socioeconomic status (SEIFA quintile), and additionally adjusting for age in the overall prevalence analysis . We founOverall prevalence and risk of all-cause cancers were significantly higher for male HIV-infected patients. HIV-infected males aged 30–44 were three times more likely to have had a diagnosis of cancer than the age-matched patients from the general practice population . Cancer rates were also significantly higher in HIV-infected males aged between 45 and 74 years. Although not statistically significant, there was also a higher risk of cancer in HIV-infected patients aged 18–29 years . Cancer prevalence increased with age in both HIV-infected and general practice population male patients.There was a small but significant difference in overall CVD prevalence between male HIV-infected and male general practice population patients . When stratified by age, there was a significantly higher risk of CVD in the younger male HIV-infected patients aged 18–29 years and in male HIV-infected patients aged from 45–64 years and 65–74 years.The overall risk of osteoporosis was higher in male HIV-infected patients compared to the male general practice population . Rates of osteoporosis were also significantly higher in male HIV-infected patients aged 30–74 years, and HIV-infected patients aged 30–44 and 45–64 years were at least six times more likely to have been diagnosed with osteoporosis than males in the general practice population .T2DM was the only NICM investigated for which the overall risk was significantly lower in the HIV-infected males compared to the male general practice population . While T2DM was relatively uncommon in younger male patients from either the HIV-infected or general practice population groups, prevalence and risk increased with age, and risk was significantly lower in the HIV-infected population aged 45–64 and 65–75 years compared to the general practice population . CKD diagnosis rates were also low in younger age groups, but were significantly higher in HIV-infected males in the 45–64- and 65–74-year age groups .Our results also show that 28% of HIV-infected males had 2 or more of the NICMs investigated here compared with only 17% of the male general practice population, while only 41.6% of male HIV-infected patients had no NICMs, compared with 63.2% of the male general practice population . The risIn this study of patients attending primary care practices in Australia we found a higher prevalence of depression, anxiety, cancer, osteoporosis and CKD in HIV-infected males compared with the male general practice population. We found a lower prevalence of T2DM in the HIV-infected cohort. While the overall prevalence of CVD was no different between the groups, after adjusting for age, socioeconomic status and rurality HIV-infected males had a significantly higher risk of CVD than the general practice population. Our findings confirm a higher burden of NICM among HIV-infected men in Australia.Data on the prevalence and risk of depression among people living with HIV are restricted to small methodologically limited studies that estimate the point-prevalence of depression to be between 20% and 30% , 20. In The antiretroviral era has seen a fall in the incidence of AIDS-defining cancers like Kaposi sarcoma and non-Hodgkin lymphoma . While tHIV-infected patients have a 1.5 to 2.5-fold increased risk for major cardiovascular events . We repoThe association between HIV infection and osteoporosis is well described . AntiretIt is well established that HIV-infected patients are at increased risk of chronic kidney disease , 28. In An association between HIV infection and risk of T2DM has not been confirmed. Published series report either an increase in risk or no difference in risk among HIV-infected subjects , 14, 28.In conclusion, the increasing prevalence of NICMs among people living with HIV has led to increased complexity of care and has implications for the screening, diagnosis and effective management of these chronic conditions in healthcare settings more accustomed to the management of acute HIV-related presentations. Raising awareness of the increased prevalence of NICMs among HIV-infected men and their care providers and placing a greater focus on reduction of risk and preventive care in the primary care setting may be effective strategies to further narrow the gap in health outcomes between people living with HIV and their non-infected counterparts.


Levetiracetam (LEV) is effective for focal and generalized epilepsy and is used worldwide because of its relatively few drug interactions and favorable tolerability. However, some psychiatric adverse events (PAEs) have been reported, resulting in drug withdrawal. The pathophysiology of LEV-induced PAE has not yet been elucidated. In this study, we investigated the relationship between PAEs and human leukocyte antigen (HLA) genes. Eleven epilepsy patients, who developed PAEs after the administration of LEV and spontaneously improved after drug withdrawal, were enrolled retrospectively. Genomic DNA from the peripheral blood was extracted, and four-digit allele genotyping of HLA genes was performed. The genotype frequencies of HLA genes were compared to those of 80 patients in which LEV was well tolerated, as well as to 485 individuals from the general Korean population. The frequency of the HLA-A*1101 allele was significantly higher in the LEV-induced PAEs group compared to both the LEV-tolerant group  and the general Korean population . This study is the first attempt at investigating the relationship between the HLA system and LEV-induced PAE. The results of this study suggest that the HLA-A*1101 allele could be a risk factor for the development of PAEs. Levetiracetam (LEV) is a broad-spectrum antiepileptic drug (AED)–3, whichThe known risk factors for PAEs during LEV treatment are previous psychiatric history, family psychiatric history and history of febrile convulsions., 8 HowevMany studies have demonstrated the associations of the human leukocyte antigen (HLA) in patients with various psychiatric diseases, including schizophrenia, schizoaffective disease, and mood disorders.–11 SinceIn the current study, we attempted to investigate the HLA-related risk factors for PAEs of LEV for the first time.We retrospectively enrolled 11 patient who experienced significant psychiatric symptoms after the administration of LEV among the patients with epilepsy who were treated with LEV at Seoul National University Hospital. A diagnosis of LEV-induced PAE was made when psychiatric symptoms or behavioral changes occurred after administration of LEV and when those symptoms were improved spontaneously after drug withdrawal. Psychiatric symptoms included hallucinations, delusions and behavioral changes consisting of aggression, irritability, and nervousness. When psychiatric symptoms were clinically confirmed, LEV administration was discontinued immediately to prevent serious adverse events. Patients with the following conditions were excluded: (1) previous history of mental illness, (2) severe mental retardation, (3) incomplete clinical data, or (4) uncertainty of LEV as the causative drug. Eighty epilepsy patients who were tolerant to LEV (LEV-tolerant group) were included as a control group. The LEV-tolerant group consisted of patients who did not exhibit any psychiatric symptoms while taking over 2000 mg/day of LEV. Additionally, we used 485 individuals from the general Korean population as the other control group. This stuAfter genomic DNA extraction from the peripheral blood of all patients belonging to the LEV-induced PAEs group (LEV-PAEs group) and the LEV-tolerant group, HLA genotyping was performed. Four-digit allele genotyping of the HLA class I and class II genes, including HLA-A, HLA-B, HLA-C, HLA-DRB1 and HLA-DQB1, was performed using direct DNA sequence analysis, according to the established protocols ., 16, 18 http://www.hmdb.ca) and used a computational program, Autodock Vina,[In silico docking was performed as previously described, using thock Vina, to calcuThe statistical analysis was performed using SPSS Statistics for Windows, version 22.0 . Fisher’s exact test was used to identify differences in the HLA frequencies among the following three groups: the LEV-PAEs group, the LEV-tolerant group and the general Korean population. Odds ratios (ORs) and 95% confidence intervals (CIs) were obtained. A two-tailed p-value < 0.05 was considered statistically significant.A total of 11 patients  who experienced LEV-induced PAEs and 80 patients who tolerated LEV treatment were included in this study. Demographics and clinical characteristics of the patients with LEV-induced PAEs are shown in The results of the genotyping of the LEV-PAEs group patients are presented in The binding affinity of the LEV molecule to HLA-A*1101 was compared to the binding affinity obtained from docking with seven HLA-A alleles whose crystallographic structures are available. The LEV molecule was predicted to be docked into the P1 pocket of HLA-A*1101  with a dThis is the first study to investigate the HLA-related risk factors for LEV-induced PAEs in epilepsy patients. We identified significant associations between LEV-induced PAEs and the HLA-A*1101 allele. The in-silico analysis revealed that the LEV molecule was predicted to dock into the P1 pocket of the MHC class I heterodimer including A*1101. This finding provides a new insight into the pathogenesis of LEV-induced PAEs, and further studies using larger numbers of patients are required.LEV is a second-generation antiepileptic drug that has been proven to be effective in focal and generalized epilepsy,–3 and isIn recent studies which investigated the possible associations among the location of brain lesion, the use of AEDs and the development of PAEs in patients with brain tumor-related epilepsy, frontal lobe tumors were highly associated with the development of PAEs after LEV treatment., 28 In aPAEs of LEV occur unexpectedly and independently of the dose, occurring even at doses below 1000 mg/day, which suIn this study, the frequency of the HLA-A*1101 allele was significantly higher in the LEV-PAE group than in the LEV-tolerant group or the general Korean population, which suggests that LEV-induced PAEs may also be related to the HLA genes. Previous studies have shown that immune responses are involved in the development of psychiatric diseases, including schizophrenia and bipolar disorders, and have suggested several HLA genes related to the pathogenesis of these diseases. Two previous studies have shown that patients with schizophrenia had higher rates of the HLA-DRB1*0101 genes than the controls in the Japanese population., 21 In aHLA-A*1101 is the dominant serotype of HLA-A11 (A11), which is one of the most common HLA class I genotypes in the world. HLA-A foLEV-induced idiosyncratic reactions may be caused by directly triggering the CTL response via the HLA system. The in-silico docking analysis has demonstrated that the LEV molecule binds within the P1 pocket of the peptide binding groove of HLA-A*1101, with a relatively stronger affinity than other HLA subtypes. Several reports have shown that drug molecules can directly interact with HLA molecules in combination with specific peptides and trigger unexpected immune reactions. AbacavirOne of the limitations of our study is the small number of patients included in the LEV-PAE group. Additionally, an objective psychiatric assessment scale was not used to evaluate the PAEs, which were mainly assessed by the clinician’s judgement. Therefore, we have excluded all the patients with unclear symptoms or unclear causality with LEV administration from the larger number of patients suspected to have PAEs. Despite the reduction in the number of patients, we made great efforts to select definite LEV-induced PAEs, which were supported by the spontaneous improvement of the psychiatric symptoms after drug removal.In conclusion, we suggest for the first time that the HLA system is associated with LEV-induced PAEs, and the HLA-A*1101 allele could be a risk factor for the development of psychiatric symptoms. Identifying the genetic risk factors for LEV-induced PAEs, along with other known risk factors, will be significantly beneficial to clinicians when prescribing LEV. To strengthen the findings obtained from the current study, additional studies using objective assessment instruments of psychiatric symptoms in larger numbers of patients or in different ethnic groups will be required in the near future.


Background: Late-type sensitizations against wood are rare and are mostly seen in patients with occupational contact with saw dust. Generally, commercial wooden products only lead to sensitization or dermatitis in cases when contact to the unprotected skin is direct, intensive and of longer duration, i.e. by wearing wooden jewelry or by playing wooden instruments. Causative for the sensitization process are ingredients of the wood core such as alkaloids, glycosides, anthrachinones, saponines, phenols, catechols, flavonoids, cumarins, and benzo-, naphtho-, furano-, and phenanthrenquinones and their precursors. Case report: We report the case of a 70-year-old patient who developed cheilitis after playing different wooden recorders made of African blackwood, rosewood, cedar, olive, and pear. Methods: Patch testing with baseline series of contact allergens and saw dust of the recorders were performed as well as skin prick tests with common inhalant allergens and saw dust of the recorders. Results: Patch testing showed sensitizations against African blackwood, rosewood, fragrance mix 2 and hydroxyisohexyl-3-cyclohexene carboxaldehyde. Skin prick testing was without pathological results. Conclusion: African blackwood and rosewood contain (S)-4’-hydroxy-4-methoxydalbergione and (S)-4-methoxydalbergione. As a result of the chemical affinity between the dalbergiones, cross-reactions between different woods are observed. The case presented shall show the diagnostic procedure in cases in which sensitizations against wood components are suspected.  The dense heart wood is of a purple-blackish color with black stripes and can be mistaken for ebony. Due to its nice color and to its hardness it is frequently used for wooden instruments like clarinets, oboes and recorders, but also for chess pieces and combs , 4. A 70-year-old patient presented to our allergology department with a 2-year history of relapsing erosive cheilitis . The cha® on Scanpor  and attached with Fixomull Stretch Tape . The allergens were removed 24 hours later to avoid irritant reactions. The test results were checked after 24, 48 and 72 hours according to the guidelines published by the International Dermatitis Research Group (ICDRG).We carried out patch tests using the standard allergens  and saw dust from the mouthpieces of his recorders (10% in petrolatum). The allergens were applied on his upper back using Finn ChambersInhalable allergens  were tested in a routine prick test using commercially available test solutions . Furthermore a skin prick test with saw dust from the different mouthpieces of his recorders (1% in water) was carried out. The results of this skin prick test were checked after 20 minutes and after 24 hours. The patch testing and the prick testing were also carried out in 7 control persons who had presented to our hospital for diagnostic work-up of occupation-related sensitizations. These control persons were informed about the aim of the testing and about the possibility of an active sensitization and gave their written consent to the additional testing.The patch testing showed positive reactions to African blackwood and rosewood  as well Dalbergia melanoxylon or African blackwood is a tropical hardwood that can easily be mistaken for ebony (Diospyros species) due to its color. The heart wood is very hard and dense and is used for the production of wooden recorders due to its nice color as well as its temperature and moisture resistance. All Dalbergia species can induce irritant or allergic reactions in skin and mucosa . IrritanThe reason for allergic reactions after contact with African blackwood are, like in the case of other Dalbergia species, the quinones (S)-4’-hydroxy-4-methoxydalbergione and (S)-4-methoxydalbergion. As a result of the structural similarity of all dalbergiones cross-reactions between different woods are observed . This waThe case presented here is supposed to remind dermatologists of the fact that, in rare cases, wooden instruments can cause contact allergic skin reactions. When sensitization to certain woods is suspected, patch and prick tests with alcohol or water extracts of the suspected woods should be carried out , 5. As tNone.


Undernutrition impairs child development outcomes and growth. In this follow-up study of an open cluster-randomized intervention trial we examined the effects of an education package delivered to mothers in rural Uganda on their children’s development, growth and gut microbiota at 36 months of age.The parental trial included 511 mother-child pairs recruited when the children were 6-8 months. In that trial, a nutrition, stimulation and hygiene education was delivered to mothers in the intervention group while the control group received routine health care. A follow-up sample of 155 pairs  were re-enrolled when the children were 24 months. Developmental outcomes were assessed with the Bayley Scales of Infant and Toddler Development (BSID-III) composite scores for cognitive (primary endpoint), language and motor development. Development outcomes were also evaluated using the Ages and Stages Questionnaire (ASQ) and the Mullen Scales of Early Learning (MSEL). Other outcomes included growth and gut microbiota composition.P > 0.05) between the intervention and control groups and similar to those of the parental study. The intervention group had higher BSID-III scores than controls, with mean difference 10.13 : 3.31-17.05, P = 0.002); 7.59 ; 9.00 , for cognitive, language and motor composite scores, respectively. An improvement in the intervention compared to the control group was obtained for both the ASQ and the MSEL scores. The mean difference in height-for-age z-score was higher in the intervention compared to the control group: 0.50 . Gut microbiota composition did not differ significantly between the two study groups.The demographic characteristics were not different (The maternal education intervention had positive effects on child development and growth at three years, but did not alter gut microbiota composition. This intervention may be applicable in other low-resource settings.ClinicalTrials.gov registration number NCT02098031. Undernutrition among children in developing countries is a major, global health challenge causing more than one-third of under-five deaths . About 2The underlying causes of chronic undernutrition are complex. In addition to inadequate food quantity and quality of food, a combination of poor sanitation and hygiene resulting in sustained exposure to enteric pathogens plays an important role . PregnanIn 2013 we initiated a cluster-randomized controlled trial to examine the effect on growth and development of a 6-month intervention comprised of nutrition, stimulation and hygiene education among impoverished mothers of children aged 6-8 months in rural districts of Uganda . The intThis is a follow-up study of a two-armed, open cluster-randomized education intervention regarding nutrition, stimulation and hygiene among impoverished mothers of children aged 6-8 months in the Kisoro and Kabale districts of South-Western Uganda. Details of the intervention have recently been published . All motFor the parental trial we used proportionate sampling, 10 sub-counties  were obtained  to participate in the study. We used a three-stage procedure to identify households for the study. First, by simple random sampling, three sub-counties in Kabale were allocated to the intervention group and the other three to the control group. Similarly, two sub-counties were allocated to the intervention and the other two to the control group in Kisoro district. Second, all the villages in each participating sub-county (intervention or control) were listed alphabetically and assigned numbers in an ascending order. By use of computer-generated random numbers, villages to whose assigned number matched with the random numbers were selected. The intervention villages did not share common geographical boundaries with control villages to minimize contamination of the intervention contents between the two study groups. Third, by complete enumeration, all consenting households with children aged 6-8 months within a participating village were recruited to the study. If a household had more than one eligible child, the youngest was selected, and in the case of twins, we randomly selected one for evaluation. We finally enrolled 511 mother-child pairs in the parental study and they were randomised to the intervention (n = 263) or the control (n = 248) group. The intervention group received the nutrition, hygiene and stimulation education in addition to routine health care while the control group received only routine health care.The child had to be 20-24 months during the period of January-May 2015 to be included in the current follow-up study since age dynamic gut microbial shifts occur at this age resulting in an adult-like, stable composition , and devThe intervention was conducted by the study team at three group meetings over a period of 6 months to 26 groups of mothers (6-10 mothers per group), and was detailed recently . BrieflyThe child development assessments were performed by three bachelor degree holders in psychology whereas two graduates of laboratory technology collected stool samples. Two bachelor degree holders in nutrition collected the anthropometric data. These three data collection teams participated in training sessions to ensure uniform and standardized procedures. Assessments were administered in the local language and conducted in hired, secluded rooms in the villages without interruptions to minimize distractions. To promote reliability, the child development assessments were administered first, followed by anthropometric measurements, stool sampling and then interviews with the mothers.P = 0.0001) for BSI-III, 0.79 (P = 0.0001) for ASQ and 0.77 (P < 0.001) for MSEL.The Bayley Scales of Infant and Toddler Development-III (BSID-III), the Ages and Stages Questionnaire (ASQ) and the Mullen Scales of Early Learning  were used . The BSIWeight, height, and head circumference (HC) were measured as recommended by WHO , with a We collected stool samples using sterile cotton swabs  and frozen at -20°C within 24 hours of collection. The samples were then air-dried and shipped to the Netherlands for further processing and analyses (Supplementary information). These storage conditions have a very limited effect on the microbial composition [P < 0.05. We used a mixed effect linear regression to compare the intervention with the control group and estimated ICC. Differences between the two study groups are given as mean (SD or 95% CI).The primary outcome in the current follow-up study was cognitive development assessed with the BSID-III at 36 months. Previous intervention studies in similar low-resource-settings report a mean difference of about 0.5 SD in child development score between intervention and control groups . To deteAll statistical analysis of gut microbiota on the 16S rRNA amplicon sequencing data was performed using R version 3.3.2  . The 16SOne hundred and fifty-five mother-child pairs were included at 20-24 months  based on the BSID-III at 36 months  and at the phylum (P = 0.001) level, but that there was no significant effect (P = 1) of the intervention on the overall gut microbiota composition.The intervention did not lead to any significant changes in the gut microbiota diversity compared with the control group at the phylum level (This is probably the first randomized education intervention trial incorporating gut microbiota analysis in rural Sub-Saharan Africa. In the parental trial the 6-month education intervention led to significant improvements in development outcomes when the children reached 20-24 months, without affecting growth . We now Our effect sizes on child development outcomes were comparable/higher than those obtained in previous studies that included nutrition supplementation and child stimulation intervention ,29,30. SThe baseline stunting levels observed in the two study groups compare favorably with those of previous surveys in Uganda . CorroboEmerging data suggest links between gut microbiota composition and stunting as well as cognition in childhood ,35, possOur baseline data on maternal and household characteristics were in line with previous reported data from Uganda -42. Our In this study we adopted a multidisciplinary approach combining aspects of nutrition, hygiene, psychology, microbiology and validated research instruments. Of note, the children were followed for several years. Despite that only about one-third of the mother-child pairs of the parental trial could be re-enrolled for this follow-up study, the latter cohort was well balanced with the baseline characteristics of the parental cohort. A limitation of our study was lack of baseline data of gut microbiota composition, and we have no information about body composition, dietary intakes or relevant biomarkers among the children or if the mothers in the intervention group continued to stimulate their children in the period between end of intervention and when the children reached the age of three years. ASQ is a maternal report and could possibly be biased. Furthermore, we do not report on maternal mental health which may impact on development and growth of small children, in particular in low-resource settings .This nutrition, hygiene and stimulation education intervention among mothers of 6-8 months old children had a positive effect on child development and growth until 36 months. We found no significant effects of the intervention on gut microbiota composition. The positive effects from this intervention would call for further research of such an intervention before consideration of scale-up and implementation in other low-income rural settings.


Elevated plasma branched-chain amino acids (BCAA) levels are often observed in obese insulin-resistant (IR) subjects and laboratory animals. A reduced capacity of the adipose tissues (AT) to catabolize BCAA has been proposed as an explanation, but it seems restricted to obesity models of genetically modified or high fat–fed rodents. We aimed to determine if plasma BCAA levels were increased in a model of IR without obesity and to explore the underlying mechanisms. Rats were fed with a standard diet, containing either starch or fructose. BCAA levels, body weight and composition were recorded before and after 5, 12, 30, or 45 days of feeding. Elevated blood BCAA levels were observed in our IR model with unaltered body weight and composition. No changes were observed in the liver or the AT, but instead an impaired capacity of the skeletal muscle to catabolize BCAA was observed, including reduced capacity for transamination and oxidative deamination. Although the elevated blood BCAA levels in the fructose-fed rat seem to be a common feature of the IR phenotype observed in obese subjects and high fat–fed animals, the mechanisms involved in such a metabolic phenomenon are different, likely involving the skeletal muscle BCAA metabolism. The place of amino acids  has been recently questioned in the metabolic dysfunctions and plasma metabolites alterations associated with insulin resistance (IR) occurrence . Indeed,Considering that an alteration of the catabolic capacities of the adipose tissues may not be the only explanation for the increased BCAA plasma level observed in IR development , the aimEight-week-old Sprague-Dawley male rats  were housed individually and kept in a controlled environment .n = 40) or fructose , during 45 days. Rats had free access to fresh tap water. The control group was pair-fed with respect to the fructose group in order to match the daily food intake. The animals were handled according to the recommendations of the Regional Ethics Committee (agreement number CE-2811). All procedures were in accordance with the guidelines formulated by the European Community for the use of experimental animals .After one week adaptation, animals were divided into two groups and fed experimental diets (based on AIN 93G diet), containing as source of carbohydrates (~63%), either starch  and after 5, 12, 30 or 45 days of feeding blood was withdrawn at the postabsorptive (overnight fasting) and postprandial  state from the tail vein. To avoid the effect of stress by sampling too often , eight different animals per diet (control or fructose) were randomly assigned to the five groups and sampled only at one time. Body weight and body composition  were evaluated at the same days than the blood sampling. Those animals assigned to the d45 sampling were also euthanized under pentobarbital anesthesia in the post-absorptive state (overnight fasting). The liver, the subcutaneous and epididymal adipose tissues, and the gastrocnemius muscle were rapidly removed, weighed, freeze-clamped into liquid nitrogen, and stored at −80 °C for further analyses. Two days before the end of the trial, the eight rats per group sampled at d0 were further subjected to an oral glucose tolerance test, in which they received orally 1 g of glucose/kg of body weight. Blood glucose levels were then assessed before (0 min) and 15, 30, 60, 90, and 120 min after the test using a glucometer . The areas under the curves (AUC) of the glucose excursion was calculated by the trapezoid method.www.OCDEM.ox.ac.uk) as in Levy et al. [+ (4 mM). The amount of BCAA was calculated based on the NADH formation using a standard leucine curve as in [Plasma glucose and triglycerides concentrations were enzymatically measured using commercial kits . Plasma insulin levels were assessed using a commercial ELISA kit . Glucose homeostasis was further estimated using the homeostasis model assessment-estimated insulin resistance using the HOMA2-IR model  were subjected to SDS-PAGE, electrotransferred on a PVDF membrane, and probed with the indicated antibodies: total BCKDH (branched-chain α-ketoacid dehydrogenase complex) and phosphorylated BCKDH-α at serine 293 (Abcam); total S6  and phosphorylated S6 at Ser240/244 antibodies . Bands were visualized by infrared fluorescence using the Odyssey imaging system. Phosphorylated proteins were corrected for the respective total proteins. The signal of each protein was further normalized by the total protein content of the gel  .®  and mRNA levels were determined by RT-PCR. cDNA was generated from 1 μg RNA using High Capacity cDNA Reverse Transcription Kit . Real-time PCR was performed in the CFX96 Touch™ Real-Time PCR Detection System  as in [Total RNA was extracted from livers and gastrocnemius muscles using RNEasy Mini KitA) as in . Primersp-value significance threshold was set at p < 0.05.The differences in BCAA levels during the time-course experiment and glucose levels in the oral glucose tolerance test were analyzed using a two-way ANOVA test, followed by post-hoc Holm-Sidak comparison . The differences between groups in the parameters evaluated at the end of the trial were performed using a t-test analysis. The The plasma BCAA levels during the time-course fructose feeding in both, the fasting and postprandial states are shown in Blood glucose and insulin levels were also assessed at the fasting state . All theBased on the well-established relationship between fructose feeding and IR on rodent models , and betTaking into account the major role of the adipose tissue in BCAA metabolism described in the literature ,6,7, we p-value for diet was 0.94, 0.43, and 0.69 respectively).Body weight, fat mass, and lean mass increased significantly with the time, but none of these parameters was affected by the IR induced by the fructose feeding  and oxidative deamination (phosphorylation status of BCKDH) potentials  expression was affected at the hepatic levels, with a 1.3-fold increase in the fructose-fed rats. In the skeletal muscle, this transcription factor was instead reduced by 1.5-fold. Further, both ppargc1a and sirt1 (sirtuin 1) were also down-regulated (about 1.5-fold) in the IR animals in comparison to the control group  [Over the last years, IR phenotype has been demonstrated to be associated with higher circulating levels of plasma fasting BCAA . Furtherdb mice)  or nutridb mice) ,29. Heredb mice) ,31,32. Fdb mice) .Very few data are available concerning BCAA levels in an IR model induced by fructose: most of the studies dealing with fructose impact on AA (and BCAA) metabolism have focused on the nitrogen sparing effect of fructose supplementation  and not db/db mice [Among the potential mechanisms able to alter the circulating levels of BCAA, their utilization and oxidation by the different targeted tissues should be considered. In several models of obesity and IR, it has been shown that the increased BCAA levels in blood were the consequence of a reduced capacity of the adipose tissue (importantly developed) to transaminate BCAA and oxidize their ketoacids ,6. In th/db mice . The facAs explained above, as BCAA can be potentially transaminated by several tissues, their whole-body homeostasis is highly dependent on the different organs cross-talk. Thus, previous studies have shown that in diet-induced obesity models liver BCKDH activity could be actually increased and compensate the reduced activity observed in the adipose tissues . We therIn order to fully characterize the model, we also explored the mTOR signaling pathway, which has been suggested to be related to the IR observed in this kind of phenotype. We observed that the mTOR signaling pathway (S6 phosphorylation) was enhanced in the liver and skeletal muscle of fructose-fed rats. Today it is well known that persistent nutrient signaling like elevated BCAA levels might cause IR due to a continuous activation of the mTORC1 signaling pathway . Severaldb/db), that suggests a minor role of the adipose tissue on BCAA homeostasis, at least in this particular model (IR but not obese). Our results also showed that particularly at the skeletal muscle level a reduction in both transamination and oxidative deamination potentials operates, suggesting that these features may participate to the elevated blood BCAA profile. The phenotype of these animals was also characterized by a reduction in several mitochondrial function markers  and an enhanced mTOR signaling pathway  in the IR animals, opening the door to the exploration of further mechanisms linking high circulating BCAA levels and IR. Finally, the fact that after 45 days of fructose feeding rats did develop IR but not obesity makes this model a very interesting tool to further elucidate the specific role of the different tissues  and the relationship between obesity and IR in the context of elevated BCAA levels.As a whole, we showed for the first time that the installation of whole body fructose-induced IR was associated with increased fasting BCAA levels. Further, in the fructose-fed rats BCAA increase took place in the absence of significant changes in body weight and fat mass, a major difference with other models (high fat, Zucker,